Envisioning the next human genome reference

Summary: We provide an Editorial perspective on approaches to improve ethnic representation in the human genome reference sequence, enabling its widespread use in genomic studies and precision medicine to benefit all peoples.

This year marks the 20th anniversary of the announced completion of the draft human genome sequence. The reference genome was a transformative accomplishment for human biological and medical research and is often referred to as biology''s moonshot. Over the past 20 years, the availability of this reference, and its refinement, has had the predicted transformative impact on our understanding of human genetic diseases, and, in many ways, has revolutionized the practice of medicine and medical diagnosis. These advances are mainly due to the emergence and refinement of rapid sequencing technology, which has facilitated our ability to generate genomic data, and to corresponding advances in computational analysis of these data, which have solidified the significant role of genomic alterations in disease etiology. Along these lines, large international projects have enriched our understanding of human genomic diversity in the context of cancer (Campbell et al., 2020), psychiatric genetics (Bipolar Disorder and Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2018; Cleynen et al., 2021), autism (Satterstrom et al., 2020; Trost et al., 2020) and many other diseases. Such studies have mainly catalogued individual and ancestry-based variation in human genomes, albeit to a limited extent. The inherent limitation in scope has been reflective of a predominant focus on populations of European ancestry in the earliest studies, such as the 1000 Genomes Project. More recent attempts to address these disparities have illuminated the challenges in recruiting diverse populations that either have a justifiable lack of trust in medical research or have cultural complexities regarding consent to participate. By improving diversity and inclusion in these studies, there would be increased hope that genomic studies will more broadly benefit populations. In order for the human genome and genomics to have a more significant and equitable impact in the future, accordingly, there is much more to be done. Medical and scientific communities need to consult with and listen to diverse populations and cultures to understand their concerns and needs, and, importantly, to make corresponding changes in our research practices that ensures accountability to these groups. Open in a separate windowImage reproduced under the terms of the Pixabay License.Ongoing large-scale population studies that link with individual clinical information are beginning to shape newer capabilities in predicting complex genetic disease susceptibility, primarily focused on developing and testing polygenic risk scores (Shen et al., 2020; Vujkovic et al., 2020; Riveros-Mckay et al., 2021; Weale et al., 2021). These efforts have the potential to radically change the practice of medicine, from a reactive to a proactive model of care delivery, based on an individual''s likelihood to experience predicted health challenges during their life course. As such, not only is current genomics-based diagnosis being imperiled by a lack of understanding of genomic variation based on ancestry (Sirugo et al., 2019), but future health care aspects also will be compromised in an ancestry-specific way, further compounding the impact of systemic racism that continues to make ethnic minorities more vulnerable in this setting (Yudell et al., 2020). In aggregate, this means that health disparities in many underserved populations will continue. In addition to these disparities in genomic representation, we also still lack a fundamental understanding across all ancestries, of what genotypes define as ‘healthy’ relative to our significant and growing understanding of ‘diseased’.There is renewed hope as newer projects strive to improve inclusivity, and here we highlight several examples arising in the United States. The National Institutes of Health (NIH)’s All of Us Research Program has set laudable goals for inclusion of diverse ethnic groups based on recent metrics, wherein over 50% of the currently enrolled 394,000 participants are ethnic minorities. This is a promising beginning, and, with a planned recruitment of 1 million participants, it will be interesting to see the final percentages toward the stated goal. More recently, the NIH posted a request for applications in support of research leading to the creation of best practices for the study of population identifiers. Local projects that focus on specific populations are also emerging, with a variety of funding mechanisms. For example, a study funded by the New York Genome Center plans to enroll minority participants from the broad diversity found throughout the New York City metropolitan area, with an aim to sequence whole genomes and collect health-related information, as discussed by Harold Varmus, one of the study''s leaders (Varmus, 2019). In Columbus, Ohio, the Institute for Genomic Medicine has opened an Institutional Review Board-approved study to consent, produce and database whole-genome sequences from unrelated individuals of Somali descent, to better inform our genomics-based diagnostic efforts for Somali children. Similarly, at Yale University, the Generations Project aims to increase diversity by recruiting in the Connecticut area, which is closely aligned with diversity in US consensus metrics. The exome sequencing and genotyping data from this project will be linked to electronic health records, allowing an opportunity to study and advance genomic health in under-represented minorities. Olufunmilayo Olopade, at the University of Chicago, has also discussed her work investigating genetic risk factors for breast cancer in Black women in studies based in Chicago and West Africa that aim to improve early detection, prevention and treatment in these populations (Olopade, 2021). Similar efforts outside the United States include the Human Heredity and Health in Africa Initiative (H3Africa), GenomeAsia 100K, ChileGenomico and the oriGen Project based in Mexico, among others.Technology continues to impact our human genome reference, predominantly using long-read single-molecule sequencing technologies to generate data, and algorithms capable of assembling these reads into long stretches of human chromosomes, permitting a more complete understanding of structural variation and unique content. Recently, an ‘end-to-end’ assembly of a complete hydatidiform mole cell line was reported, providing contiguity across centromeric and other complex repeats in the human genome, as described in a preprint (Nurk et al., 2021). However, the ancestry of this sample is European. Importantly, the Pangenome Project will aim to produce high-quality long-read sequencing for 300 individuals originally profiled in the 1000 Genomes Project. These comprehensive reference genomes will also include sequences of highly repetitive regions, including centromeres, segmental duplications and ribosomal DNA (rDNA) arrays on telomeres. In addition, the population diversity provided by these genomes will give researchers the option to choose a reference that is more closely related to any given sequenced individual, resulting in improved variant discovery.In addition to the influence of genomic technology, we have branched out from sole focus on DNA sequence to cataloguing RNA expression, isoforms and other types of characterization by applying next-generation and single-molecule sequencing, which, when integrated with DNA information, can provide significant insights into the sequences actively being expressed in tissues, as well as those being silenced by chromatin conformation or methylation. Such studies reveal that there is much more to be learned, and emphasize the importance of cataloguing normal tissue gene expression, which is available at GTEx, the Allen Brain Atlas and other internet resources. Exquisite new knowledge of gene expression profiles at the single-cell level from normal and diseased human tissue is emerging from the Human Cell Atlas projects, revealing the intricacies of human biology at high resolution (Ponting, 2019; Lindeboom et al., 2021).Yet, the concern about inclusion persists, even for these newest technological avenues that may indeed reveal important, ancestry-relevant differences with respect to disease susceptibility, physiologic specificity, pharmacogenomics and other pertinent areas (Okada et al., 2018). Without broadening the scope of diversity, we are concerned that individuals and populations will be left behind in many aspects of genomic medicine, effectively broadening disparities. Considering worldwide disparities that exist, such as poor access to health care, even in countries with high income and/or universal health care, the question remains about how to effectively foster inclusion and ensure that under-represented populations will benefit from expanded genomic research. Several strategies for increasing diversity and inclusion have been published (Cooke Bailey et al., 2020; Essien and Ufomata, 2021; Rotimi and Adeyemo, 2021; Nature Editorial, 2021). Here, we would like to highlight the following approaches. First, diversity in research subjects and samples starts with a diverse workforce at all levels, including leadership of major consortium efforts. To enable the creation of a diverse workforce, researchers need to engage with these communities to encourage and support them in pursuing such career paths and overcoming institutional barriers to achieve these goals. Researchers from under-represented groups that truly understand the communities being studied should have the opportunity to participate at all levels of a project, including leading the project and consenting participants (Bonham and Green, 2021). This facilitates surmounting socio-economic and cultural barriers that make it difficult to recruit under-represented minorities and engages the study team to meet diversity recruitment goals, while ensuring that the interpretation and outcomes of research are broadly beneficial. Second, there needs to be increased funding of institutions with diverse staff and students, such as Historically Black Colleges and Universities or community colleges with 2-year associate programs, as well as internship programs that bring minority students from inner-city high schools into genomics laboratories to learn about genomics research and its applicability to human health. Such programs ensure that leadership opportunities and training in genomics will directly benefit the communities that we intend to recruit. Equally important in this regard is education directed at researchers and medical providers that illuminates ongoing issues concerning racism, diversity and inclusion in science and health care. Third, long-term funding must be dedicated to build infrastructure and collaborative networks to enable the facile recruitment of diverse cohorts. H3Africa is a good model that could be replicated in other under-represented regions across the world, including diverse populations in large inner-city settings, emphasizing needed focus on consultation within these communities to understand their wants, needs and concerns regarding genetics and genomics. Lastly, funding agencies need to switch mindsets from having diversity as an optional goal to being a measurable milestone that must be met. The Human Cell Atlas in their recent funding round for the Pediatric Cell Atlas provides one example of explicit ancestry recruitment goals. Longer term, we must take responsibility to put in place mechanisms that both ensure accessibility to data and quantify the benefit of these studies to all populations.In reflecting on the 20 years since the published draft human genome, it is time to recognize that the combination of technologies, computational algorithms, and the diversity and inclusion of participants gives us the opportunity, this time around, to design cohort studies to benefit ALL of us. Certainly, there is a responsibility for journals, such as DMM, to address the issue of diversity and inclusion, by encouraging the publication of research that advances our understanding of diseases over-represented in individuals of diverse ancestries, and also encouraging reviewers to be conscious that access to technology is not equivalent in all countries, when requesting revisions for publication. DMM''s policies include aims to engage diverse and inclusive groups of authors, reviewers, Editors, Editorial Board members, readers and the communities being studied, and the journal is a signatory of the Royal Society of Chemistry''s initiative ‘Joint commitment for action on inclusion and diversity in publishing’. Journals can also seek out and publish pieces that address these important (and sometimes difficult) conversations, and openly discuss the ongoing challenges as well as approaches employed by others, as we strive to identify solutions that benefit everyone.     

Inhibition of alcohol dehydrogenases by thiol compounds

2-Mercaptoethanol is a strong inhibitor of LADH. The inhibitory effect is likely due to the binding of the SH group to the enzymatic zinc ion. Various thiol compounds do not inhibit YADH and it is suggested that the zinc atoms involved in the catalytic mechanism of LADH and YADH may have different structural arrangements and that these zinc atoms in YADH may not be blocked by thiol compounds. Thiol compounds also quench the enhanced fluorescence of LADH-NADH in a pH-dependent manner. At pH 9.2, the binding of coenzyme to LADH is replaced by 2-mercaptoethanol, whilst at pH 7.3, it further quenches the fluorescence of NADH-LADH. This quenching of fluorescence is likely attributed to a conformational change and energy transfer upon binding of 2-mercaptoethanol to the LADH-NADH complex. Complete reversal of the inhibitory effect of thiol compounds on LADH can be obtained by dialysis.     

Diets of reef‐dwelling labrids (Choerodon species) vary with body size,season and habitat: influence of foraging ability,specialization and opportunism

Contemporary multivariate statistics were used to test the hypotheses that the dietary compositions of three populations of labrids on the west Australian coast are related to body size and undergo seasonal changes and to elucidate the relative extents and basis for any dietary differences within and between those populations. Gut content analyses determined the dietary compositions of Choerodon rubescens in marine waters of the outer reefs in the World Heritage Area of Shark Bay (26° S; 114° E) and of Choerodon schoenleinii in inner protected reefs of that large embayment. The dietary compositions of C. rubescens and C. schoenleinii differed significantly among length classes, progressed serially with increasing body size, both overall and almost invariably in each season and were more closely related to body size than season, whose effect was at best minimal. The size‐related dietary change in C. rubescens involved, in particular, a shift from crustaceans and non‐mytilid bivalves to mytilid bivalves and echinoid echinoderms. Although the diet of C. schoenleinii followed similar size‐related changes, it contained a greater volume of gastropods when the fish were small and mytilids when large and only a small volume of echinoids. The dietary composition of C. rubescens in the Abrolhos Islands, 300 km to the south of Shark Bay, was related both to length class and season and differed from that of this labrid in Shark Bay with the ingestion of lesser volumes of mytilids and greater volumes of echinoids. The size‐related changes in diet imply that these species shift from foraging over soft substrata to over reefs as their very well‐developed jaws become sufficiently strong to remove attached and larger prey. The dietary compositions of C. rubescens and C. schoenleinii in Shark Bay and of C. rubescens at the Abrolhos Islands were related far more to habitat–locational differences than to length class and season. The above intraspecific and interspecific differences in diet are consistent with qualitative accounts of the relative abundances of the main prey in their respective environments, supporting the view that, despite specializations in their feeding apparatus, these labrids can feed opportunistically to a certain extent and could thus potentially respond to moderate changes in the composition of their prey caused by climate change and other anthropogenic effects.     

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase,Cause Retinal Dystrophy

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs^∗2];[Glu529Valfs^∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs^∗45(;)Trp1118^∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543^∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.     

Association of Maternal Vitamin D Status with Glucose Tolerance and Caesarean Section in a Multi-Ethnic Asian Cohort: The Growing Up in Singapore Towards Healthy Outcomes Study

Objective

Epidemiological studies relating maternal 25-hydroxyvitamin D (25OHD) with gestational diabetes mellitus (GDM) and mode of delivery have shown controversial results. We examined if maternal 25OHD status was associated with plasma glucose concentrations, risks of GDM and caesarean section in the Growing Up in Singapore Towards healthy Outcomes (GUSTO) study.

Methods

Plasma 25OHD concentrations, fasting glucose (FG) and 2-hour postprandial glucose (2HPPG) concentrations were measured in 940 women from a Singapore mother-offspring cohort study at 26–28 weeks’ gestation. 25OHD inadequacy and adequacy were defined based on concentrations of 25OHD ≤75nmol/l and >75nmol/l respectively. Mode of delivery was obtained from hospital records. Multiple linear regression was performed to examine the association between 25OHD status and glucose concentrations, while multiple logistic regression was performed to examine the association of 25OHD status with risks of GDM and caesarean section.

Results

In total, 388 (41.3%) women had 25OHD inadequacy. Of these, 131 (33.8%), 155 (39.9%) and 102 (26.3%) were Chinese, Malay and Indian respectively. After adjustment for confounders, maternal 25OHD inadequacy was associated with higher FG concentrations (β = 0.08mmol/l, 95% Confidence Interval (CI) = 0.01, 0.14), but not 2HPPG concentrations and risk of GDM. A trend between 25OHD inadequacy and higher likelihood of emergency caesarean section (Odds Ratio (OR) = 1.39, 95% CI = 0.95, 2.05) was observed. On stratification by ethnicity, the association with higher FG concentrations was significant in Malay women (β = 0.19mmol/l, 95% CI = 0.04, 0.33), while risk of emergency caesarean section was greater in Chinese (OR = 1.90, 95% CI = 1.06, 3.43) and Indian women (OR = 2.41, 95% CI = 1.01, 5.73).

Conclusions

25OHD inadequacy is prevalent in pregnant Singaporean women, particularly among the Malay and Indian women. This is associated with higher FG concentrations in Malay women, and increased risk of emergency caesarean section in Chinese and Indian women.     

The Spontaneously Adhesive Leukocyte Function-associated Antigen-1 (LFA-1) Integrin in Effector T Cells Mediates Rapid Actin- and Calmodulin-dependent Adhesion Strengthening to Ligand under Shear Flow

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5–20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.     

Water quality assessment using diatom assemblages and advanced modelling techniques

1. Two types of artificial neural networks procedures were used to define and predict diatom assemblage structures in Luxembourg streams using environmental data. 2. Self‐organising maps (SOM) were used to classify samples according to their diatom composition, and multilayer perceptron with a backpropagation learning algorithm (BPN) was used to predict these assemblages using environmental characteristics of each sample as input and spatial coordinates (X and Y) of the cell centres of the SOM map identified as diatom assemblages as output. Classical methods (correspondence analysis and clustering analysis) were then used to identify the relations between diatom assemblages and the SOM cell number. A canonical correspondence analysis was also used to define the relationship between these assemblages and the environmental conditions. 3. The diatom‐SOM training set resulted in 12 representative assemblages (12 clusters) having different species compositions. Comparison of observed and estimated sample positions on the SOM map were used to evaluate the performance of the BPN (correlation coefficients were 0.93 for X and 0.94 for Y). Mean square errors of 12 cells varied from 0.47 to 1.77 and the proportion of well predicted samples ranged from 37.5 to 92.9%. This study showed the high predictability of diatom assemblages using physical and chemical parameters for a small number of river types within a restricted geographical area.     

Modelling roach (Rutilus rutilus) microhabitat using linear and nonlinear techniques

• 1 Multiple linear regression (MLR), generalised additive models (GAM) and artificial neural networks (ANN), were used to define young of the year (0+) roach (Rutilus rutilus) microhabitat and to predict its abundance.
• 2 0+ Roach and nine environmental variables were sampled using point abundance sampling by electrofishing in the littoral area of Lake Pareloup (France) during summer 1997. Eight of these variables were used to set up the models after log₁₀ (x+ 1) transformation of the dependent variable (0+ roach density). Model training and testing were performed on independent subsets of the whole data matrix containing 306 records.
• 3 The predictive quality of the models was estimated using the determination coefficient between observed and estimated values of roach densities. The best models were provided by ANN, with a correlation coefficient (r) of 0.83 in the training procedure and 0.62 in the testing procedure. GAM and MLR gave lower prediction in the training set (r = 0.53 for GAM and r = 0.32 for MLR) and in the testing set (r = 0.48 for GAM and r = 0.43 for MLR). In the same way, samples without fish were reliably predicted by ANN whereas GAM and MLR predicted absence unreliably.
• 4 ANN sensitivity analysis of the eight environmental variables in the models revealed that 0+ roach distribution was mainly influenced by five variables: depth, distance from the bank, local slope of the bottom and percentage of mud and flooded vegetation cover. The nonlinear influence of these variables on 0+ roach distribution was clearly shown using nonparametric lowess smoothing procedures.
• 5 Non‐linear modelling methods, such as GAM and ANN, were able to define 0+ fish microhabitat precisely and to provide insight into 0+ roach distribution and abundance in the littoral zone of a large reservoir. The results showed that in lakes, 0+ roach microhabitat is influenced by a complex combination of several environmental variables acting mainly in a nonlinear way.

     

Patterns of endemism in riverine fish of the Northern Hemisphere

Loss of endemic species represents a symptom of general degrading ecosystem conditions that is the indirect result of biodiversity alteration. Here, we developed a predictive model relating species richness of endemic riverine fishes to measured biological, climatic, and historical variables using data from 118 rivers distributed all over the Northern Hemisphere. In a minimally adequate multiple general least square model, total riverine fish species richness, historical biogeography (Pleistocene glaciations), and comtemporary climate accounted for 63% of the variability in endemic species richness; the strongest correlate being riverine fish species richness. Our findings suggest that (i) endemism and richness patterns are generally similar (fish diversity "hot-spots" areas sustain higher endemic species richness); (ii) glaciation in the Pleistocene have had a significant negative influence on endemic species richness in the more septentrional areas; and (iii) certain basins situated in desertic areas (subtropical dry-zone of deserts) have unusually high numbers of endemics. These last areas should not be overshadowed when setting conservation priorities.