Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition.

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four 'omes' to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

CHRNA5 gene variation affects the response of VTA dopaminergic neurons during chronic nicotine exposure and withdrawal.

Nicotine is the principal psychoactive component in tobacco that drives addiction through its action on neuronal nicotinic acetylcholine receptors (nAChR). The nicotinic receptor gene CHRNA5, which encodes the α5 subunit, is associated with nicotine use and dependence. In humans, the CHRNA5 missense variant rs16969968 (G > A) is associated with increased risk for nicotine dependence and other smoking-related phenotypes. In rodents, α5-containing nAChRs in dopamine (DA) neurons within the ventral tegmental area (VTA) powerfully modulate nicotine reward and reinforcement. Although the neuroadaptations caused by long-term nicotine exposure are being actively delineated at both the synaptic and behavioral levels, the contribution of α5-containing nAChRs to the cellular adaptations associated with long-term nicotine exposure remain largely unknown. To gain insight into the mechanisms behind the influence of α5-containing nAChRs and the rs16969968 polymorphism on nicotine use and dependence, we used electrophysiological approaches to examine changes in nAChR function arising in VTA neurons during chronic nicotine exposure and multiple stages of nicotine withdrawal. Our results demonstrate that CHRNA5 mutation leads to profound changes in VTA nAChR function at baseline, during chronic nicotine exposure, and during short-term and prolonged withdrawal. Whereas nAChR function was suppressed in DA neurons from WT mice undergoing withdrawal relative to drug-naïve or nicotine-drinking mice, α5-null mice exhibited an increase in nAChR function during nicotine exposure that persisted throughout 5-10 weeks of withdrawal. Re-expressing the hypofunctional rs16969968 CHRNA5 variant in α5-null VTA DA neurons did not rescue the phenotype, with α5-SNP neurons displaying a similar increased response to ACh during nicotine exposure and early stages of withdrawal. These results demonstrate the importance of VTA α5-nAChRs in the response to nicotine and implicate them in the time course of withdrawal.

ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal-dominant disorder caused by an expanded pentanucleotide repeat in the ATXN10 gene. This repeat expansion, when fully penetrant, has a size of 850-4,500 repeats. It has been shown that the repeat composition can be a modifier of disease, e.g., seizures. Here, we describe a Mexican kindred in which we identified both pure (ATTCT)n and mixed (ATTCT)n-(ATTCC)n expansions in the same family. We used amplification-free targeted sequencing and optical genome mapping to decipher the composition of these repeat expansions. We found a considerable degree of mosaicism of the repeat expansion. This mosaicism was confirmed in skin fibroblasts from individuals with ATXN10 expansions with RNAScope in situ hybridization. All affected family members with the mixed ATXN10 repeat expansion showed typical clinical signs of spinocerebellar ataxia and epilepsy. In contrast, individuals with the pure ATXN10 expansion present with Parkinson's disease or are unaffected, even in individuals more than 20 years older than the average age at onset for SCA10. Our findings suggest that the pure (ATTCT)n expansion is non-pathogenic, while repeat interruptions, e.g., (ATTCC)n, are necessary to cause SCA10. This mechanism has been recently described for several other repeat expansions including SCA31 (BEAN1), SCA37 (DAB1), and three loci for benign adult familial myoclonic epilepsy BAFME (SAMD12, TNRC6A, RAPGEF2). Therefore, long-read sequencing and optical genome mapping of the entire genomic structure of repeat expansions are critical for clinical practice and genetic counseling, as variations in the repeat can affect disease penetrance, symptoms, and disease trajectory.

Sentinel lymph node biopsy for early oral cancer - accuracy and considerations in patient selection.

Sentinel lymph node biopsy (SLNB) for staging oral squamous cell carcinoma (OSCC) patients presenting with early (T1 and T2 N0) disease in preference to elective neck dissection (END) remains controversial worldwide. A retrospective analysis of 145 patients who underwent sentinel lymph node biopsy for a previously untreated early oral cancer between 2010 and 2020 was performed. The primary outcome measures were predictors of occult metastases, accuracy of SLNB and disease specific plus overall survival. The negative predictive value, the false negative rate, and sensitivity for SLNB were 97%, 7.8%, and 92%, respectively. Depth of invasion (DOI) was a significant predictor of N status, overall survival, and disease specific survival. There was a significant difference in the incidence of the neck node metastasis in patients with DOI <5mm compared to those with DOI >5mm. For tumours >5mm there was a moderate to good correlation between radiological depth on contrast enhanced computed tomography (CECT) and histopathological DOI. Preoperative estimation of DOI may be a useful tool in the counselling of patients in the selection of either SLNB or END for N staging purposes in early OSCC.

MR1 encompasses at least six allele groups with coding region alterations.

Unlike classical HLA class I genes, MR1 is assumed to have limited polymorphic positions. We developed a MR1 specific PCR assay and sequenced 56 DNA samples from cells with a diverse set of HLA genotypes. In this relatively small panel we found six allele groups encoding for different MR1 proteins. The two most frequent allele groups found in this panel had a frequency of 71% (MR1*01) and 25% (MR1*02), respectively. Moreover, the panel contained many intronic SNPs and silent variants, with individual samples containing up to 15 heterozygous positions. The data presented here is consistent with marked variation in MR1.

Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases.

To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10-4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10- 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases.

Characterisation of 99mTc EDDA/HYNIC-TOC (Tektrotyd) physiological and neuroendocrine tumour uptake using SPECT/CT standardised uptake values: initial experience.

99mTc Ethylene diamine N,N'-diacetic acid hydrazinonicotinamide-conjugated Tyr3-octreotide (99mTc EDDA/HYNIC-TOC) single photon emission tomography/computed tomography (SPECT/CT) imaging of somatostatin receptors is used in the assessment of neuroendocrine tumours (NETs). The objective of this study was to characterise quantitative standardised uptake value (SUV) SPECT/CT of normal physiological uptake and NET disease. Forty-four patients (22 female and 22 male) referred for 99mTc EDDA/HYNIC-TOC SPECT/CT imaging for diagnosis/primary staging (n = 28) or the assessment of residual/recurrent disease (n = 16) were included. SPECT/CT SUVmax values were determined for normal physiological uptake (spleen, kidney, liver and bone) and NET disease (liver metastases, metastatic lymph nodes, bone metastases and intrapulmonary lesions). Statistical testing was performed to compare normal uptake and NET disease uptake in liver and bone (Student's t-test). The highest normal physiological uptake was observed in the spleen (mean SUVmax 29.8, SD 13.7), with lower uptake in the kidneys (16.7, 3.2) and liver (7.3, 2.1). Increased SUVmax values were observed in primary tumour and metastatic disease, greatest in liver metastases (21.8, 13.3), with lower, similar values obtained for metastatic lymph nodes (16.3, 7.5) and intrapulmonary lesions (17.5, 16.8). SUVmax in bone metastases averaged 12.9 (7.0). Significant differences were observed between normal and metastatic SUVmax in the liver and bone (P < 0.01). SPECT/CT SUV quantification is feasible in a manner similar to PET/CT. 99mTc EDDA/HYNIC-TOC SPECT/CT SUVmax has been characterised in NET disease, demonstrating high target to non-target ratios for primary tumours and metastatic lesions.

Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease.

BACKGROUND: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 'GGGGCC' (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences' (PacBio) and Oxford Nanopore Technologies' (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9. RESULTS: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual's repeat region was > 99% G4C2 content, though we cannot rule out small interruptions. CONCLUSIONS: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

The medial habenula and interpeduncular nucleus circuitry is critical in addiction, anxiety, and mood regulation.

Abstinence from chronic use of addictive drugs triggers an aversive withdrawal syndrome that compels relapse and deters abstinence. Many features of this syndrome are common across multiple drugs, involving both affective and physical symptoms. Some of the network signaling underlying withdrawal symptoms overlaps with activity that is associated with aversive mood states, including anxiety and depression. Given these shared features, it is not surprising that a particular circuit, the dorsal diencephalic conduction system, and the medial habenula (MHb) and interpeduncular nucleus (IPN), in particular, have been identified as critical to the emergence of aversive states that arise both as a result and, independently, of drug addiction. As the features of this circuit continue to be characterized, the MHb-IPN axis is emerging as a viable target for therapeutics to aid in the treatment of addiction to multiple drugs of abuse as well as mood-associated disorders. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Tobacco industry responsibility for butts: a Model Tobacco Waste Act.

Cigarette butts and other postconsumer products from tobacco use are the most common waste elements picked up worldwide each year during environmental cleanups. Under the environmental principle of Extended Producer Responsibility, tobacco product manufacturers may be held responsible for collection, transport, processing and safe disposal of tobacco product waste (TPW). Legislation has been applied to other toxic and hazardous postconsumer waste products such as paints, pesticide containers and unused pharmaceuticals, to reduce, prevent and mitigate their environmental impacts. Additional product stewardship (PS) requirements may be necessary for other stakeholders and beneficiaries of tobacco product sales and use, especially suppliers, retailers and consumers, in order to ensure effective TPW reduction. This report describes how a Model Tobacco Waste Act may be adopted by national and subnational jurisdictions to address the environmental impacts of TPW. Such a law will also reduce tobacco use and its health consequences by raising attention to the environmental hazards of TPW, increasing the price of tobacco products, and reducing the number of tobacco product retailers.

Nicotine withdrawal.

An aversive abstinence syndrome manifests 4-24 h following cessation of chronic use of nicotine-containing products. Symptoms peak on approximately the 3rd day and taper off over the course of the following 3-4 weeks. While the severity of withdrawal symptoms is largely determined by how nicotine is consumed, certain short nucleotide polymorphisms (SNPs) have been shown to predispose individuals to consume larger amounts of nicotine more frequently--as well as to more severe symptoms of withdrawal when trying to quit. Additionally, rodent behavioral models and transgenic mouse models have revealed that specific nicotinic acetylcholine receptor (nAChR) subunits, cellular components, and neuronal circuits are critical to the expression of withdrawal symptoms. Consequently, by continuing to map neuronal circuits and nAChR subpopulations that underlie the nicotine withdrawal syndrome--and by continuing to enumerate genes that predispose carriers to nicotine addiction and exacerbated withdrawal symptoms--it will be possible to pursue personalized therapeutics that more effectively treat nicotine addiction.

Reducing tobacco use and access through strengthened minimum price laws.

Higher prices reduce consumption and initiation of tobacco products. A minimum price law that establishes a high statutory minimum price and prohibits the industry's discounting tactics for tobacco products is a promising pricing strategy as an alternative to excise tax increases. Although some states have adopted minimum price laws on the basis of statutorily defined price "markups" over the invoice price, existing state laws have been largely ineffective at increasing the retail price. We analyzed 3 new variations of minimum price laws that hold great potential for raising tobacco prices and reducing consumption: (1) a flat rate minimum price law similar to a recent enactment in New York City, (2) an enhanced markup law, and (3) a law that incorporates both elements.

Modulation of methylphenidate effects on wheel running and acoustic startle by acute food deprivation in commercially and selectively bred rats.

Behavioral effects of the same dose of the same drug can vary in degree and direction between and within individuals. The present study examines behavioral base rates, feeding status, and dispositional differences as sources of inter- and intra-individual heterogeneity in drug response. Modulation of the effects of methylphenidate (MPD) on wheel running and acoustic startle by food deprivation was examined in three experiments. Freely fed or food deprived Harlan Sprague-Dawley rats (running study) or rats selectively bred for low (LoS) and high (HiS) saccharin intake (running and startle studies) were given MPD (10 mg/kg) or saline before testing. Overall drug effects and predictors of drug response were assessed. MPD increased running and startle amplitude and disrupted prepulse inhibition; systematic variation among rats of these effects and their modulation by food deprivation was observed. Deprivation-induced running predicted MPD's effect in Harlan SD and LoS rats. Observation of this relationship among commercial rats suggests that acute deprivation sensitivity has utility as a noninvasive marker for drug responses. Its observation in rats selected on a taste phenotype with known correlates points to fruitful avenues of research on stimulant drugs' mechanisms, especially in dopaminergic pathways, and may be relevant to their clinical usage.