Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. All animals have evolved in a bacterial world, and interactions with microbes are known to affect many aspects of animal biology . One of The mammalian gut typically hosts hundreds of bacterial species, where the taxonomic composition varies both within and between host species . Based oDespite variability in species composition, the mammalian gut microbiota is dominated by relatively few bacterial phyla, including Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucomicrobia . InteresFigure 1). Analysis of intra-species diversity is therefore needed to shed light on how horizontal gene transfer, competition and selection shape the evolution of gut-colonizing bacteria. In this review, we will outline current knowledge on intra-species diversity within the gut microbiota, discuss methodological approaches and propose possible future research avenues in this exciting new field.Overall, 16S rRNA profiling studies have provided invaluable insights into the taxonomic composition of the gut microbiota, which in turn has facilitated the inference of broad evolutionary patterns. However, species diversity may only present the tip of the iceberg of microbial complexity in the gut. Recent studies applying methods that go beyond the typical 16S rRNA profiling approach have provided compelling evidence that the bacterial species of the gut microbiota are composed of a multitude of strains, which are likely to influence gut microbiota function  . These groups are subsequently referred to as “OTUs” , thereby circumventing the need for the term “species.” Nevertheless, the 97% cut-off was originally proposed as a proxy for bacterial species, as it was found to correspond well to the 70% cut-off in DNA-DNA hybridization analysis  and true diversity becomes non-trivial when the informative number of SNPs is small . 16S rRNBox 1), our current knowledge on the extent and distribution of intra-species diversity in the gut microbiota is scarce. However, pioneering studies have been conducted on the human gut microbiota, which have provided exciting new insights. Pilot studies have also been done on the honeybee, likewise unraveling the presence of extensive intra-species diversity. Thus, in the following, we will describe what is known about intra-species diversity in the gut microbiota, focusing primarily on these two model organisms.Since standard 16S rRNA profiling provides little resolution for inferring intra-species diversity : what is the functional relevance of intra-species diversity in the gut? And how are host-specific profiles generated? On one hand, the host could potentially select strains with specific functional profiles. In fact, since the gut microbiota strain profile tends to be more similar among related individuals, it is even possible that strains could adapt to specific host backgrounds. On the other hand, host-specific strain profiles could also result from colonization bottlenecks early in life  has been shown to harbor 8–10 bacterial species groups in the gut, which usually constitute more than 98% of the total bacterial community .The simplest explanation for stable co-existence within the gut would arguably be that bacterial strains belonging to the same species group do in fact occupy distinct niches, despite being closely related. Indeed, several studies have shown that strains of the same species-group can be ecologically distinct within a shared habitat . Within The animal gut harbors a wide range of microbial habitats that vary in physiological parameters such as pH, oxygen and concentration of antimicrobial compounds . Thus, dFigure 2A). For example, strain diversity within Bacteroides spp. is associated with PULs . Assuming that members of the gut microbiota can tolerate unfavorable conditions, at least for some time, a variable diet could potentially support a large number of specialized strains. For example, a large proportion of the human gut microbiota was recently shown to have spore-forming capabilities . For example, both mammals and insects secrete antimicrobial peptides in the gut, which affect bacteria differentially . Phages have previously been proposed to play an important role in maintaining strain diversity in natural bacterial populations, via “kill-the-winner” dynamics .According to classic ecological principles, co-operation can be expected to be common among bacteria of the same genotype, whereas competition should prevail among different genotypes, i.e., strains of the same species-group . Neverth-culture . Therefoin vitro among members of the Bacteroidetes genus , but their relative importance for shaping and maintaining intra-species diversity is largely unknown. Is the gut simply an environment, which supports a high degree of micro-niche differentiation? Or do bacterial strains depend on or benefit from each other’s presence? And to what extent is the host involved in controlling and manipulating these highly complex communities? In order to answer these questions, we need to be able to quantify and follow the abundance of bacterial strains over time and in response to experimental manipulation, directly from bacterial communities. In the following sections, we will describe current methods employed to study intra-species diversity and dynamics directly from metagenomic samples.Several different mechanisms have been proposed to be involved in the maintenance of diversity within the gut microbiota (Box 1). To address this problem, Taxonomic profiling of the gut microbiota is most commonly done based on PCR amplification of the 16S rRNA gene. However, for the study of intra-species diversity, the 16S rRNA profiling method provides limited resolution, and is susceptible to biases introduced by sequencing and PCR artifacts ; Box 1. Given the low information content of the 16S rRNA gene, primers targeting alternative genes have also been developed for studies on intra-species diversity. Although such primers can only target a subset of any community, they can provide a high level of resolution, and have been successfully applied to demonstrate the presence of intra-species diversity and co-diversification on shorter evolutionary time-scales in several recent papers . HoweverAside from PCR-associated artifacts, the targeting of single genes for diversity and evolutionary analyses is also affected by the variable rates of gene evolution in different bacterial species. For example, a comparison of the 16S rRNA identity and ANI (average nucleotide identity) scores between species showed that the core genome and the 16S rRNA gene evolve at different relative rates in different species . TherefoUsing shotgun metagenomics, sequencing data is obtained from complete genomes within a sample. Thus, although shotgun metagenomics has mostly been used to derive functional profiles of bacterial communities, this approach also has the potential to provide a high level of resolution for intra-species diversity analysis.de novo assembled metagenomic contigs . Thereby, the extent of intra-species diversity can be estimated, and genes under positive or diversifying selection may be identified . Moreover, when samples are available for multiple time-points, the abundance of strains  can be followed over time.To reduce this dependency, In conclusion, SNP-based analysis of shotgun metagenomic data provides a powerful framework for quantifying intra-species diversity, selection and abundance dynamics. However, since all current SNP-based methods are based on core genes or marker genes, the functional profiles of quantified strains are not known, complicating the interpretation of abundance fluctuations.Given the lack of functional information provided by PCR profiling and SNP calling methods, several recent methods have been developed to study intra-species gene content variation, based on shotgun metagenomics data.de novo or mapped against a reference database . De novo assembly is desirable, since it allows for the discovery of novel genes or genomes in a sample. However, the assembly of short reads from complex bacterial communities is highly challenging. Firstly, the use of short reads is a general problem for genome assembly, since any repetitive sequences will hinder the assembly. This problem is exacerbated in samples from complex bacterial communities, where different species will have different optimal assembly parameters. Moreover, sequences from related strains may assemble into single or multiple contigs, depending on the conservation of the genomic region. Therefore, although several assemblers have by now been developed specifically for metagenomic data .In order to determine the gene content within a sample, the sequences can either by assembled mic data , metagende novo assembly, read mapping represents a much less complex computational problem and several mapping tools are available that can efficiently handle even very large datasets .If a representative database of previously sequenced genomes is available, reference genome mapping can be a viable alternative. Relative to datasets . Howeverdatasets . Thus, tFigure 3). Similarly, when a suitable reference genome database is available, the pan genome can be estimated and genes with higher or lower coverage relative to the core genes identified .In conclusion, all current methods for inferring intra-species gene content variation in metagenomic samples are based on mapped read coverage on genes, and therefore depend on the availability of a suitable reference database or a representative terminus . FinallyFigure 4), the development of sequencing technologies that can generate longer reads has the potential to greatly advance the field. Several novel technologies are currently under development, some of which have already become commercially available, with demonstrated improvements on applications such as de novo genome assembly  sequencing  and NanoFinally, metagenomic analysis targeting complete genomes also require very high throughput. Even with Illumina Hiseq sequencing, which is currently the most cost-effective and large-scale sequencing platform available, insufficient coverage is frequently a problem for strain-level analysis. For example, in a re-analysis of publicly available metagenomic data from the Human Microbiome Project, intra-species gene content variation was investigated for only 11 bacterial species, since all other species did not have sufficiently high coverage for reliable strain quantification .Based on several recent studies, it is now clear that intra-species diversity is omnipresent in the gut microbiota, also within host individuals . Howeverin vitro and in vivo. Thirdly, a large number of genomes isolated from the honeybee gut have by now been sequenced and analyzed, paving the way for future bioinformatic studies . In addition to providing distinct technical advantages, the use of diverse animal models will be of particular importance for understanding differences and commonalities of gut microbiota evolution across the metazoa. Likewise, a better understanding of fundamental mechanisms involved in maintaining and establishing diverse gut bacterial communities will be indispensable for establishing safe and effective probiotics to cure disease. For example, a recent study found that the success of colonization after fecal microbiota transplantation differed between hosts when using the same donor sample (A combination of shotgun metagenomics and experimental data from animal models has the potential to provide novel insights into many of the currently outstanding questions concerning intra-species diversity in the gut microbiota (r sample . Moreover sample , highlig• Evolution◦How do strains diversify in the gut?◦How do strains adapt to specific host environments?◦What is the impact of selection pressure exerted by the host versus other bacterial community members?• Function◦Do specific strains contribute distinct complementary functions in the gut?◦Does the functional contribution of specific strains depend on host-specific factors or the presence of other community members?◦Does a high level of intra-species diversity benefit the host, i.e., by increased productivity or by providing robustness against disturbances?• Community assembly and interactions◦How are communities with high levels of intra-species diversity established and maintained?◦Do strains of the same species group occupy distinct niches in the gut?◦Do strains of the same species group compete or collaborate?◦Which mechanisms facilitate stable co-existence within the gut?All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy with a prevalence of 1 in 500 in the general population. Since the first pathological case series at post mortem in 1957, we have come a long way in its understanding, diagnosis and management. Here, we will describe the history of our understanding of HCM including the initial disease findings, diagnostic methods and treatment options. We will review the current guidelines for the diagnosis and management of HCM, current gaps in the evidence base and discuss the new and promising developments in this field. In 1957, Robert Donald Teare, an English pathologist at St. George’s Hospital in London, reported the autopsy findings of eight patients with asymmetrical hypertrophy of the heart, seven of whom died suddenly . It is cHypertrophic cardiomyopathy was first described by Vulpian, as “idiopathic hypertrophic subaortic stenosis” in 1868, after its anatomical anomaly ,8 in theIn the same year, Bercu et al. published their post-mortem findings of a patient who was diagnosed clinically as having aortic stenosis and was referred for aortic valvulotomy . During By 1961, although still poorly understood, this disease entity was recognised to be distinct from aortic stenosis and termed idiopathic hypertrophic subaortic stenosis (IHSS). As two-dimensional echocardiography was not yet possible, diagnosis was made primarily during cardiac catheterisation. Since the aortic valve could not be directly visualised, distinguishing HCM from aortic stenosis remained challenging, even with invasive haemodynamics. Brockenbrough described the effect of post-premature ventricular contractions on aortic stenosis and HCM as a means of distinguishing the two conditions . Frank–SIn HCM, the obstruction in the LVOT is dynamic (orifice narrows during systole and relaxes during diastole), whereas, in aortic stenosis, the obstruction is constant. Brockenbrough et al.  hypothesIn 1963, the nonobstructive form of HCM was first described. Braunwald et al. found 14 patients, who, despite having left ventricular hypertrophy, brisk arterial pulses, a fourth heart sound and an ejection systolic murmur, did not have evidence of LVOTO (Left ventricular outflow tract obstruction) and were largely asymptomatic . This waLeft ventricular outflow tract obstruction (LVOTO) is defined as a peak LVOT gradient of ≥30 mmHg. LVOTO has been shown to be associated with an increased risk of SCD ,30,31,32Two factors contribute to LVOTO; hypertrophy of the basal septum and systolic anterior motion (SAM) of the mitral leaflet. In HCM, the mitral valve architecture is abnormal, characterised by leaflet elongation, anterior displacement of the apparatus and papillary muscle abnormalities A. These When the mitral valve touches the septum, it transiently but significantly narrows the LVOT, leading to a greater obstruction and a greater pressure difference across the LVOT. This pressure difference further drags the mitral leaflets against the septum, narrowing the orifice further, resulting in a reinforcing feedback loop that persists until the end of systole. This significantly reduces cardiac output and contributes to coronary flow abnormalities . Where cIn its nascent days, the diagnosis of HCM revolved mainly around identifying dynamic LVOTO, a characteristic we now know only exists in approximately one-third of patients at rest. This was initially done via cardiac catheterization, which allowed pressure gradients across the LVOT to be measured. Although a reliable diagnostic tool, it was invasive, time-consuming, caused discomfort and was not without radiation and procedural risks.Echocardiography has transformed diagnostic testing in cardiology. In 1969, Moreyra et al. pioneered the use of echocardiography in diagnosing HCM . The ideTwo-dimensional (2D) echocardiography has long been the mainstay initial modality of choice in HCM diagnosis. It is used to outline cardiac structures and assess valvular and cardiac function. Doppler echocardiography at rest and during Valsalva manoeuvre is recommended to detect evidence of resting or latent LVOTO C. Left vToday, the diagnosis of HCM relies on the identification of increased left ventricular wall thickness on imaging. This is most commonly via echocardiography. The European Society of Cardiology/American College of Cardiology Foundation (ESC/ACCF) diagnostic criteria for HCM is a wall thickness of ≥15 mm in at least one LV myocardial segment, in the absence of abnormal loading conditions such as hypertension and valvular disease ,45. A loCMR is a helpful diagnostic and risk stratification tool in HCM. It possesses excellent spatial and temporal resolution, allowing accurate assessment of ventricular volumes and anatomical structures . Unlike PET is the gold-standard imaging modality for the non-invasive detection of ischaemia and is particularly valuable in identifying microvascular dysfunction through perfusion abnormalities. In the absence of epicardial coronary disease, coronary flow resistance is predominantly determined by the coronary microvasculature. In patients with normal or minimal coronary artery disease, a blunted increase in myocardial blood flow during cardiac stress may be present and is a strong independent predictor of heart failure, SCD, LV remodelling and cardiovascular mortality in HCM. Although perfusion abnormalities predict adverse outcomes, there is a lack of targeted treatment options for these abnormalities and so PET is not recommended for risk stratification ,45.The first pathogenic mutation implicated in HCM was discovered by Geisterfer-Lowrance et al. in 1990 . They foTo date, nine genes have been definitively implicated in HCM ,55,56,57Historically, management of HCM has focussed on symptom relief and reducing the risk of fatal arrhythmic events. Prevention of SCD in HCM is complex. Various risk stratification schemes have been proposed. Risk stratification helps guide clinicians as to whether implantable cardioverter defibrillator (ICD) implantation should be recommended. The ICD detects sustained ventricular arrhythmias (ventricular fibrillation or ventricular tachycardia) and either attempts to use anti-tachycardia pacing to terminate ventricular tachycardia or delivers a cardioversion (“shock”).ICD implantation will effectively treat VT or VF in HCM. However, determining an appropriate and acceptable threshold for ICD implantation is complicated. ICDs can adversely affect patients’ quality of life and psychological wellbeing through inappropriate discharges and driving restrictions, and have periprocedural risks such as infection, pneumothorax, pericardial effusion, lead displacement and death. The first ICD use in humans was pioneered by Mirowski et al. in 1980 . An HCM Maximal LV wall thickness,Left atrial diameter,Maximal LVOT gradient ,Family history of SCD,Unexplained syncope,Non-sustained ventricular tachycardia (NSVT), andAge.Currently, two main models guiding ICD implantation are advocated by the ESC and American College of Cardiology Foundation/American Heart Association (ACCF/AHA), respectively ,45. The 2S2-VASc model used for estimating stroke risk in AF. Using the risk scores from this model, the ESC states that ICD implantation in patients with a 5-year SCD risk of; ≥6% should be considered, ≥4% to <6% may be considered, and <4% to be considered only in the presence of clinical features proven to have prognostic importance. All patients receiving ICD should have a life expectancy of >1 year. Using these risk factors, they produced a formula predicting 5-year SCD risks with a discriminatory ability (C-statistic 0.7) that is comparable to the widely used CHADNSVT,family history of SCD,maximal LV wall thickness ≥30 mm,unexplained syncope, andabnormal blood pressure response during exercise (defined as a decrease in blood pressure or inability to increase blood pressure by ≥20 mmHg during exercise).The second model, advocated by the ACCF/AHA, states that ICD implantation can be considered in the presence of ≥1 of the following risk factors:The implantation rates of both models differ quite significantly. Use of ACCF/AHA model resulted in an ICD implantation rate of 40–60% ,60 of HCPatients with LVOTO are advised to avoid factors that reduce the end diastolic volume  or increase myocardial contractility and hence the degree of outflow tract obstruction . Simple lifestyle measures such as weight loss and staying well hydrated are appropriate. Medical therapy comprises largely of negatively inotropic drugs and is aimed at reducing left ventricular wall contraction, reducing the narrowing of the outflow tract. Both ESC and ACCF/AHA have similar recommendations ,45: Non-Invasive therapy aims at reducing interventricular septum thickness and contractility, thus reducing LVOT gradient. Septal reduction therapy should be considered in symptomatic patients (NYHA III-IV) with a resting or maximum provoked LVOT gradient of ≥50 mmHg, despite optimal medical therapy ,45. It sSurgical myectomy involves the removal of a small amount (5–10 g) of hypertrophied tissue from the basal interventricular septum. Many centres now also incorporate an extended myectomy down to the level of the mid-septum. Morrow performed the first surgical myectomy in 1964 [Surgical myectomy has been the gold standard for septal reduction since its introduction in 1964. It obliterates or significantly reduces LVOT gradient in 90% of patients when performed in expert centres ,66,67,68Three decades after the first myectomy in HCM, Sigwart performed the first ever non-surgical septal reduction  using suBoth surgical myectomy and ASA may provide substantial symptomatic improvement, increased exercise capacity and reduction or obliteration of an LVOT gradient ,72,73,74Septal reduction therapy decisions should be jointly made by a multidisciplinary team of expert cardiologists, cardiothoracic surgeon, a heart care team and the patient. Septal reduction therapies should be performed at dedicated HCM centres, as this is associated with lower rates of complications. Atrial fibrillation has a prevalence of over 20% in the HCM population  comparedThromboembolism is a common consequence of atrial fibrillation, with an estimated prevalence of up to 30% in the HCM population with AF . It is o2DS2VASc score should not be used in risk stratification of HCM with AF as the risk of stroke is already high. Restoration of sinus rhythm should be considered in new onset AF after at least three weeks of sufficient anticoagulation or exclusion of left atrial appendage thrombus. Non-invasive management of AF in HCM focuses on rate control, thromboprophylaxis and restoration of sinus rhythm. Rate control is achieved with beta blockers or non-dihydropyridine calcium channel antagonists. Oral anticoagulation should be started in all HCM patients with AF where possible. Warfarin remains the anticoagulant recommended by the ESC/ACCF ,45. HoweAF ablation may be considered as a method to restore sinus rhythm. There are limited reports of AF ablation outcomes in HCM, although procedural success rate seems to be lower in the HCM population compared to the general population  . HoweverIschaemia is a common finding in HCM. Up to half ,89,90 ofThe impaired relaxation of hypertrophied and fibrotic ventricles during diastole will also lead to an increased intramural end diastolic pressure, while contraction of the hypertrophied ventricle during systole will result in greater compression of the intramyocardial vessels and reduced myocardial blood flow. LVOTO may further impair myocardial blood flow during systole . While the presence of ischaemia is well-recognised in HCM, its effects on outcomes were largely understudied until 2003 when Cecchi et al. showed that extent of microvascular dysfunction measured using PET strongly predicted clinical deterioration and death in HCM . PatientGuidelines for the management of microvascular dysfunction are largely absent. Data on the beneficial effects of treating perfusion abnormalities on long-term outcomes such as heart failure, mortality and SCD are currently limited. Therapies that reduce myocardial contractility  and prolong diastole, such as beta blockers and calcium channel blockers, are probably beneficial. LGE in CMR is an excellent tool in identifying myocardial fibrosis. However, the LGE technology is based on a nulling method, which means it requires normal surrounding myocardium to detect areas of fibrosis. This constitutes a challenge in patients who have diffuse interstitial fibrosis. T1 mapping in CMR may provide a solution . Recent Diffusion tensor CMR (DT-CMR) studies the diffusion of water across microstructures. This is based on the concept that water molecules diffuse at different rates along microstructures of different anisotropy and orientation. Water molecules diffuse more easily along the direction that the microstructures are aligned in, and diffuse slower when going in a direction perpendicular to the alignment. Using DT-CMR, recent work has shed some light on the behaviour and orientation of cardiomyocyte sheetlets (the secondary organisation of cardiomyocytes) in HCM during systole and diastole . In norm(1)Fibrotic development occurs when the equilibrium between collagen synthesis and degradation is disrupted(2)Sarcomeric mutations may directly induce the development of fibrosis, irrespective of the presence of ischaemia.Myocardial fibrosis has long been a hallmark of HCM and has been assumed to be a result of repetitive ischaemic injury and consequent remodelling of LV tissue. A recent biomarker study looking at the C-terminal propeptide of type I procollagen (PICP), a by-product of type I collagen synthesis, have found that this biomarker was significantly raised in sarcomeric mutation carriers compared to their mutation-negative counterparts . PICP waIf confirmed by subsequent studies, this may allow clinicians to identify HCM patients with high levels of PICP who may be at elevated risk of aggressive disease progression before the development of overt hypertrophy. Currently, genetic testing has little role to play in risk stratification in HCM and is primarily used for family screening. However, patients with multiple sarcomeric gene mutations have an increased risk of cardiovascular death, SCD and end-stage progression ,109. AltSpironolactone has been demonstrated in an animal study to have an incremental effect in reducing cardiac hypertrophy when combined with captopril in rat models with HCM . Its effNaL) in cardiomyocytes. This prolongs the action potential and increases intracellular Ca2+ accumulation, increasing myocyte contraction, and thus greater energy expenditure and ischaemia. By inhibiting late sodium currents, Ranolazine is thought to reduce myocardial energy expenditure and potentially slow disease progression in HCM. The latter was shown to be possible in mice models but is yet to be demonstrated in human models [Ranolazine has shown promising results in improving angina, heart failure symptoms, and quality of life in symptomatic HCM patients in a proof-of-concept study . In HCM,n models .Angiotensin receptor blockers (ARBs) have been postulated to be effective at inducing phenotypic regression in transgenic HCM ,115,116.Endocardial radiofrequency ablation (ERA) is a relatively new interventional technique that offers an alternative to ASA in the treatment of LVOTO. It involves the application of radiofrequency lesions to the hypertrophied septum, inducing necrosis and the development of fibrosis within the septum. Although the effects on septal size are limited, the damaged myocardium does not contract and systolic excursion of the basal septum into the LVOT is reduced, reducing LVOTO ,118. ERAMYK-461 is a novel molecular inhibitor of sarcomeric contraction. Discovered recently in a screen for direct inhibitors of the sarcomere , MYK-461Our understanding and management of HCM has come a long way since its first modern description in the 1950s. Management of HCM is complex and involves risk assessment of SCD and the treatment of complications resulting from the disease process, such as LVOTO, AF, ischaemia and heart failure. The developing role of genetic and molecular therapy to selectively target the defects of the sarcomere directly is particularly exciting since this may prevent the development of the HCM phenotype altogether. Developing new treatment options for microvascular ischaemia and studying its beneficial effects on long-term outcomes may support the call for early, aggressive anti-ischaemic treatment in HCM.


Some engineered nanomaterials (ENMs) may have the potential to cause damage to the genetic material in living systems. The mechanistic machinery functioning at the cellular/molecular level, in the form of DNA repair processes, has evolved to help circumvent DNA damage caused by exposure to a variety of foreign substances. Recent studies have contributed to our understanding of the various DNA damage repair pathways involved in the processing of DNA damage. However, the vast array of ENMs may present a relatively new challenge to the integrity of the human genome; therefore, the potential hazard posed by some ENMs necessitates the evaluation and understanding of ENM-induced DNA damage repair pathways. This review focuses on recent studies highlighting the differential regulation of DNA repair pathways, in response to a variety of ENMs, and discusses the various factors that dictate aberrant repair processes, including intracellular signalling, spatial interactions and ENM-specific responses. The unique properties of engineered nanomaterials (ENMs), intentionally manufactured particles or objects with at least one dimension in the size range of 1 nm to 100 nm (with 50% or more particles in the number size distribution), have contributed to the exponential growth and innovative advances in nanotechnology ,3. MonodOngoing research on evaluating the safety of ENMs has highlighted the potential of these nano-entities to cause perturbation of various cellular pathways and functional processes ,3. The aThe integrity of the genome is maintained at three levels. Level 1 involves phase I  and/or phase II  metabolizing enzymes that can process, inactivate or intercept the mechanistic processes that lead to ENM-induced DNA damage . For exaDNA repair is a complex process: it is comprised of >168 genes (that encode for proteins) involved in numerous, diverse processes encompassing intracellular signalling, cell cycle checkpoints, enzymatic reactions and chemical and structural modifications and transformations which eventually culminate in DNA repair . Each paDNA damage signalling (DDS): this pathway is induced in response to DNA damage caused by various agents including environmental, ENM and endogenous. DDS pathways are programmed to induce several cellular responses including checkpoint activity, triggering of apoptotic pathways and DNA repair .Direct reversal repair (DRR): reverses/eliminates the DNA damage caused by chemical reversal or modification by restoring the original nucleotide. It is also known as direct DNA damage reversal.Base-excision repair (BER): this repair mechanism is initiated by the excision of modified bases from DNA by DNA glycosylases. The length of the DNA that needs to undergo re-synthesis can be variable; thus, the pathway can be subdivided into short-path or long-path BER. Although various pathways are involved in this repair process, one of the most widely studied mechanisms that triggers the BER pathway is oxidatively induced damage. Since oxidative stress is one of the most common mechanisms of ENM-induced DNA damage, oxidatively induced DNA lesions are predominantly repaired by the BER pathway see . The keyNucleotide excision repair (NER): is involved in removing bulky DNA adducts. The damage from the active strand of transcribed DNA and DNA damage elsewhere in the genome is removed in this pathway by transcription-coupled repair and global genome repair, respectively. Silver and cadmium based ENMs have been shown to interfere with the NER pathway .Mismatch repair (MMR): this pathway is involved in post-replicational DNA repair that removes errors including mismatched nucleotides, insertions, deletions, etc.Homologous recombination repair (HRR): this pathway involves repair of DSBs using the homologous DNA strand as a template for re-synthesis.Non-homologous end joining repair (NHEJ): helps to ligate the DNA ends resulting from DSBs.Translesion synthesis (TLS): this pathway employs specialized polymerases that use damaged DNA as templates, to finish replication across lesions. Although the mechanism is error-prone, and cell survival may be associated with an increased risk of mutagenesis/carcinogenesis, it helps to prevent a stalled replication fork.There are multiple DNA repair pathways targeting various levels and extent of DNA damage. Researchers over the last few decades have progressively deciphered and revealed responses to DNA damage (including ENM-induced); currently, the DNA damage repair pathways can be divided into eight major categories:DNA repair pathways/proteins seldom work in isolation in the cell, i.e., the repair pathways are interdependent and interconnected via shared proteins and components of the DNA repair system. More than one pathway may be up-regulated/down-regulated in response to cellular exposure by a given ENM. Moreover, repair pathways (genes/proteins/enzymes) induced because of DNA damage do not follow a similar trend with respect to being up-regulated or down-regulated; different studies on ENM exposure have shown varied DNA damage responses, i.e., the same gene/protein is up-regulated in one study, while being down-regulated in another ENM-exposure study. For example, several studies have shown apurinic/apyrimidinic endonuclease (APEX), involved in the BER pathway to be either up-regulated or down-regulated in response to exposure by ENMs . This la2 NPs) induce the activation of serine/threonine kinase ATM/Chk2, involved in the DDS signalling pathway pyrene-7,8-diol-9,10-epoxide,  on zebrafish liver cells revealed that exposure to soluble Cd2+  significantly reduced NER repair capacity. This observation was attributed to Cd2+ interacting with DNA and hindering interactions, that lead to BPDE adduct formation, cellular uptake, intracellular distribution and possibly metabolism [2+-inhibition of DNA repair of BPDE-induced DNA lesions has been studied previously [2+ in the nucleus [Release of ions governs repair capacity: co-exposure studies using Cdtabolism . Indeed,eviously ,34. Ther nucleus .2 NPs [2 NPs. The authors showed inhibition of both BER and NER pathway, as evaluated using a DNA repair microarray assay [Apart from Cd ions, studies have shown that Fe ions can inhibit APE1’s enzymatic activity . McNeill2 NPs ,14. Moreay assay .The evolving development of ENMs has enabled innovative breakthroughs in a variety of sectors such as healthcare, manufacturing, agriculture and transportation ,39. The Besides the adverse impact on genome integrity, it is worth mentioning that any defects in DNA repair genes involving aberrant regulation may impact a range of physiological functions and cellular processes. This is because the DNA repair pathway genes/proteins are also involved in apoptosis, transcriptional regulation, migration, telomere maintenance, chromatin remodelling and dNTP synthesis . AlteredCharacterisation of induced DNA damage lesions: a given ENM may have a primary mechanism for the induction of DNA damage, which triggers the initiation of a specific repair pathway. For example, metal and metal oxide based ENMs tend to cause oxidatively induced DNA damage, which is mainly repaired via the BER pathway. Therefore, characterising the type of DNA damage is critically important in future studies, as it will enable predictive models to be developed that can be used to predict which types of ENMs might affect specific DNA repair pathways.2+, Fe3+, Zn2+, and Ag+ and hence influence the upregulation/downregulation (measured as excision activity) of various pathways. Additionally, metal ions released from ENMs have been shown to interact/bind with protein domains and amino acids of DNA repair proteins  resulting in distorted protein structure and inefficient DNA repair activity [Role of ions: inorganic NPs could via corrosion and dissolution release metal ions such as Cdactivity . TherefoDose-dependent DNA damage response: presently, the doses of ENMs administered in in vitro studies/test species to generate dose-response analysis may not mimic a potential human exposure level. This is because concentration-dependent activation of genes/pathways as well as transition in gene changes can be highly dose dependent. Therefore, dose ranges that are relevant to true exposure levels of ENMs need to be included when studying DNA damage responses pertaining to repair pathways. However, ENM exposure assessment currently presents a technical challenge and more work is needed to evaluate emissions of ENMs into the environment . For exaMethod/technique: various techniques and methods with different endpoints are utilized for evaluating DNA damage repair and/or DNA damage responses, e.g., Western blots for translational changes/modifications and/or phosphorylation events; RT-PCR for transcriptional alterations; excision or incision assays for DNA repair enzyme activity; mass spectrometry methods for measuring adduct or lesion formation and multiplexed excision/synthesis assays for DNA repair enzyme inhibition activity . Each meTissue specific detection /expression: different tissues and cell types  exhibit varying DNA repair responses, which may correlate with the degree of DNA damage and susceptibility following exposure to some ENMs. Hence, it is imperative to measure the levels and activity of DNA repair genes and proteins, respectively, in all relevant cells, tissues or organs of interest as their expression and responses are largely “site-specific”.Effect of acute vs. chronic exposure of ENMs: the type of exposure may affect the severity of the DNA damage and the resultant activation of specific DNA repair pathway(s). The human population may be exposed to natural, environmental or ENMs in a cumulative manner . On the Effect of potential ENM artefacts on the interpretation of DNA damage repair or DNA damage response: as described in previous reports, the solution state physico-chemical properties of ENMs are not like the solution state physicochemical properties of chemicals . DependiGiven the importance of DNA repair, it is vital to understand the role it plays in the genotoxic responses observed, and its specific impact on molecular interactions necessary for maintaining genomic stability. This review of the current literature demonstrates that there are several different factors that control DNA damage-induced mechanistic repair pathways. Important aspects to consider in the future, in order to more fully understand the potential impact of ENMs on DNA repair fidelity include, but are not limited to:Although DNA repair process(es) are highly regulated and multifaceted involving cross-talk and overlapping functions, their evaluation in ENM-based studies contributes to an additional tier of complexity. Also, it is worth mentioning that it is extremely difficult at this stage to extrapolate from the in vitro/test species data discussed in this review, to actual ENMs exposure in vivo, e.g., outcomes on human health. The impact of ENM exposure on the fidelity of DNA repair processes is not fully understood at present and requires more in-depth investigation to elucidate the pathways of importance and the intrinsic or extrinsic ENM features that interfere with the maintenance of genomic stability.


Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin’s roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation. Figure 1A). It also binds to transcription regulators to control their activities . In addition to these functions of actin to support fundamental nuclear processes, nuclear actin influences cellular phenotypes through transcriptional regulation and chromatin alternation, exemplified by transcriptional activation of the osteogenic genes in differentiating mesenchymal stem cells . In this review, we summarize recent findings of functional roles of nuclear actin in transcriptional activation and chromatin remodeling. Currently, discovered functions of nuclear actin are executed in many different crucial cellular events such as transcription (Dynamic changes between globular (G-) and filamentous (F-) forms of nuclear actin affect these nuclear events. Nuclear actin polymerization is regulated by multiple mechanisms; more than 30 of actin-binding proteins that affect its polymerized states have been discovered in nuclei . It is ols MSCs;  or by rels MSCs;  . As a consequence, the amount of nuclear actin, regulated by IMPORTIN 9  . Even transcriptional reprogramming can be induced in the oil GV system after nuclear transplantation of somatic nuclei into oil GVs  . Hereaft oil GVs . Using tpression . Furtherpression . The knopression . These ron . Translocation of actin to nuclei is also observed when HL-60 cells differentiate toward macrophages after phorbol 12-myristate 13-acetate (PMA) treatment . Another recent study shows that nuclear actin is involved in lineage commitment of epidermal progenitor cells (EPCs) through the regulation of the transcriptional activity . In addition to these roles of nuclear actin in cell differentiation and lineage commitment, nuclear actin-binding proteins also serve as a key player for cell differentiation. In hematopoietic cells, WASP is localized in nuclei during T cell differentiation (H1 genes, such as IFNG and TBX21, and is important for differentiation (Considering nuclear roles of actin in transcriptional regulation, it is reasonable to speculate that nuclear actin is involved in cellular differentiation and development, which entail changes in transcriptional programs. Accumulating evidence indicates that this is indeed true at least in several different cell types. Induced accumulation of nuclear actin in MSCs causes transcriptional activation of the osteogenic genes osterix and osteocalcin, leading to the osteogenic phenotype. Mechanistically, YAP protein binds to and inhibits the osteogenic transcription factor RUNX2. The accumulation of nuclear actin induces nuclear export of YAP protein. As a result, RUNX2 is relieved from its repressive interaction with YAP and osteogenesis is enhanced in a RUNX2-dependent manner  (Figure reatment . In thisactivity . Upon inntiation . WASP suntiation . It is nntiation , althougntiation . DiffereXenopus laevis (Drosophila (Drosophila, nurse cells and GVs contain nuclear actin during oogenesis and nuclear actin rod formation is regulated by Fascin (Xenopus, a large amount of nuclear actin in GVs contribute to mechanical stability of the giant nucleus (Xenopus oocytes, is important for early embryonic development. Knockdown of maternal WAVE1 protein induces downregulation of Hox genes in gastrula embryos, which is rescued by nuclear WAVE1 expression (HoxB expression upon retinoic acid treatment has been shown in cultured cells (Hox gene expression and embryonic development (Nuclear actin is found in female germ cells of s laevis , Drosophosophila , and aviosophila . In the y Fascin . In Xeno nucleus , stabili nucleus  and tran nucleus . The amo nucleus . The abupression . In gooded cells . Howeverelopment . It is pelopment . Furtherin vivo cells and/or in the context of pathogenesis warrants further investigation.Recent studies have uncovered the implication of nuclear actin in a variety of nuclear processes, including basal transcription , transcrAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Immunodeficiencies reveal the crucial role of the immune system in defending the body against microbial pathogens. Given advances in genomics and other technologies, this is currently best studied in humans who have inherited monogenic diseases. Such investigations have provided insights into how gene products normally function in the natural environment and have opened the door to new, exciting treatments for these diseases. However, the immune system mainly evolved to defend against microbial pathogens encountered in early life. Thus, understanding how the human immune system is fundamentally regulated requires understanding how humans respond to infections. This is most directly accomplished by studying immunodeficient patients.The immune system impacts human health in many ways. Some obvious ways are causing autoimmune disease or allergic disease, contributing to immune surveillance against or killing off certain cancers, and determining whether transplanted organs will be accepted or rejected. More recently, the immune system has been recognized as influencing seemingly non-immune conditions. These include the obesity-related metabolic syndrome secondary to chronic low-level inflammationIn this brief review, I will define immunodeficiencies and argue that with newer genomics and other technologies, human studies of immunodeficiencies can offer insights equal to or surpassing those previously obtained from mice. In step with the expectation that basic research should lead to improvements in diagnosis and treatment, the ability to study these diseases in humans offers a concrete step towards the development of new immunomodulatory therapies.in vivo. In the past, immunodeficiencies were recognized by a pattern of recurrent, persistent, or severe infections. Often these included infections caused by unusual microbes: for example, opportunistic infection withPneumocystis jirovecii, which is a hallmark of severe combined immunodeficiency. However, with advances in medical care  and improvements in hygiene and other societal factors that are keeping patients alive longer, patterns of infection susceptibility in individual patients can be better characterized. This has led to increased recognition of functional redundancy in the immune system that results in some immunodeficiencies presenting with a much narrower infection susceptibility. An example is a group of inherited diseases that primarily present with an isolated susceptibility to Epstein-Barr virus (EBV), manifesting either with a fulminant course including hemophagocytic lymphohistiocytosis or with severe complications such as lymphoma. As this example illustrates, counterintuitively, immunodeficiencies can be accompanied by apparently hyperactive forms of immunodysregulation, including autoimmunity, allergy, lymphoproliferation, and autoinflammation, because of the regulatory complexity of the immune system.Inherited human immunodeficiencies are experiments of nature in which gene defects compromise immune function and thereby illuminate how the human immune system worksTraditionally, immunodeficiencies have been divided into primary immunodeficiencies, which reflect defects intrinsic to the immune system, or secondary immunodeficiencies, which reflect defects extrinsic to the immune system. Although some secondary immunodeficiencies can be caused by environmental factors such as drugs or irradiation, others can be indirectly caused by non-immune conditions. An example of the latter, primary ciliary dyskinesia, is caused by one of a number of autosomal recessive mutations that affect the structure of cilia on respiratory epithelial cells; by compromising ciliary function, mucus clearance is impaired, leading to bacterial stasis and recurrent sinopulmonary infections.3. Because altered glycosylation patterns can affect protein structure and function, this class of disorders typically causes widespread abnormalities in multiple organ systems, including the nervous system and the immune system. One recently discovered example is caused by autosomal recessive hypomorphic mutations in the phosphoglucomutase-3 (PGM3) gene6. PGM3 encodes an enzyme required for the generation of UDP-Glc-NAc, a building block forO- andN-linked glycosylation as well as O-Glc-NAc modification involved in signaling. PGM3 deficiency causes a combined immunodeficiency, with neutropenia, autoimmunity, and atopy, although the specific molecules targeted to cause this phenotype have yet to be identified.As shown by the above example, the distinction between primary and secondary immunodeficiencies caused not by environmental factors but by non-immune conditions has increasingly been breaking down with the recognition that non-immune cells can participate in innate immune processes or that global effects more obvious in non-immune cells can occur in parallel in the immune system. The latter is exemplified by many congenital disorders of glycosylationOverall, these and other examples have broadened our definition of what constitutes an immunodeficiency and have also rendered fuzzier the distinction between primary and secondary causes of inherited immunodeficiencies due to non-immune conditions.Immunodeficiencies have often been studied using genetically mutant mice that have arisen spontaneously, after ENU mutagenesis, or after targeted induced mutagenesis (knockouts and knockins). The forward genetics approach has been very successful in uncovering the role of individual gene products in regulating immunity in intact animals. However, laboratory mice are inbred and phenotypes can differ markedly depending upon the genetic background. For example, lpr mutation in mice causes exuberant autoimmunity on the MRL but not the C57BL/6 genetic background. Despite this knowledge, typically only one strain is tested by mouse researchers for any given study, mainly for cost reasons, thus limiting the generalizability of findings to humans, who have diverse genetic backgrounds. Furthermore, it is a concern whether mouse phenotypes tested under highly controlled environments accurately reflect what happens in the natural environment in which people are constantly exposed to different microbes.in vivo. This trend has been led by advances in genomics. In particular, whole exome sequencing of patients with monogenic disorders has facilitated the unbiased identification of their underlying responsible rare genetic variants. Furthermore, immune cells are easily isolated and tractable to manipulation using genome editing , gene silencing , or lentiviral transduction. In the case of transient progenitors of immune cells or non-immune cells that are not easily accessible, induced pluripotent stem cells from patients can be generated and differentiated into relevant cell types for further study8. These experimental technologies can be used to establish a causal link between the genetic defect andin vitro cellular phenotype, even in single-patient studies9. This functional validation of any given candidate disease-causative mutation is an important step required for these types of studies, given that each human is estimated to carry 76 to 190 rare non-synonymous and as many as 20 loss-of-function genetic variants that are potentially deleterious10.For these reasons, humans can be considered a more relevant model for understanding how the immune system is regulatedBCL1011,CD7013,CTPS114,DOCK215,MSN16,NIK17,RASGRP118,RLTPR19, andTRFC20; haploinsufficient mutations inIKAROS21 orNFKB122; as well as a heterozygous dominant negative mutation inBCL11B8. Other examples include humoral immunodeficiency accompanying multi-organ autoimmunity caused by haploinsufficient mutations inCTLA424 or gain-of-function mutations inSTAT325; as well as neutrophil dysfunction caused by autosomal recessive mutations inJAGN126 orWDR127. For the most part, these discoveries have dovetailed with findings previously made in mice but have revealed important aspects of the infectious context in which humans manifest disease. For example, human genetic diseases revealed that the CD27–CD70 co-stimulatory pathway has a non-redundant role for EBV control but required identification of patients with corresponding loss-of-function mutations because mice cannot be infected with EBV28. Because of similar experimental limitations, defects in antiviral immunity that confer selective susceptibility to influenza virus or measles/mumps/rubella  or defects in cytokine production that confer simultaneous susceptibility to Mycobacterial and Candida infections  were first unveiled from studies in humans. In some cases, the work from human patients has also extended work in mice: for example, showing that RASGRP1 regulates cytoskeletal dynamics in immune cells18 or that BCL11B regulates the migration of prethymic hematopoietic progenitors during T cell development8. Finally, with the accumulated discovery of patients having many different gene mutations, larger studies examining the relative roles of these genes involved in signaling for the development of T cell subsets in humans have also been made possible32.In the past three years, the pace of discovery has accelerated, especially in patients with combined immunodeficiencies or common variable immunodeficiency. Examples of these discoveries are those caused by autosomal or hemizygous recessive mutations in34. This trend was more prominent in older adults, reflecting changes in the adaptive immune system in response to cumulative prior infection history. Systems biology and genomics approaches have made these studies in humans possible, providing new insights into the “healthy” human immune system in the natural environment that could not be obtained from traditional mouse studies. Whether common genetic variants having smaller individual effects together can contribute to milder differences in infection susceptibility in the general population is unknown. If so, increased susceptibility to infection in some situations could be viewed as a complex genetic disease.In contrast to monogenic immunodeficiencies, which typically present in early childhood with severe and often lethal outcomes, the genetic contributions to immune responses in older individuals may be less influential, as these would have already been selected out in the general population. Indeed, recent twin studies have established that genetic variations in healthy populations are a minor contributor to immune differences as compared to non-inheritable environmental factorsTPP2) gene, which causes a combined immunodeficiency, autoimmunity, and neurodevelopmental delay36. Although TPP2 had been previously recognized as an enzyme involved in protein degradation and antigen processing and presentation, the initial genetic discovery and immunophenotyping experiments in the patients led to biochemical studies showing a broader principle governing the immune system : that intracellular amino acid homeostasis is inextricably linked, through aerobic glycolysis, to immune effector functions35.The unbiased discovery approach to human disease genes has led to exciting investigations of the important physiological functions of previously uncharacterized genes that turn out to have major roles in immunity. One recent example is the discovery of autosomal recessive mutations in the tripeptidyl peptidase-2  cell activity against EBV-infected cells and immunodeficiency by impairing magnesium-dependent calcium signaling in T cells39. With regard to the latter point, that magnesium can more broadly function as a second messenger for signal transduction was revealed only by delving deeply into understanding these patients. Thus, these three examples demonstrate that studying the immune system through genetic studies in humans yields conceptual scientific advances that go beyond the specific disease being studied.Finally, a third example of a new discovery coming from studying humans with immunodeficiencies was that of X-linked recessive mutations inOTULIN, a methionine-1-specific deubiquitinase that normally limits NF-κB activation and hence TNF-associated systemic inflammation40. Using mouse models of the human disease, the authors established the central pathogenic role of dysregulated TNF production when they were able to improve disease by treating with anti-TNF neutralizing antibodies. Importantly, similar effects were obtained when the OTULIN-deficient patients were treated with the TNF antagonist infliximab.One of the ways to establish a proposed mechanism of disease involves not only observation but also therapeutic intervention. This is done routinely in mouse studies of the immune system. A good example of this is from a recently reported autoinflammatory disease resulting from autosomal recessive mutations inIn contrast to hyperactive immune disorders, immunodeficiencies are traditionally cured by hematopoietic stem cell transplantation or gene therapy to restore the missing function, although these modalities can sometimes be associated with mortality and long-term sequelae such as chronic graft-versus-host disease. However, specific knowledge of the pathway affected can also suggest alternative and potentially less risky treatment approaches, even if unlikely to be curative.44. This disease is caused by heterozygous gain-of-function mutations in eitherPIK3CD orPIK3R1, which encode catalytic or regulatory phosphatidylinositol 3-kinase (PI3K) subunits that are highly expressed in lymphocytes. The mutations hyperactivate mTOR signaling, causing increased senescent CD8+ T cells at the expense of T cell and B cell memory generation, concurrent lymphoproliferation and hypogammaglobulinemia, and increased infections including poor control of EBV and cytomegalovirus infections. With the knowledge of the signaling pathway affected, the mTOR inhibitor sirolimus has been used in these patients and clinical improvement observed. Fortuitously, targeted therapy using small molecular inhibitors of PI3Kδ are being developed for the treatment of cancers, in which somatic gain-of-function mutations ofPIK3CD are sometimes found. Thus, PASLI as an orphan disease provides the perfect opportunity to demonstrate precision medicine in action by using the same drugs designed for another purpose. Clinical trials are ongoing to test their efficacy in this disease.A fine example is PASLI  diseaseLRBA deficiency with autoantibodies, regulatory T [Treg] cell defects, autoimmune infiltration, and enteropathy”), due to genetic deficiency of the LRBA adaptor protein, causes a mixed picture of autoimmunity and humoral immunodeficiency and responds to CTLA4-Ig (abatacept) treatment45. This therapeutic intervention helped to establish the mechanism by which LRBA deficiency phenocopies CHAI disease (for “CTLA4 haploinsufficiency with autoimmune infiltration”)24. Both molecules are interconnected in the same pathway whereby LRBA normally regulates the intracellular vesicle trafficking of, and is required for, cell surface expression of CTLA4. CTLA4 expression, especially on Treg cells, in turn restrains lymphocyte responses and indirectly regulates B cell responses. Furthermore, by defining that LRBA normally helps prevent CTLA4 deposition and degradation in lysosomes, treatment with hydroxychloroquine, a lysosome blocker that is already clinically used for the treatment of lupus, could also be helpful in the treatment of LRBA deficiency.A second example comes from recent work elucidating the molecular mechanism by which LATAIE disease (for “Together, these two examples illustrate that the unbiased identification of causative gene mutations in human patients leads to knowledge of potential mechanistic targets with immediate clinical implications. Translation of this knowledge has sped up because of the increased development of new biological immunomodulators and repurposing of previously approved medications for new uses. Additionally, knowing what infections result from particular gene mutations can give clues to potential adverse effects in healthy people that might be associated if a new immunomodulatory therapy directed at that molecular target were to be developed.in vivo the central importance of the proposed pathogenic mechanisms.Research into immunodeficiencies in humans is now generating exciting new knowledge that is directly relevant for understanding the healthy and diseased immune system and, in some cases, has led to new treatment approaches. Whole exome sequencing technologies are currently driving this trend but have not been able to solve a subset of patients with inherited immunodeficiencies. In this unsolved group of patients, the use of whole genome sequencing in conjunction with transcriptome profiling in relevant cell types could reveal how mutations in non-coding regions such as promoters, enhancers, or other regulatory sequence domains can contribute to human disease. As these could involve temporal as well as tissue-dependent expression during development, functional validation will increasingly require the use of induced pluripotent stem cells or be secondarily complemented by mouse, zebrafish, or other animal models. Finally, because of their immediate relevance to the sick patient, these types of studies are likely to become more closely integrated with clinical interventions that directly demonstrate