Original Mutation Research Articles

Role of mutational reversions and fitness restoration in Zika virus spread to the Americas

Zika virus (ZIKV) emerged from obscurity in 2013 to spread from Asia to the South Pacific and the Americas, where millions of people were infected, accompanied by severe disease including microcephaly following congenital infections. Phylogenetic studies have shown that ZIKV evolved in Africa and later spread to Asia, and that the Asian lineage is responsible for the recent epidemics in the South Pacific and Americas. However, the reasons for the sudden emergence of ZIKV remain enigmatic. Here we report evolutionary analyses that revealed four mutations, which occurred just before ZIKV introduction to the Americas, represent direct reversions of previous mutations that accompanied earlier spread from Africa to Asia and early circulation there. Our experimental infections of Aedes aegypti mosquitoes, human cells, and mice using ZIKV strains with and without these mutations demonstrate that the original mutations reduced fitness for urban, human-amplifed transmission, while the reversions restored fitness, increasing epidemic risk. These findings include characterization of three transmission-adaptive ZIKV mutations, and demonstration that these and one identified previously restored fitness for epidemic transmission soon before introduction into the Americas. The initial mutations may have followed founder effects and/or drift when the virus was introduced decades ago into Asia.

Whole-genome assembly and resequencing reveal genomic imprint and key genes of rapid domestication in narrow-leafed lupin.

Shifting from a livestock-based protein diet to a plant-based protein diet has been proposed as an essential requirement to maintain global food sustainability, which requires the increased production of protein-rich crops for direct human consumption. Meanwhile, the lack of sufficient genetic diversity in crop varieties is an increasing concern for sustainable food supplies. Countering this concern requires a clear understanding of the domestication process and dynamics. Narrow-leafed lupin (Lupinus angustifolius L.) has experienced rapid domestication and has become a new legume crop over the past century, with the potential to provide protein-rich seeds. Here, using long-read whole-genome sequencing, we assembled the third-generation reference genome for the narrow-leafed lupin cultivar Tanjil, comprising 20 chromosomes with a total genome size of 615.8Mb and contig N50=5.65Mb. We characterized the original mutation and putative biological pathway resulting in low seed alkaloid level that initiated the recent domestication of narrow-leafed lupin. We identified a 1133-bp insertion in the cis-regulatory region of a putative gene that may be associated with reduced pod shattering (lentus). A comparative analysis of genomic diversity in cultivars and wild types identified an apparent domestication bottleneck, as precisely predicted by the original model of the bottleneck effect on genetic variability in populations. Our results identify the key domestication genetic loci and provide direct genomic evidence for a domestication bottleneck, and open up the possibility of knowledge-driven denovo domestication of wild plants as an avenue to broaden crop plant diversity to enhance food security and sustainable low-carbon emission agriculture.

PBMC-derived integration-free iPSCs line SDQLCHi039-A from a patient with X-linked agammaglobulinemia carrying a novel 9-bp in-frame deletion in BTK gene.

X-linked agammaglobulinemia (XLA, OMIM #300755) is one of the most common pediatric primary immunodeficiencies characterized by failure to produce mature B lymphocytes and hypogammaglobulinemia, caused by mutation of the gene encoding Bruton's tyrosine kinase (BTK, OMIM *300300), a key regulator in B-cell development. Patientssuffering XLA are prone to recurrentbacterial infection. We established an induced pluripotent stem cells (iPSCs) line from a 3-year-5-month-old boy with XLA caused by a hemizygous in-frame 9-bp deletion in BTK (c.1530-1538delATACCTGGA, p.Y510_E513delEYLEinsE). The iPSCs was verified based on pluripotency markers, original gene mutation and demonstrated trilineage differentiation potential in vitro.

Generation and characterization of an induced pluripotent stem cell line SDQLCHi018-A from a congenital myasthenic syndrome patient carrying compound heterozygote mutations in RAPSN gene

Mutations in RAPSN are an important cause of congenital myasthenic syndrome (CMS). In this study, we generated an induced pluripotent stem cell line (iPSC) derived from a 14-day-old male CMS patient carrying compound heterozygote mutations (c.532-2A>G and c.264C>A/p.Asn88Lys) in RAPSN gene. The established iPSC line harboring the original mutations, possessing a normal karyotype, is able to differentiate into all three germ layers in vitro and expresses pluripotency markers.

T(8;21) Acute Myeloid Leukemia as a Paradigm for the Understanding of Leukemogenesis at the Level of Gene Regulation and Chromatin Programming.

Acute myeloid leukemia (AML) is a heterogenous disease with multiple sub-types which are defined by different somatic mutations that cause blood cell differentiation to go astray. Mutations occur in genes encoding members of the cellular machinery controlling transcription and chromatin structure, including transcription factors, chromatin modifiers, DNA-methyltransferases, but also signaling molecules that activate inducible transcription factors controlling gene expression and cell growth. Mutant cells in AML patients are unable to differentiate and adopt new identities that are shaped by the original driver mutation and by rewiring their gene regulatory networks into regulatory phenotypes with enhanced fitness. One of the best-studied AML-subtypes is the t(8;21) AML which carries a translocation fusing sequences encoding the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO gene. The resulting oncoprotein, RUNX1/ETO has been studied for decades, both at the biochemical but also at the systems biology level. It functions as a dominant-negative version of RUNX1 and interferes with multiple cellular processes associated with myeloid differentiation, growth regulation and genome stability. In this review, we summarize our current knowledge of how this protein reprograms normal into malignant cells and how our current knowledge could be harnessed to treat the disease.

Full-coverage TP53 deep sequencing of recurrent head and neck squamous cell carcinoma facilitates prognostic assessment after recurrence.

This study aims to evaluate whether the accumulation of TP53 mutations is associated with clinical outcome by comparing full-coverage TP53 deep sequencing of the initial and recurrent head and neck squamous cell carcinoma (HNSCC). Medical records and surgical specimens of 400 patients with HNSCC surgically treated with curative intent, of which 95 patients developed local or locoregional recurrence, were reviewed. Of these patients, 63 were eligible for genomic analysis. Full-coverage TP53 deep sequencing of 126 paired initial and recurrent tumor samples was examined using next-generation sequencing (NGS). Temporal changes in the mutation status, molecular characterization, and clinical outcome were compared. Fisher's exact test, Kaplan-Meier method, log-rank test, and Cox regression models were used for statistical analysis. Of the recurrent tumors, 22% harbored accumulation of TP53 mutations, and 16% lost the original mutation. The accumulation of TP53 mutations was significantly more frequent in oral cancer than in pharyngeal or laryngeal cancer (33% vs. 7%, p=0.016). Two-year post-recurrence survival (PRS) was associated with TP53 status for recurrent tumors, but not for initial tumors. The TP53 status for recurrent tumors was an independent risk factor in multivariate analysis (hazard ratio, 5.76; 95% confidence interval, 1.86-17.8; p=0.0023). Approximately one-third of the recurrent HNSCC cases showed a different TP53 status from the initial tumor. Temporal changes in the mutation status differed by primary site. Full-coverage TP53 deep sequencing of recurrent tumors was useful in predicting post-recurrence prognosis.

PATH-42. COMBINED NEXT GENERATION SEQUENCING AND IMMUNOHISTOCHEMISTRY IN THE DIFFERENTIAL DIAGNOSIS OF A PROGRESSIVE NEURONAL-GLIAL TUMOR: A COMPOSITE NEOPLASM

Abstract BACKGROUND Neuronal-Glial tumors (NGT) are rare neoplasms typically benign, and characterized by various admixtures of neuronal and glial components. Occasionally these tumors transform to higher grades and rarely develop other histologic features inconsistent with a neuroepithelial origin, raising the question of histogenesis. We present a case of World Health Organization (WHO) grade I ganglioglioma (GG) diagnosed in a 28 year old man whose tumor, over a period of 8 years, progressed to anaplastic ganglioglioma (AGG, WHO grade III) and ultimately AGG with high grade sarcomatoid features. The sarcomatoid component eventually outgrew the NGT components, resulting in the patient’s death. We used next generation sequencing (NGS) and immunohistochemistry to characterize the genetic changes associated with these events. METHODS Tissues representing NGT and sarcomatoid components were macrodissected from 6 samples representing the tumor during its evolution from low to higher grades. DNA and RNA were extracted from these samples and NGS performed using the Oncomine Comprehensive Panel. Whole exome sequencing was performed using DNA from the 6 tumor samples and adjacent non-neoplastic tissue. RESULTS The original tumor, WHO grade I ganglioglioma had a BRAF V600E mutation. Seven years later, the AGG component had the BRAF V600E, absent in the sarcomatoid component, which had a CDKN2A homozygous deletion. The following year, the sarcomatoid component appeared to predominate and had an additional PIK3CA activating mutation. The NGT component retained the original BRAF mutation but no others. Hierarchical clustering of genome-wide variants identified two distinct cell populations emerging over time: GG progressing to AGG and sarcomatoid tumor. CONCLUSIONS Based on these results, we propose two distinct tumor cell populations: GG/AGG and sarcomatoid component, evolving over time and resulting in what is commonly considered a collision tumor, two independent tumors adjacent to each other.

Cytokine-Induced Memory-like NK Cells Exhibit Massive Expansion and Long-Term Persistence after Infusion Post-Haploidentical Stem Cell Transplantation: A Report of the First Three Cases in a Phase I Trial

Cytokine-Induced Memory-like NK Cells Exhibit Massive Expansion and Long-Term Persistence after Infusion Post-Haploidentical Stem Cell Transplantation: A Report of the First Three Cases in a Phase I Trial

RUNX1/ETO and mutant KIT both contribute to programming the transcriptional and chromatin landscape in t(8;21) acute myeloid leukemia

Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.

Identification of a novel gene controlling homothallism in the yeast Kazachstania naganishii isolated in Japan.

A novel gene controlling homothallic life cycle was identified in the yeast Kazachstania naganishii isolated in Japan. This gene was isolated by means of complementing a mutation, mti1, which had led to heterothallism from original homothallism in the yeast. The configuration of original mutation in MTI1 gene revealed that a truncated product is formed due to occurrence of a stop codon by a nucleotide insertion. When the gene was disrupted with a marker, the disruptant spore clone was haploid and stably heterothallic. Disfunction of the gene caused inability to self-diploidize due to defect of mating-type interconversion. The gene MTI1 (for Mating Type Interconversion) is a weak homolog of the Saccharomyces cerevisiae VID22/ENV11, which has been reported to function in vacuolar protein processing. K. naganishii has a gene representing significant homology with the HO gene of S. cerevisiae on chromosome V, which has not been clarified to be involved in regulation of life cycle in K. naganishii. The MTI1 gene defined in this study is located on K. naganishii chromosome IV and does not represent significant homology to the above ScHO-like gene and any other genes concerning life cycles of yeasts. From the viewpoint of gene evolution, it is extremely interesting that the MTI1 gene is a new type of gene controlling homothallism in addition to an HO-type gene, leading to discovery of an unknown mechanism regulating life cycles in yeasts.

Genetic diagnostic features after failure of initial treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors among non-small-cell lung cancer patients harboring EGFR mutations

BackgroundOsimertinib, a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), can be used as second-line treatment for lung cancer patients harboring the T790M substitution. Although osimertinib is more effective than the first-generation EGFR-TKIs used for first-line treatment, its efficacy with respect to long-term patient survival remains unclear even upon the administration of a complete sequence of EGFR-TKI therapy. Moreover, limited information is available regarding genetic diagnostic approaches after the treatment of EGFR-TKI–naïve patients. This study investigated the clinical characteristics of EGFR-mutated lung cancer patients harboring the T790M substitution resistant to EGFR-TKIs, as well as the advantages of rebiopsy and liquid biopsy for these patients.MethodsThe medical records of patients screened for EGFR mutations were reviewed. Upon failure of naïve treatment with EGFR-TKIs, except for osimertinib, single-plexus cobas version 2 was repeatedly used to detect the T790M substitution in EGFR via tissue or liquid biopsy.ResultsFrom April 2016 through May 2019, 113 patients were found to harbor EGFR mutations. Sixty patients were treated with EGFR-TKIs, among whom 46 underwent tissue or liquid biopsy. Twenty-nine of these 46 (63%) patients harbored the T790M substitution. In total, 141 rebiopsies were performed. The T790M substitution was detected in 24 of 43 tissue biopsies and 11 of 98 liquid biopsies. If patients displayed an EGFR exon 19 deletion, had a new lesion, and were administered gefitinib as first-line therapy, they were suspected to harbor the T790M substitution. Furthermore, the T790M substitution was detected through rebiopsy in patients with coexisting original mutations, brain metastases, tumor enlargement by ≥12 mm, or metastases at minor sites.ConclusionAmong patients with positive factors associated with the T790M mutation, repeated tissue or liquid biopsies are useful to maximize the detection rate of the T790M substitution. Furthermore, these biopsies need to be repeated numerous times in order to reduce “detection overlook” among such patients.

Homoplasmic deleterious MT-ATP6/8 mutations in adult patients

To address the frequency of complex V defects, we systematically sequenced MT-ATP6/8 genes in 512 consecutive patients.We performed functional analysis in muscle or fibroblasts for 12 out of 27 putative homoplasmic mutations and in cybrids for four. Fibroblasts, muscle and cybrids with known deleterious mutations underwent parallel analysis. It included oxidative phosphorylation spectrophotometric assays, western blots, structural analysis, ATP production, glycolysis and cell proliferation evaluation.We demonstrated the deleterious nature of three original mutations. Striking gradation in severity of the mutations consequences and differences between muscle, fibroblasts and cybrids implied a likely under-diagnosis of human complex V defects.

Generation of four induced pluripotent stem cell lines (FHUi003-A, FHUi003-B, FHUi004-A and FHUi004-B) from two affected individuals of a familial neurohypophyseal diabetes insipidus family.

Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms.

EGFR circulating tumour DNA testing: identification of predictors of ctDNA detection and implications for survival outcomes.

BackgroundEGFR T790M testing is the standard of care for activating EGFR mutation (EGFRm) non-small cell lung cancer (NSCLC) progressing on 1st/2nd generation TKIs to select patients for osimertinib. Despite sensitive assays, detection of circulating tumour deoxyribonucleic acid (ctDNA) is variable and influenced by clinical factors. The number and location of sites of progressive disease at time of testing were reviewed to explore the effect on EGFR ctDNA detection. The prognostic value of EGFR ctDNA detection on survival outcomes was assessed.MethodsFollowing extraction of cell-free DNA from plasma using the QIAamp Circulating Nucleic Acid Kit, custom droplet digital polymerase chair reaction (ddPCR) assays were used to assess EGFR ctDNA using the Bio-Rad QX200 system. The ddPCR assay has a limit of detection of ≤0.15% variant allele fraction. Baseline characteristics and imaging reports at time of EGFR ctDNA testing were reviewed retrospectively for a 1 year period.ResultsThe study included 177 patients who had an EGFR ctDNA test. Liver (aOR 3.13) or bone (aOR 2.76) progression or 3–5 sites of progression (aOR 2.22) were predictive of EGFR ctDNA detection. The median OS from first ctDNA test after multiple testing iterations was 12.3 m undetectable EGFR ctDNA, 7.6 m for original EGFR mutation only and 24.1 m with T790M (P=0.001).ConclusionsPatients with liver or bone progression and 3–5 progressing sites are more likely to have informative EGFR ctDNA testing. Detection of EGFR ctDNA is a negative prognostic indicator in the absence of a T790M resistance mutation, potentially reflecting the disease burden in the absence of targeted therapy options.

Abstract 431: Probing the Pathogenic Mechanisms Underlying Ca V 1.2 Channelopathies

The Ca V 1.2 channel is essential to cardiac function. It is the sentinel that translates the electrical signal on the surface of the cell to the intracellular calcium cascade that leads to contraction. To allow the precise tuning of that is essential for cardiac function, Ca V 1.2 is subject to two forms of feedback regulation, voltage dependent inactivation (VDI) and calcium dependent inactivation (CDI). The first mutations described in Ca V 1.2 were causative of Timothy Syndrome (TS), a multisystem disorder with neurological, developmental and life-threatening cardiac symptoms, defined as long-QT type 8 (LQT8). These single point mutations occur in the S6, a region commonly associated with channel activation. In fact, the original TS mutations were shown to perturb channel activation as well as inactivation. Since this first description of TS, multiple additional mutations have been discovered in Ca V 1.2 and are associated with either TS-like or cardiac specific phenotypes. This phenotypic variance suggests that these mutations disrupt channel mechanisms in divergent ways. Moreover, for patients harboring mutations in Ca V 1.2, the cardiac symptoms have proven recalcitrant to conventional therapy including calcium channel blockers (CCBs). This therapeutic inadequacy reinforces the need for a better understanding of how mutations in the S6 disrupt channel function and how this affects therapeutic options for patients that carry them. Here we show that LQT8 mutations that cluster in the S6 of Ca V 1.2 do indeed have a variety of mechanistic underpinnings. This may account for the gradient of symptoms seen in patients with these mutations. In addition, we demonstrate that many of these mutant channels are resistant to block by CCBs, and this resistance strongly correlates to specific mechanistic perturbations of the channel. Thus, reliance on a CCB based therapeutic strategy is likely to leave many in a growing population of patients without a viable treatment option.

Increased ratio of galactolipid MGDG:DGDG induces jasmonic acid overproduction and changes chloroplast shape.

Summary Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG‐synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown.We introduced an inducible microRNA (ami‐MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA‐biosynthesis and JA‐responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated.Expression of JA‐biosynthesis and JA‐responsive genes were reduced in amiR‐MGD1‐transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued.Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.

Defining relative mutational difficulty to understand cancer formation

Most mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here, we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN, and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such a parameter, we can bring new angles to understanding cancer formation.

CtDNA analysis reveals different molecular patterns upon disease progression in patients treated with osimertinib

BackgroundSeveral clinical trials have demonstrated the efficacy and safety of osimertinib in advanced non-small-cell lung cancer (NSCLC). However, there is significant unexplained variability in treatment outcome.MethodsObservational prospective cohort of 22 pre-treated patients with stage IV NSCLC harboring the epidermal growth factor receptor (EGFR) p.T790M resistance mutation and who were treated with osimertinib. Three hundred and twenty-six serial plasma samples were collected and analyzed by digital PCR (dPCR) and next-generation sequencing (NGS).ResultsThe median progression-free survival (PFS), since the start of osimertinib, was 8.9 [interquartile range (IQR): 4.6–18.0] months. The median treatment durations of sequential gefitinib + osimertinib, afatinib + osimertinib and erlotinib + osimertinib treatments were 30.1, 24.6 and 21.1 months, respectively. The p.T790M mutation was detected in 19 (86%) pre-treatment blood samples. Undetectable levels of the original EGFR-sensitizing mutation after 3 months of treatment were associated with superior PFS (HR: 0.2, 95% CI: 0.05–0.7). Likewise, re-emergence of the original EGFR mutation, alone or together with the p.T790M mutation was significantly associated with shorter PFS (HR: 8.8, 95% CI: 1.1–70.7 and HR: 5.9, 95% CI: 1.2–27.9, respectively). Blood-based monitoring revealed three molecular patterns upon progression to osimertinib: sensitizing+/T790M+/C797S+, sensitizing+/T790M+/C797S–, and sensitizing+/T790M–/C797S–. Median time to progression in patients showing the triplet pattern (sensitizing+/T790M+/C797S+) was 12.27 months compared with 4.87 months in patients in whom only the original EGFR sensitizing was detected, and 2.17 months in patients showing the duplet pattern (sensitizing+/T790M+). Finally, we found that mutations in exon 545 of the PIK3CA gene were the most frequent alteration detected upon disease progression in patients without acquired EGFR-resistance mutations.ConclusionsDifferent molecular patterns identified by plasma genotyping may be of prognostic significance, suggesting that the use of liquid biopsy is a valuable approach for tumor monitoring.

The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release.

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.

Causal Attributions in an Australian Aboriginal Family With Marfan Syndrome: A Qualitative Study.

Causal attributions are important determinants of how health threats are processed and affect health-related behaviors. To date, there has been no research on causal attributions in genetic conditions in Aboriginal Australians. Forty members of a large Aboriginal Australian family with Marfan syndrome (MFS) were invited to participate in an ethically approved study exploring causal attributions, including perceived causes of phenotypic variability within the family. Eighteen individuals consented to conduct semi-structured qualitative interviews, which were recorded, transcribed verbatim and analyzed thematically. Most participants knew that MFS was genetic, but there were diverse theories about inheritance, including beliefs that it skipped generations, was affected by birth order and/or gender, and that it co-occurred with inheritance of blue eyes within this family. The mutation was thought to have been inherited from British settlers and initially triggered by disease or diet. Factors believed to modify disease severity included other genes and lifestyle factors, particularly alcohol and substance abuse and stress. Generally, this family did not endorse “blaming” chance or a higher power for phenotypic variability, though some felt that the spirits or a deity may have played a role. In conclusion, although participants knew MFS was a genetic condition, many speculated about the role of non-genetic causes in initiating the original mutation; and the gene-environment interaction was thought to affect severity. This study demonstrates a successful approach for exploring causal attributions in other genetic conditions in First Australians.