Human Genetic Diseases Research Articles

Sir David John Weatherall. 9 March 1933—8 December 2018

Sir David Weatherall was the outstanding, internationally recognized, clinician scientist of his generation. His lifelong interest in the haemoglobinopathies first came from studying a child that he recognized as having beta thalassaemia while working as an army physician in Malaya. His interest in these diseases further developed as he undertook his PhD in the Department of Haematology under the supervision of Lockard Conley and Victor McKusick in Baltimore. There, together with John Clegg (FRS 1999), he developed the technology to directly measure the synthesis of the alpha and beta globin chains of haemoglobin, and his subsequent work at the University of Liverpool showed that thalassaemia resulted from imbalanced globin chain synthesis. Importantly, using the globin genes as their model, David's laboratory went on to make many of the first discoveries of how expression of the globin genes and mammalian genes in general could be perturbed to cause human genetic diseases. As professor of medicine in Oxford, he established one of the first institutes to develop the use of molecular biology in medicine and he trained many of the world's leading clinician scientists in this area of biomedical research. He also established worldwide networks to apply molecular biology to the haemoglobinopathies and infectious diseases, making a unique contribution to global medicine. He had outstanding qualities as a clinician, a scientist, and a leader, all of which he achieved with great humility and humanity.

Targeted, Safe, and Efficient Gene Delivery to Human Hematopoietic Stem and Progenitor Cells In Vivo Using Engineered Avid Adenovirus Vector Platform

Precise editing of human genome using CRISPR-Cas nucleases, base editors, prime editors, or other technologies represents a promising approach for ameliorating or correcting many human genetic diseases. Although numerous pre-clinical and clinical proof-of-concept studies demonstrated feasibility of gene correction in the desired regions of the human genome ex vivo, targeted delivery to and cell-type-specific expression of gene editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of AVIDs, a novel adenovirus-based gene delivery platform, that allows for a highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. The AVID vectors attach to human HSPCs via engineered fibers that bind to either CD46 or DSG2 receptors, which are highly expressed on human HSPCs. The efficient entry of AVIDs into these cells is mediated via a mutated penton that was engineered to interact with CD49f, α6 integrin class, whose expression is restricted to cells with long-term repopulating and stem cell capacity. To improve virus safety after intravenous administration, as well as to increase the efficacy of virus production for a cost-effective high-yield vector manufacturing, AVIDs further comprise mutations in hypervariable loops of the major capsid protein hexon. While in vitro infection with AVIDs leads to about 30% transduction of a pool of human CD34 + cells, the efficacy of transduction of CD34 +CD38 -CD45RA -CD90 + subsets, highly enriched for hematopoietic stem cells (HSCs), ranged from 80% to 100% for individual donors. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34 + cells, after mobilization led to up to 20% transduction of CD34 +CD38 -CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34 +CD38 -CD45RA -CD90 -CD49f + subsets, highly enriched for human HSCs, reached up to 19%, which represented a 1900-fold selectivity in gene delivery to HSC-enriched over lineage committed CD34-negative cell populations in the bone marrow. Because the AVID platform allows for regulated, cell type-specific expression of gene editing technologies as well as for expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of novel curative therapies through safe and effective in vivo correction of disease-causing mutations in human HSCs after a single intravenous administration.

Comparison of transcriptome-wide N6-methyladenosine profiles from healthy trio families reveals regulator-mediated methylation alterations.

The N6-methyladenosine (m6A) modification is a highly conserved RNA modification found in eukaryotic messenger RNAs (mRNAs). It plays a vital role in regulating various biological processes. Dysregulation of m6A modifications has been linked to a range of complex genetic diseases in humans. However, there has been a lack of comprehensive characterization and comparison of m6A modifications at the transcriptome-wide level within families. To address this gap, we profiled transcriptome-wide m6A methylation in 18 individuals across 6 Yoruba trio families. The m6A methylomes of these 18 individuals revealed that m6A modifications in children showed greater similarity to each other than to their parents. This suggests that m6A modifications are influenced by multiple factors rather than solely determined by genetic factors. Additionally, we found that mRNAs exhibiting m6A modifications specific to children were enriched in cell cycle control processes, while those with m6A modifications specific to parents were associated with chromatin modifications. Furthermore, our analysis on the interactions between differentially expressed m6A-related regulatory genes and age-related genes suggested that age might be one of the factors influencing m6A modifications. In summary, our study provided a valuable dataset that highlighted the differences and functional diversity of m6A modifications within and between trio families.

Zebrafish as a Model for Osteoporosis: Functional Validations of Genome-Wide Association Studies.

GWAS, as a largely correlational analysis, requires in vitro or in vivo validation. Zebrafish (Danio rerio) have many advantages for studying the genetics of human diseases. Since gene editing in zebrafish has been highly valuable for studying embryonic skeletal developmental processes that are prenatally or perinatally lethal in mammalian models, we are reviewing pros and cons of this model. The true power for the use of zebrafish is the ease by which the genome can be edited, especially using the CRISPR/Cas9 system. Gene editing, followed by phenotyping, for complex traits such as BMD, is beneficial, but the major physiological differences between the fish and mammals must be considered. Like mammals, zebrafish do have main bone cells; thus, both in vivo stem cell analyses and in vivo imaging are doable. Yet, the "long" bones of fish are peculiar, and their bone cavities do not contain bone marrow. Partial duplication of the zebrafish genome should be taken into account. Overall, small fish toolkit can provide unmatched opportunities for genetic modifications and morphological investigation as a follow-up to human-first discovery.

The impact of Fisher's reproductive compensation on raising equilibrium frequencies of semidominant, nonlethal mutations under mutation/selection balance.

Fisher's reproductive compensation (fRC) occurs when a species' demography means the death of an individual results in increased survival probability of his/her relatives, usually assumed to be full sibs. This likely occurs in many species, including humans. Several important recessive human genetic diseases cause early foetal/infant death allowing fRC to act on these mutations. The impact of fRC on these genetic conditions has been previously calculated and shown to be substantial as quantified by ω, the fold increase in equilibrium frequencies of the mutation under fRC compared with its absence, i.e. ω = 1.22 and ω = 1.33 for autosomal and sex-linked loci, respectively. However, the impact of fRC on the frequency of the much larger class of semidominant, nonlethal mutations is unknown. This is calculated here as ω = 2 - h*s for autosomal loci and ω up to 2 for sex-linked loci where h is dominance (varied between 0.05 and 0.95) and s is selection coefficient (varied between 0.05 and 0.9). These results show that the actions of fRC can almost double the equilibrium frequency of deleterious mutations with low values of h and/or s (noting that "low" is s∼0.05 to 0.1). It is noted that fRC may act differentially across the genome with genes expressed early in life being fully exposed to fRC while those expressed later in life may be unaffected; this could lead to systematic differences in deleterious allele frequency across the genome.

A model-based clustering via mixture of hierarchical models with covariate adjustment for detecting differentially expressed genes from paired design

The causes of many complex human diseases are still largely unknown. Genetics plays an important role in uncovering the molecular mechanisms of complex human diseases. A key step to characterize the genetics of a complex human disease is to unbiasedly identify disease-associated gene transcripts on a whole-genome scale. Confounding factors could cause false positives. Paired design, such as measuring gene expression before and after treatment for the same subject, can reduce the effect of known confounding factors. However, not all known confounding factors can be controlled in a paired/match design. Model-based clustering, such as mixtures of hierarchical models, has been proposed to detect gene transcripts differentially expressed between paired samples. To the best of our knowledge, no model-based gene clustering methods have the capacity to adjust for the effects of covariates yet. In this article, we proposed a novel mixture of hierarchical models with covariate adjustment in identifying differentially expressed transcripts using high-throughput whole-genome data from paired design. Both simulation study and real data analysis show the good performance of the proposed method.

45 Genome Editing of Livestock for Production Agriculture and Biomedical Applications

Abstract Continued population growth and clear evidence of climate change are placing unprecedented strain on food production and health care systems worldwide. Agricultural production goals that highlight environmental sustainability are at the forefront of social dialog, with animal agriculture routinely targeted for inefficiencies, welfare concerns and impact on climate. Genomic selection has transformed animal production and the rate of genetic gain in each generation continues to increase. Nonetheless, it is apparent that the rate of production gains will fall short of the necessary goals of increased efficiency and adaptation to the changing environment. Genetic engineering may have an important role in enhancing livestock production as well as providing new models for research in human health. The CRISPR/Cas and related systems for targeted gene editing has revolutionized livestock genetic engineering by increasing the rate of targeted mutation, thus drastically reducing the cost of producing founder animals with the desired trait. Intentional insertion of well-known mutations into genes such as myostatin have been shown to impart the expected increase in meat production, but have also resulted in undesirable associated traits affecting fertility, marbling and the skeleton. Thus, the approach to increasing meat production may require nuances such as promotor targeting and multiple gene targeting to obtain the desired production characteristics. Efforts to increase disease resistance could have dramatic impacts on animal welfare and production rates, potentially saving millions of animals from morbidity and mortality. However, confronting a single disease with each genetic alteration will be costly and inefficient. Considerations on how broad scale disease resistance could be achieved through genetic engineering will be instrumental in developing livestock with advantages to avoid infection and/or transmission of multiple pathogens. Gene editing also allows correction of mutations that arise in livestock through natural mechanisms while under intense genomic selection for increased production. Removing disease causing mutations in otherwise high value livestock will allow continued production from these lines without concern for inheriting the causative mutation. The same process can be utilized to insert genetic mutations known to phenocopy human conditions providing valuable large animal models of human genetic disease. Increased milk, meat, or eggs per animal unit will sustainably improve high quality, nutrient dense food production while large animal models of human disease will provide a more translational biomedical model for accelerated development of drugs and treatments. These goals are achievable, but require continued investment in innovation, clear regulatory framework for commercialization, education, and public engagement to ensure success.

Applying the CRISPR/Cas9 for Treating Human and Animal Diseases – Comprehensive Review

Abstract Recently, genome editing tools have been extensively used in many biomedical sciences. The gene editing system is applied to modify the dnA sequences in the cellular system to comprehend their physiological response. A developing genome editing technology like clustered regularly short palindromic repeats (CRISPR) is widely used in medical sciences. CRISPR and CRISPR-associated protein 9 (CRISPR/Cas9) system is being exploited to edit any DNA mutations related to inherited ailments to investigate in animals (in vivo) and cell lines (in vitro). Remarkably, CRISPR/Cas9 could be employed to examine treatments of many human genetic diseases such as cystic fibrosis, tyrosinemia, phenylketonuria, muscular dystrophy, Parkinson’s disease, retinoschisis, hemophilia, β-thalassemia and atherosclerosis. Moreover, CRISPR/Cas9 was used for disease resistance such as tuberculosis, Johne’s diseases, chronic enteritis, and brucellosis in animals. Finally, this review discusses existing progress in treating hereditary diseases using CRISPR/Cas9 technology and the high points accompanying obstacles.

Genetic variations in G-quadruplex forming sequences affect the transcription of human disease-related genes.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4s). G4s folded in proximal promoter regions (PPR) are associated either with positive or negative transcriptional regulation. Given that single nucleotide variants (SNVs) affecting G4 folding (G4-Vars) may alter gene transcription, and that SNVs are associated with the human diseases' onset, we undertook a novel comprehensive study of the G4-Vars genome-wide (G4-variome) to find disease-associated G4-Vars located into PPRs. We developed a bioinformatics strategy to find disease-related SNVs located into PPRs simultaneously overlapping with putative G4-forming sequences (PQSs). We studied five G4-Vars disturbing in vitro the folding and stability of the G4s located into PPRs, which had been formerly associated with sporadic Alzheimer's disease (GRIN2B), a severe familiar coagulopathy (F7), atopic dermatitis (CSF2), myocardial infarction (SIRT1)and deafness (LHFPL5). Results obtained in cultured cells for these five G4-Vars suggest that the changes in the G4s affect the transcription, potentially contributing to the development of the mentioned diseases. Collectively, data reinforce the general idea that G4-Vars may impact on the different susceptibilities to human genetic diseases' onset, and could be novel targets for diagnosis and drug design in precision medicine.

Hemoglobin E Prevalence among People Residing in Malaria Areas

Background: Malaria can infect erythrocytes and hence cause different pathogenesis episodes leading to death mostly in pregnant women and children under the age of 5 years. The selective pressure of these parasites leads to the production of new human genetic diseases. The most prevalent genetic alterations in the human genome are thalassemia and hemoglobinopathies (Hb E, Hb S), which are recognized throughout the world, including Saudi Arabia. Methods: From May 2018 to August 2019, 13972 Saudi citizens from King Fahd Central Hospital and premarital facilities in the Saudi Arabian province of Jazan participated in this study. This study aims to compare the prevalence of Hb E and other hemoglobinopathies in positive versus negative cases of malaria. So, CBC, malaria test, Hb-electrophoresis, and molecular study were investigated. Result: For thalassemias and Hb disorders, 36% with abnormal Hb (47% of them) carried Hb S in their blood, 37% with á-thalassemia, 11% for â-thalassemia and 4% of Hb E. Significant variations in CBC parameters were observed in Hb E patients. There was significant decrease in MCV, MCH and MCHC and slightly increase in WBCs, RBCs, RDW and PLT as compared to controls.

Human hepatic organoids for the analysis of human genetic diseases.

Human hepatic organoids for the analysis of human genetic diseases.

Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria

Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant Tcells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ.

Drosophila melanogaster as an organism model for studying cystic fibrosis and its major associated microbial infections.

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene that encodes a chloride channel. The most severe clinical manifestation is associated with chronic pulmonary infections by pathogenic and opportunistic microbes. Drosophila melanogaster has become the invertebrate model of choice for modeling microbial infections and studying the induced innate immune response. Here, we review its contribution to the understanding of infections with six major pathogens associated with CF (Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, Mycobacterium abscessus, Streptococcus pneumoniae, and Aspergillus fumigatus) together with the perspectives opened by the recent availability of two CF models in this model organism.

TMEM63 mechanosensitive ion channels: Activation mechanisms, biological functions and human genetic disorders

The transmembrane 63 (TMEM63) family of proteins are originally identified as homologs of the osmosensitive calcium-permeable (OSCA) channels in plants. Mechanosensitivity of OSCA and TMEM63 proteins are recently demonstrated in addition to their proposed activation mechanism by hyper/hypo-osmolarity. TMEM63 proteins exist in all animals, with a single member in Drosophila (TMEM63) and three members in mammals (TMEM63 A/B/C). In humans, monoallelic variants of TMEM63A have been reported to cause transient hypomyelination during infancy, or severe hypomyelination and global developmental delay. Heterozygous variants of TMEM63B are found in patients with intellectual disability and abnormal motor function and brain morphology. Biallelic variants of TMEM63C are associated with hereditary spastic paraplegias accompanied by mild or no intellectual disability. Physiological functions of TMEM63 proteins clearly recognized so far include detecting food grittiness and environmental humidity in Drosophila, and supporting hearing in mice by regulating survival of cochlear hair cells. In this review, we summarize current knowledge about the activation mechanisms and biological functions of TMEM63 channels, and provide a concise reference for researchers interested in investigating more physiological and pathogenic roles of this family of proteins with ubiquitous expression in the body.

Global analysis of suppressor mutations that rescue human genetic defects

BackgroundGenetic suppression occurs when the deleterious effects of a primary “query” mutation, such as a disease-causing mutation, are rescued by a suppressor mutation elsewhere in the genome.MethodsTo capture existing knowledge on suppression relationships between human genes, we examined 2,400 published papers for potential interactions identified through either genetic modification of cultured human cells or through association studies in patients.ResultsThe resulting network encompassed 476 unique suppression interactions covering a wide spectrum of diseases and biological functions. The interactions frequently linked genes that operate in the same biological process. Suppressors were strongly enriched for genes with a role in stress response or signaling, suggesting that deleterious mutations can often be buffered by modulating signaling cascades or immune responses. Suppressor mutations tended to be deleterious when they occurred in absence of the query mutation, in apparent contrast with their protective role in the presence of the query. We formulated and quantified mechanisms of genetic suppression that could explain 71% of interactions and provided mechanistic insight into disease pathology. Finally, we used these observations to predict suppressor genes in the human genome.ConclusionsThe global suppression network allowed us to define principles of genetic suppression that were conserved across diseases, model systems, and species. The emerging frequency of suppression interactions among human genes and range of underlying mechanisms, together with the prevalence of suppression in model organisms, suggest that compensatory mutations may exist for most genetic diseases.

Detection of Mosaic Sequence Variants Associated with Human Genetic Diseases

Detection of Mosaic Sequence Variants Associated with Human Genetic Diseases

Efficient repair of human genetic defect by CRISPR/Cas9-mediated interlocus gene conversion

Abstract DNA double-strand breaks (DSBs) induced by gene-editing tools are primarily repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR) using synthetic DNA templates. However, error-prone NHEJ may result in unexpected indels at the targeted site. For most genetic disorders, precise HDR correction using exogenous homologous sequence is ideal. But, the therapeutic application of HDR might be especially challenging given the requirement for the codelivery of exogenous DNA templates with toxicity into cells, and the low efficiency of HDR could also limit its clinical application. In this study, we efficiently repair pathogenic mutations in HBB coding regions of hematopoietic stem cells (HSCs) using CRISPR/Cas9-mediated gene conversion (CRISPR/GC) using the paralog gene HBD as the internal template. After transplantation, these edited HSCs successfully repopulate the hematopoietic system and generate erythroid cells with significantly reduced thalassemia propensity. Moreover, a range of pathogenic gene mutations causing β-thalassemia in HBB coding regions were effectively converted to normal wild-type sequences without exogenous DNA templates using CRISPR/GC. This highlights the promising potential of CRISPR/GC, independent of synthetic DNA templates, for genetic disease gene therapy.

Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.

External genitalia phenotypes of a Mab21l1-null mouse model for cerebellar, ocular, craniofacial, and genital (COFG) syndrome.

The cerebellar, ocular, craniofacial, and genital (COFG) syndrome is a human genetic disease that is caused by MAB21L1 mutations. A COFG mouse model with Mab21l1-null mutation causes severe microphthalmia and fontanelle dysosteogenesis, similar to the symptoms in human patients. One of the typical symptoms is scrotal agenesis in male infants, while male Mab21l1-null mice show hypoplastic preputial glands, a rodent-specific derivative of the cranial scrotal fold. However, it is still unclear where and how MAB21Ll acts in the external genitalia in both mice and humans. Here we show that, at the neonatal stage, MAB21L1 expression in the external genitalia was restricted to two mesenchymal cell populations-underneath the scrotal and labial skin and around the preputial and clitoral glands (PG/CG). Morphometric analyses of the Mab21l1-/- pups revealed a significant reduction in the external size of the scrotum, vulva, and CG, as well as PG. In the periglandular region around PG and CG, the periglandular mesenchymal cells showed a drastic reduction in both cell density and immunoreactive signals for several extracellular matrix proteins (e.g., collagen I, fibronectin, and proteoglycans), together with their reduced Ki67-positive cell proliferation index. In the Mab21l1-/- PG/CG, together with reduced vascularization, the glandular epithelia displayed atrophy with discontinuous basal lamina along the basal surface and defective glycogen accumulation in their cytoplasm. Under a 5-day organ culture of the isolated PG, the Mab21l1-/- explants showed poor outgrowth and retention of the glandular structure in vitro. However, the addition of exogenous Matrigel could partially rescue such tissue-autonomous phenotypes, showing glandular morphology similar to that of the wild-type explants. These findings suggest that MAB21L1+ mesenchymal cells play a crucial role in providing nutrient ECM support for glandular outgrowth and morphogenesis in the peripheral external genitalia.

Genome-wide CNV analysis uncovers novel pathogenic regions in cohort of five multiplex families with neurodevelopmental disorders

Structural reorganization of chromosomes by genomic duplications and/or deletions are known as copy number variations (CNVs). Pathogenic and disease susceptible CNVs alter gene dosage and its phenotypic expression that often leads to human genetic diseases including Neurological disorders. CNVs affecting same common genes in multiple neurodevelopmental disorders can better explain the shared clinical and genetic aetiology across brain diseases. Our study presents the novel copy number variations in a cohort of five multiplex consanguineous families with intellectual disability, microcephaly, ASD, epilepsy, and neurological syndromic features. Cytoscan HD microarray suite has revealed genome wide deletions, duplications and LOH regions which are co-segregating in the family members for the rare neurodevelopmental syndromic phenotypes. This study identifies 1q21.1 microduplication, 16p11.2 microduplication, Xp11.22 microduplication, 4p12 microdeletion and Xq21.1 microdeletion that significantly contribute to primary disease onset and its progression for the first time in Pakistani families. Our study has potential impact on the understanding of pathogenic genetic predisposition for appearance of complex and heterogeneous neurodevelopmental disorders with otherwise unexplained syndromic features. Identification of altered gene dosage across the genome is helpful in improved diagnosis, better disease management in day-to-day life activities of patients with cognitive impairment and genetic counselling of families where consanguinity is a tradition. Our study will contribute to expand the knowledge of genotype-phenotype expression and future gateways in therapeutics and precision medicine research will be open in Pakistan.