Addressing Diagnostic Gaps and Priorities of the Global Rare Diseases Community: Recommendations from the IRDiRC Diagnostics Scientific Committee.

The International Rare Diseases Research Consortium (IRDiRC) Diagnostic Scientific Committee (DSC) is charged with discussion and contribution to progress on diagnostic aspects of the IRDiRC core mission. Specifically, IRDiRC goals include timely diagnosis, use of globally coordinated diagnostic pipelines, and assessing the impact of rare diseases on affected individuals. As part of this mission, the DSC endeavored to create a list of research priorities to achieve these goals. We present a discussion of those priorities along with aspects of current, global rare disease needs and opportunities that support our prioritization. In support of this discussion, we also provide clinical vignettes illustrating real-world examples of diagnostic challenges.

The effect of underwater massage during hot water immersion on acute cardiovascular and mood responses.

PURPOSE: There is emerging evidence that demonstrates the health benefits of hot water immersion including improvements to cardiovascular health and reductions in stress and anxiety. Many commercially available hot tubs offer underwater massage systems which purport to enhance many benefits of hot water immersion, however, these claims have yet to be studied. METHODS: Twenty participants (4 females) completed three, 30-min sessions of hot-water immersion (beginning at 39 °C) in a crossover randomized design: with air massage (Air Jet), water massage (Hydro Jet) or no massage (Control). Cardiovascular responses comprising; heart rate, blood pressure and superficial femoral artery blood flow and shear rate were measured. State trait anxiety, basic affect, and salivary cortisol were recorded before and after each trial. Data were analysed using a mixed effects model. RESULTS: Post immersion, heart rate increased (Δ31bpm, P < 0.001, d = 1.38), mean arterial blood pressure decreased (Δ16 mmHg, P < 0.001, d = -0.66), with no difference between conditions. Blood flow and mean shear rate increased following immersion (P < 0.001, Δ362 ml/min, d = 1.20 and Δ108 s-1, d = 1.00), but these increases were blunted in the Air Jet condition (P < 0.001,Δ171 ml/min, d = 0.43 and Δ52 s-1, d = 0.52). Anxiety and salivary cortisol were reduced (P = 0.003, d = -0.20, P = 0.014, d = -0.11), but did not vary between conditions. Enjoyment did not vary between conditions. CONCLUSION: These data demonstrate positive acute responses to hot water immersion on markers of cardiovascular function, anxiety, and stress. There was no additional benefit of water-based massage, while air-based massage blunted some positive vascular responses due to lower heat conservation of the water.

Post exercise hot water immersion and hot water immersion in isolation enhance vascular, blood marker, and perceptual responses when compared to exercise alone.

Exercise and passive heating induce some similar vascular hemodynamic, circulating blood marker, and perceptual responses. However, it remains unknown whether post exercise hot water immersion can synergise exercise derived responses and if they differ from hot water immersion alone. This study investigated the acute responses to post moderate-intensity exercise hot water immersion (EX+HWI) when compared to exercise (EX+REST) and hot water immersion (HWI+HWI) alone. Sixteen physically inactive middle-aged adults (nine males and seven females) completed a randomized cross-over counterbalanced design. Each condition consisted of two 30-min bouts separated by 10 min of rest. Cycling was set at a power output equivalent to 50% V̇o2 peak . Water temperature was controlled at 40°C up to the mid sternum with arms not submerged. Venous blood samples and artery ultrasound scans were assessed at 0 (baseline), 30 (immediately post stressor one), 70 (immediately post stressor two), and 100 min (recovery). Additional physiological and perceptual measures were assessed at 10-min intervals. Brachial and superficial femoral artery shear rates were higher after EX+HWI and HWI+HWI when compared with EX+REST (p < 0.001). Plasma nitrite was higher immediately following EX+HWI and HWI+HWI than EX+REST (p < 0.01). Serum interleukin-6 was higher immediately after EX+HWI compared to EX+REST (p = 0.046). Serum cortisol was lower at 30 min in the HWI+HWI condition in contrast to EX+REST (p = 0.026). EX+HWI and HWI+HWI were more enjoyable than EX+REST (p < 0.05). Irrespective of whether hot water immersion proceeded exercise or heating, hot water immersion enhanced vascular and blood marker responses, while also being more enjoyable than exercise alone.

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.

A Comparison Between Pre-Sleep Heart Rate Variability Biofeedback and Electroencephalographic Biofeedback Training on Sleep in National Level Athletes with Sleep Disturbances.

The current study compared the effects of heart rate variability biofeedback (HRV-BF) and electroencephalographic biofeedback (EEG-BF) on sleep, mood, and reaction time. Fourteen highly trained male athletes with sleep disturbances participated in this randomised crossover study. Participants took part in HRV-BF and EEG-BF training, with each condition consisting of eight sessions over 15 days. Polysomnography (PSG) and the Pittsburgh sleep quality index (PSQI) were used to assess sleep quality, the profile of mood states (POMS) questionnaire to monitor mood, and reaction time to measure performance pre and post intervention. HRV-BF training improved PSG sleep efficiency (SE) (P = 0.022, d = 0.35, 95% CI 0.01 to 0.16) and subjective sleep duration (P = 0.011, ES = 0.40) when compared to EEG-BF. Only HRV-BF reduced reaction time pre to post biofeedback training (P = 0.020, d = 0.75, 95% CI 0.006 to 0.059). The PSQI showed that both HRV-BF (P = 0.025, ES = 0.31) and EEG-BF (P = 0.003, ES = 0.32) resulted in improved global PSQI scores. Total mood disturbance was also reduced though HRV-BF (P = 0.001, ES = 0.40) and EEG-BF (P = 0.001, ES = 0.30). HRV-BF and EEG-BF enhanced some subjective parameters of sleep and mood. HRV-BF increased PSG SE and subjective sleep duration more than EEG-BF in highly trained athletes with sleep disturbances.

The effect of age and mitigation strategies during hot water immersion on orthostatic intolerance and thermal stress.

NEW FINDINGS: What is the central question of this study? The aim was to characterize adverse responses to whole-body hot water immersion and to investigate practical strategies to mitigate these effects. What is the main finding and its importance? Whole-body hot water immersion induced transient orthostatic hypotension and impaired postural control, which recovered to baseline within 10 min. Hot water immersion was well tolerated by middle-aged adults, but younger adults suffered from a greater frequency and severity of dizziness. Cooling the face with a fan or not immersing the arms can mitigate some of these adverse responses in younger adults. ABSTRACT: Hot water immersion improves cardiovascular health and sporting performance, yet its adverse responses are understudied. Thirteen young and 17 middle-aged adults (n = 30) were exposed to 2 × 30 min bouts of whole-body 39°C water immersion. Young adults also completed cooling mitigation strategies in a randomized cross-over design. Orthostatic intolerance and selected physiological, perceptual, postural and cognitive responses were assessed. Orthostatic hypotension occurred in 94% of middle-aged adults and 77% of young adults. Young adults exhibited greater dizziness upon standing (young subjects, 3 out of 10 arbitrary units (AU) vs. middle-aged subjects, 2 out of 10 AU), with four terminating the protocol early owing to dizziness or discomfort. Despite middle-aged adults being largely asymptomatic, both age groups had transient impairments in postural sway after immersion (P < 0.05), but no change in cognitive function (P = 0.58). Middle-aged adults reported lower thermal sensation, higher thermal comfort, and higher basic affect than young adults (all P < 0.01). Cooling mitigation trials had 100% completion rates, with improvements in sit-to-stand dizziness (P < 0.01, arms in, 3 out of 10 AU vs. arms out, 2 out of 10 AU vs. fan, 4 out 10 AU), lower thermal sensation (P = 0.04), higher thermal comfort (P < 0.01) and higher basic affect (P = 0.02). Middle-aged adults were predominantly asymptomatic, and cooling strategies prevented severe dizziness and thermal intolerance in younger adults.

Validity and reliability of short-term heart-rate variability from disposable electrocardiography leads.

Background and Aims: Single-use electrocardiography (ECG) leads have been developed to reduce healthcare-associated infection. This study compared the validity and reliability of short-term heart rate variability (HRV) obtained from single-use disposable ECG leads. Methods: Thirty healthy subjects (33 ± 10 years; 9 females) underwent 5-min resting HRV assessments using disposable (single use) ECG cable and wire system (Kendall DL™ Cardinal Health) and a standard, reusable ECG leads (CardioExpress, Spacelabs Healthcare). Results: Intraclass correlation coefficient (ICC) with 95% confidence interval (CI) between disposable and reusable ECG leads was for the time domain [R-R interval (ms); 0.99 (0.91, 1.00)], the root mean square of successive normal R-R interval differences (RMSSD) (ms); 0.91 (0.76, 0.96), the SD of normal-to-normal R-R intervals (SDNN) (ms); 0.91 (0.68, 0.97) and frequency domain [low-frequency (LF) normalized units (nu); 0.90 (0.79, 0.95), high frequency (HF) nu; 0.91 (0.80, 0.96), LF power (ms2); 0.89 (0.62, 0.96), HF power (ms2); 0.90 (0.72, 0.96)] variables. The mean difference and upper and lower limits of agreement between disposable and reusable leads for time- and frequency-domain variables were acceptable. Analysis of repeated measures using disposable leads demonstrated excellent reproducibility (ICC 95% CI) for R-R interval (ms); 0.93 (0.85, 0.97), RMSSD (ms); 0.93 (0.85, 0.97), SDNN (ms); 0.88 (0.75, 0.95), LF power (ms2); 0.87 (0.72, 0.94), and HF power (ms2); 0.88 (0.73, 0.94) with coefficient of variation ranging from 2.2% to 5% (p > 0.37 for all variables). Conclusion: Single-use Kendall DL™ ECG leads demonstrate a valid and reproducible tool for the assessment of HRV.

Pharmacogenomics: an opportunity for personalised psychotropic prescribing in adults with intellectual disabilities.

There is growing evidence for the use of pharmacogenomics in psychotropic prescribing. People with intellectual disabilities are disproportionately prescribed psychotropics and are at risk of polypharmacy. There is an urgent need for safeguards to prevent psychotropic overprescribing but it is equally crucial that this population is not left behind in such exciting initiatives. Understanding how genetic variations affect medications is a step towards personalised medicine. This may improve personalised prescribing for people with intellectual disabilities, especially given the high rate of psychiatric and behavioural problems in this population. Our editorial explores opportunities and challenges that pharmacogenomics offers for the challenges of polypharmacy and overprescribing of psychotropics in people with intellectual disabilities.

Presleep Heart-Rate Variability Biofeedback Improves Mood and Sleep Quality in Chinese Winter Olympic Bobsleigh Athletes.

PURPOSE: To evaluate the effectiveness of heart-rate variability (HRV) biofeedback in improving autonomic function, mood, and sleep in elite bobsleigh athletes. METHODS: Eight Chinese Winter Olympic bobsleigh athletes (age: 24 [2] y, body mass: 89 [15] kg, and height: 184 [5] cm) completed a randomized crossover study with and without HRV biofeedback before a single night's sleep. HRV biofeedback was provided 35 minutes prior to bedtime in the experimental condition. The assessment of HRV took place 45 and 10 minutes before bedtime. The Profile of Mood States questionnaire was completed 50 and 15 minutes prior to bedtime. Sleep duration and quality were measured through an air-mattress sleep-monitoring system. RESULTS: Sleep efficiency (P = .020; F = 7.831; CI, 0.008 to 0.072) and the percentage of deep sleep duration increased (P = .013; F = 10.875; CI, 0.006 to 0.035), while the percentage of light sleep decreased (P = .034; F = 6.893; CI, -0.038 to -0.002). Presleep HRV biofeedback increased parasympathetic and decreased sympathetic activity. Mood states of anger (P = .006, F = 7.573), panic (P = .031, F = 4.288), tension (P = .011, F = 6.284), depression (P = .010, F = 6.016), fatigue (P = .000, F = 16.901), and total mood disturbance (P = .001, F = 11.225) were reduced before sleep. CONCLUSION: Presleep HRV biofeedback improved some measures of autonomic function, mood, and sleep quality in Chinese Olympic bobsleigh athletes. Presleep HRV biofeedback provides a practical strategy that may help reduce sleep disturbances during periods of training and competition.

Unlocking sociocultural and community factors for the global adoption of genomic medicine.

Advances in genomic sequencing and genetic testing are increasingly transforming the diagnosis and treatment of diseases-specifically, rare diseases. However, the application and benefit of such technologies remain inequitable globally. There is a clear and urgent need to provide genomic sequencing to people across the global population, including people living in under-resourced areas and/or underrepresented populations. Financial considerations are the most obvious barriers to the adoption of genomic medicine, yet there are many other factors that are not so obvious, such as geography, language, communication, and culture. Herein, we use the lens of rare diseases and focus on firstly, selected socio-cultural factors, and in particular stigma; and secondly, empowering community factors such as education, advocacy and connectivity amongst people living with rare diseases globally. These are critical areas of need and opportunity if genomic medicine is to achieve equitable and global adoption in the patient best-interest across low- middle- and high-income country health systems. Furthermore, we touch on specific child health aspects and how they can point towards opportunities to build on specific infrastructures.

A systematic review of the role of heat therapy for patients with intermittent claudication due to peripheral artery disease.

Intermittent claudication (IC) is associated with impairments in quality of life and walking ability. Heat therapy is an emerging cardiovascular therapy, which may improve walking in patients with IC. We undertook a systematic review to establish current evidence for heat therapy for patients with IC. We searched five databases (Ovid Medline / PubMed, Embase, Scopus / Web of Science, Cochrane Library and Health Technology Assessment Databases). A total of 6751 records were screened with 76 full-text articles assessed for eligibility. We included three randomised control trials and three acute interventions. For chronic interventions, three different heat therapy interventions were used. The 6-minute walk distance significantly improved following whole-body immersion (p = 0.03; ES 0.94, 95% CI: 0.06-1.82), but not after Waon therapy or a water-perfused garment. Ankle-brachial pressure indices were significantly improved following whole-body immersion (p = 0.01; ES 1.10, 95% CI: 0.20-1.99) but not after other therapies. No form of heat therapy demonstrated statistical improvements in quality of life or brachial blood pressure. Acute interventions were characterised by large increases in limb blood flow and core temperature, and transient reductions in blood pressure post-heating. At present there are only three randomised controlled trials assessing heat therapy for patients with IC. Moreover, each of those randomised controlled trials utilised different heat therapies. There is also very limited study of the acute physiological responses to different heat therapy interventions in these populations. Future research should establish appropriate heat therapy protocols and implement more randomised trials to understand its effectiveness. PROSPERO: CRD42020187941.

The health benefits of passive heating and aerobic exercise: To what extent do the mechanisms overlap?

Exercise can induce numerous health benefits that can reduce the risk of chronic diseases and all-cause mortality, yet a significant percentage of the population do not meet minimal physical activity guidelines. Several recent studies have shown that passive heating can induce numerous health benefits, many of which are comparable with exercise, such as improvements to cardiorespiratory fitness, vascular health, glycemic control, and chronic low-grade inflammation. As such, passive heating is emerging as a promising therapy for populations who cannot perform sustained exercise or display poor exercise adherence. There appears to be some overlap between the cellular signaling responses that are regulated by temperature and the mechanisms that underpin beneficial adaptations to exercise, but detailed comparisons have not yet been made. Therefore, the purpose of this mini review is to assess the similarities and distinctions between adaptations to passive heating and exercise. Understanding the potential shared mechanisms of action between passive heating and exercise may help to direct future studies to implement passive heating more effectively and identify differences between passive heating and exercise-induced adaptations.

A comparison of genomic diagnostics in adults and children with epilepsy and comorbid intellectual disability.

Next generation sequencing provides an important opportunity for improved diagnosis in epilepsy. To date, the majority of diagnostic genetic testing is conducted in the paediatric arena, while the utility of such testing is less well understood in adults with epilepsy. We conducted whole exome sequencing (WES) and copy number variant analyses in an Irish cohort of 101 people with epilepsy and co-morbid intellectual disability to compare the diagnostic yield of genomic testing between adult and paediatric patients. Variant interpretation followed American College of Medical Genetics and Genomics (ACMG) guidelines. We demonstrate that WES, in combination with array-comparative genomic hybridisation, provides a diagnostic rate of 27% in unrelated adult epilepsy patients and 42% in unrelated paediatric patients. We observe a 2.7% rate of ACMG-defined incidental findings. Our findings indicate that WES has similar utility in both adult and paediatric cohorts and is appropriate for diagnostic testing in both epilepsy patient groups.

The future of genomics in Ireland - focus on genomics for health.

Genomics is revolutionizing biomedical research, medicine and healthcare globally in academic, public and industry sectors alike. Concrete examples around the world show that huge benefits for patients, society and economy can be accrued through effective and responsible genomic research and clinical applications. Unfortunately, Ireland has fallen behind and needs to act now in order to catch up. Here, we identify key issues that have resulted in Ireland lagging behind, describe how genomics can benefit Ireland and its people and outline the measures needed to make genomics work for Ireland and Irish patients. There is now an urgent need for a national genomics strategy that enables an effective, collaborative, responsible, well-regulated, and patient centred environment where genome research and clinical genomics can thrive.  We present eight recommendations that could be the pillars of a national genomics health strategy.

Re-annotation of 191 developmental and epileptic encephalopathy-associated genes unmasks de novo variants in SCN1A.

The developmental and epileptic encephalopathies (DEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for a lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 putative transcript models. Our annotations increase the transcriptional 'footprint' of these genes by over 674 kb. Using SCN1A as a case study, due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome or a similar phenotype with a panel of exon sequences representing eight established genes and identified two de novo SCN1A variants that now - through improved gene annotation - are ascribed to residing among our exons. These two (from 122 screened people, 1.6%) molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously classified SCN1A intronic Dravet syndrome-associated variant that now lies within a deeply conserved exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders.

One week of magnesium supplementation lowers IL-6, muscle soreness and increases post-exercise blood glucose in response to downhill running.

PURPOSE: Magnesium supplementation modulates glucose metabolism and inflammation, which could influence exercise performance and recovery. This study investigated the effect of magnesium intake on physiological responses and performance during eccentric exercise and recovery. METHODS: Nine male recreational runners completed a counterbalanced, double-blind, placebo-controlled, cross-over study, registered at ClinicalTrial.gov. Participants consumed low magnesium diets and were supplemented with 500 mg/day of magnesium (SUP) or placebo (CON) for 7 days prior to a 10 km downhill (- 10%) running time trial (TT), separated by a 2-week washout period. At baseline and 24 h post-TT, maximal muscle force was measured. Interleukin-6 (IL-6), soluble interleukin-6 receptor (sIL-6R) and creatine kinase (CK) were measured at rest, 0 h, 1 h and 24 h post-TT. Muscle soreness was measured at the previous times plus 48 h and 72 h post. Glucose and lactate were measured during the TT. RESULTS: The main effect of condition was detected for IL-6 (SUP: 1.36 ± 0.66 vs CON: 2.06 ± 1.14 pg/ml) (P < 0.05, η2 = 0.54), sIL-6R (SUP: 27,615 ± 8446 vs CON: 24,368 ± 7806 pg/ml) (P < 0.05, η2 = 0.41) and muscle soreness (P < 0.01, η2 = 0.67). Recovery of blood glucose and muscle soreness were enhanced in SUP post-TT. There were no differences in glucose and lactate during the TT, or post measures of CK and maximal muscle force. CONCLUSION: Magnesium supplementation reduced the IL-6 response, enhanced recovery of blood glucose, and muscle soreness after strenuous exercise, but did not improve performance or functional measures of recovery.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Genome-wide association study: Exploring the genetic basis for responsiveness to ketogenic dietary therapies for drug-resistant epilepsy.

OBJECTIVE: With the exception of specific metabolic disorders, predictors of response to ketogenic dietary therapies (KDTs) are unknown. We aimed to determine whether common variation across the genome influences the response to KDT for epilepsy. METHODS: We genotyped individuals who were negative for glucose transporter type 1 deficiency syndrome or other metabolic disorders, who received KDT for epilepsy. Genotyping was performed with the Infinium HumanOmniExpressExome Beadchip. Hospital records were used to obtain demographic and clinical data. KDT response (≥50% seizure reduction) at 3-month follow-up was used to dissect out nonresponders and responders. We then performed a genome-wide association study (GWAS) in nonresponders vs responders, using a linear mixed model and correcting for population stratification. Variants with minor allele frequency <0.05 and those that did not pass quality control filtering were excluded. RESULTS: After quality control filtering, the GWAS of 112 nonresponders vs 123 responders revealed an association locus at 6p25.1, 61 kb upstream of CDYL (rs12204701, P = 3.83 × 10-8 , odds ratio [A] = 13.5, 95% confidence interval [CI] 4.07-44.8). Although analysis of regional linkage disequilibrium around rs12204701 did not strengthen the likelihood of CDYL being the candidate gene, additional bioinformatic analyses suggest it is the most likely candidate. SIGNIFICANCE: CDYL deficiency has been shown to disrupt neuronal migration and to influence susceptibility to epilepsy in mice. Further exploration with a larger replication cohort is warranted to clarify whether CDYL is the causal gene underlying the association signal.

Genome annotation for clinical genomic diagnostics: strengths and weaknesses.

The Human Genome Project and advances in DNA sequencing technologies have revolutionized the identification of genetic disorders through the use of clinical exome sequencing. However, in a considerable number of patients, the genetic basis remains unclear. As clinicians begin to consider whole-genome sequencing, an understanding of the processes and tools involved and the factors to consider in the annotation of the structure and function of genomic elements that might influence variant identification is crucial. Here, we discuss and illustrate the strengths and weaknesses of approaches for the annotation and classification of important elements of protein-coding genes, other genomic elements such as pseudogenes and the non-coding genome, comparative-genomic approaches for inferring gene function, and new technologies for aiding genome annotation, as a practical guide for clinicians when considering pathogenic sequence variation. Complete and accurate annotation of structure and function of genome features has the potential to reduce both false-negative (from missing annotation) and false-positive (from incorrect annotation) errors in causal variant identification in exome and genome sequences. Re-analysis of unsolved cases will be necessary as newer technology improves genome annotation, potentially improving the rate of diagnosis.

Extension of human lncRNA transcripts by RACE coupled with long-read high-throughput sequencing (RACE-Seq).

Long non-coding RNAs (lncRNAs) constitute a large, yet mostly uncharacterized fraction of the mammalian transcriptome. Such characterization requires a comprehensive, high-quality annotation of their gene structure and boundaries, which is currently lacking. Here we describe RACE-Seq, an experimental workflow designed to address this based on RACE (rapid amplification of cDNA ends) and long-read RNA sequencing. We apply RACE-Seq to 398 human lncRNA genes in seven tissues, leading to the discovery of 2,556 on-target, novel transcripts. About 60% of the targeted loci are extended in either 5' or 3', often reaching genomic hallmarks of gene boundaries. Analysis of the novel transcripts suggests that lncRNAs are as long, have as many exons and undergo as much alternative splicing as protein-coding genes, contrary to current assumptions. Overall, we show that RACE-Seq is an effective tool to annotate an organism's deep transcriptome, and compares favourably to other targeted sequencing techniques.