Single Gene Defects Research Articles

What Mendel could learn from the inborn errors of immunity – harnessing the power of NGS genetic testing for diagnosis of immune disorders

The inborn errors of immunity (IEIs) include primary immunodeficiencies (PIDs) and primary immune regulatory disorders (PIRDs). Though there are somatic variations in genes that cause immunological disease, the IEIs, for the most part, describe germline gene defects. The IEIs now include 485 genes and the list continues to grow. These genes are classified into nine categories based on the component of the immune system dominantly affected (IUIS classification 2022). The explosion in the number of genes identified in the past two decades has to do with rapid advances in genomic technologies, which allow for high throughput analysis, such as next generation sequencing (NGS). NGS includes targeted gene panels (TGPs), exome (ES) and genome sequencing (GS), besides copy number variation (CNV) analysis. Since the etiological basis of most, if not all, IEIs is related to a single gene defect (monogenic) or in some cases, two genes (digenic), it stands to reason that diagnosis of these disorders requires genetic testing for confirmation of the diagnosis. However, a confirmed genetic answer is not just for diagnosis but also for selecting appropriate therapeutic and management strategies, which facilitates personalised approaches to treatment. This talk will provide an overview of the rationale for genetic testing for the diagnosis of IEIs, the most commonly used NGS methodologies and data analysis, which includes variant classification and interpretation of variants of uncertain significance (VUS). The talk will also provide information on various tools and databases used for genomic data analysis, and the concepts of incomplete penetrance, variable expressivity, somatic reversion, somatic mosaicism, hypopmoprhic variants, gain-of-function and loss-of-function.

Skin manifestations of inborn errors of NF-κB.

More than 400 single gene defects have been identified as inborn errors of immunity, including many arising from genes encoding proteins that affect NF-κB activity. We summarise the skin phenotypes in this subset of disorders and provide an overview of pathogenic mechanisms. NF-κB acts cell-intrinsically in basal epithelial cells during differentiation of skin appendages, influences keratinocyte proliferation and survival, and both responses to and amplification of inflammation, particularly TNF. Skin phenotypes include ectodermal dysplasia, reduction and hyperproliferation of keratinocytes, and aberrant recruitment of inflammatory cells, which often occur in combination. Phenotypes conferred by these rare monogenic syndromes often resemble those observed with more common defects. This includes oral and perineal ulceration and pustular skin disease as occurs with Behcet's disease, hyperkeratosis with microabscess formation similar to psoriasis, and atopic dermatitis. Thus, these genotype-phenotype relations provide diagnostic clues for this subset of IEIs, and also provide insights into mechanisms of more common forms of skin disease.

The role of HBG2, BCL11A, and HBS1L-MYB in early diagnosis of transfusion-dependent thalassemia among Egyptian children

Abstract Background Hereditary hemoglobinopathies are the most frequent diseases accountable to a single gene defect. Common mutations of the beta-globin gene are detected by PCR-based techniques. Objective To evaluate the role of HBG2, BCL11A, and HBS1L-MYB polymorphisms in addition to Thalassemia Severity Score (TSS) in the early diagnosis of transfusion-dependent thalassemia patients among Egyptian children and their impact on clinical decision. Patients and methods Thalassemia mutation analysis was performed by the Beta-Thal Modifier Strip Assay to determine the five polymorphisms associated with severity, and an automated online calculator (TSS). Results The transfusion-dependent group showed significantly higher TSS (P<0.001), with a sensitivity of 75%, specificity of 95%, positive predictive value of 93.8%, negative predictive value of 79.2%, and an accuracy of 85%. HBG2 CT and CC genotypes were significantly associated with younger age of first transfusion and higher transfusion rates. Deletion in alpha gene was significantly associated with TT genotype, followed by GG and then GT. TSS decreased gradually through wild, heterozygous, and homozygous rs7482144 and rs1427407 genotypes. Transfusion-free survival tends to decrease gradually with increased TSS severity (P<0.001). The HBS1L-MYB rs9399137 TC genotype was associated with poor transfusion-free survival by Cox regression analysis. Beta phenotype mild/mild, mild/severe, rs7482144 CT, TT, and rs1427407 GT, TT were associated with the protective effect against higher severity. Conclusion HBG2, BCL11A, and HBS1L-MYB have an important role in early diagnosis and prognosis of transfusion-dependent thalassemia among Egyptian children.

Dysregulation of the immune response in TGF-β signalopathies.

The transforming growth factor-β (TGF-β) family of cytokines exerts pleiotropic functions during embryonic development, tissue homeostasis and repair as well as within the immune system. Single gene defects in individual component of this signaling machinery cause defined Mendelian diseases associated with aberrant activation of TGF-β signaling, ultimately leading to impaired development, immune responses or both. Gene defects that affect members of the TGF-β cytokine family result in more restricted phenotypes, while those affecting downstream components of the signaling machinery induce broader defects. These rare disorders, also known as TGF-β signalopathies, provide the unique opportunity to improve our understanding of the role and the relevance of the TGF-β signaling in the human immune system. Here, we summarize this elaborate signaling pathway, review the diverse clinical presentations and immunological phenotypes observed in these patients and discuss the phenotypic overlap between humans and mice genetically deficient for individual components of the TGF-β signaling cascade.

Long-Term Improvements in Patient-Reported Outcomes in Patients with Pyruvate Kinase Deficiency Treated with Mitapivat

Background: Pyruvate kinase (PK) deficiency is a rare, hereditary hemolytic anemia caused by a single gene defect encoding the red blood cell (RBC)-specific form of PK. This condition is associated with acute and long-term complications, as well as a spectrum of signs and symptoms including jaundice, fatigue, and dyspnea, that have a profound, wide-ranging impact on health-related quality of life (HRQoL). Patients (pts) report physical limitations, negatively impacted social functioning, reduced self-esteem, and concerns for the future. Mitapivat, a first-in-class, oral, allosteric activator of PK, has demonstrated improvement in hemoglobin, hemolysis, and transfusion burden in pts with PK deficiency, and is approved by the US Food and Drug Administration for the treatment of hemolytic anemia in adults with PK deficiency. In addition, improvements in patient-reported outcomes (PROs) (measured by disease-specific PRO instruments: the PK deficiency diary [PKDD] and the PK deficiency impact assessment [PKDIA]) were observed in two global, phase 3 clinicals trials among adults with PK deficiency treated with mitapivat who were not regularly (ACTIVATE; NCT03548220) or regularly transfused (ACTIVATE-T; NCT03559699). Here, we describe PKDD and PKDIA outcomes up to Week (Wk) 84 of the long-term extension (LTE) study (NCT03853798) of mitapivat in pts with PK deficiency previously enrolled in ACTIVATE and ACTIVATE-T. Methods: In the placebo (PBO)-controlled ACTIVATE trial, pts were randomized 1:1 to receive mitapivat (5/20/50 mg twice daily) or PBO for 24 wks. In the open-label, single-arm ACTIVATE-T trial, pts were treated with mitapivat for 40 wks. Pts who completed either trial were eligible to continue in the LTE, where all pts received mitapivat. Pts from ACTIVATE were categorized into either the mitapivat-to-mitapivat arm (M/M) or the PBO-to-mitapivat arm (P/M). Pts from ACTIVATE-T continued to receive mitapivat (M). Two PK deficiency-specific PRO instruments were used: the PKDD is a 7-item measure of the core signs and symptoms of PK deficiency, and the PKDIA is a 14-item measure of the impacts of PK deficiency on pts' HRQoL. In the ACTIVATE/LTE and ACTIVATE-T/LTE analyses, changes from baseline (BL; defined as the last complete assessment before start of study treatment in ACTIVATE/ACTIVATE-T) in PKDD and PKDIA mean scores were assessed at scheduled visits up to Wk 84 of the LTE for each of the 3 treatment arms. For both PRO instruments, a lower score represents a lower disease burden, and the minimal clinically important change is defined as a reduction of 4.2 and 5.5 in PKDD and PKDIA scores, respectively. Changes in PKDD and PKDIA scores were summarized descriptively using mean and SD. Results: In total, 80 pts were included in the ACTIVATE/LTE analysis (M/M=40; P/M=40) and 27 pts were included in the ACTIVATE-T/LTE analysis. At Wk 84 of the LTE study (ie, Wk 108 for ACTIVATE pts; Wk 124 for ACTIVATE-T pts), mean (SD) change from BL in PKDD score was ‒7.19 (6.740) for M/M pts, ‒4.58 (5.758) for P/M pts, and ‒3.94 (14.087) for M pts; mean (SD) change from BL in PKDIA score was ‒6.3 (7.12) for M/M pts, ‒6.3 (9.09) for P/M pts, and ‒11.5 (11.53) for M pts. Notably, P/M pts experienced improvements in PKDD and PKDIA mean scores after starting mitapivat in the LTE that were consistent with initial improvements seen in the mitapivat-treated group in the ACTIVATE trial. Across both PRO instruments, improvements among mitapivat-treated pts were sustained over time in the LTE (Figures 1A and 1B). Conclusions: Treatment with mitapivat was associated with long-term, durable, and clinically meaningful improvements in signs, symptoms, and functional impacts based on disease-specific PRO instruments, irrespective of transfusion status. These results suggest that by improving HRQoL, treatment with mitapivat may provide meaningful pt-centric benefits. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gene Therapy Hits Its Stride in the Clinic

Gene Therapy Hits Its Stride in the Clinic

Highlights from the International Twins Congress 2021

Highlights from the International Twins Congress 2021

Disease mechanisms of monogenic congenital anomalies of the kidney and urinary tract American Journal of Medical Genetics Part C.

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) is a developmental disorder of the kidney and/or genito-urinary tract that results in end stage kidney disease (ESKD) in up to 50% of children. Despite the congenital nature of the disease, CAKUT accounts for almost 10% of adult onset ESKD. Multiple lines of evidence suggest that CAKUT is a Mendelian disorder, including the observation of familial clustering of CAKUT. Pathogenesis in CAKUT is embryonic in origin, with disturbances of kidney and urinary tract development resulting in a heterogeneous range of disease phenotypes. Despite polygenic and environmental factors being implicated, a significant proportion of CAKUT is monogenic in origin, with studies demonstrating single gene defects in 10%-20% of patients with CAKUT. Here, we review monogenic disease causation with emphasis on the etiological role of gene developmental pathways in CAKUT.

The Efficacy and Safety of Cystic Fibrosis Gene Therapy Clinical Trials: A Systematic Review and Meta-Analysis

BackgroundGene therapy has been proposed as a treatment approach for cystic fibrosis by replacing the single defective gene, cystic fibrosis transmembrane regulator (CFTR), through topical lung delivery. Relatively few studies have addressed gene therapy for cystic fibrosis.Objectives Via referral to the published literature, this study aimed to identify any success of the gene therapy approach for cystic fibrosis regarding experimental and routine clinical outcomes in different drug development stages, and to determine any adverse effects noted. MethodologyA search of the PubMed database (NCBI) for 1989–2020 was made using predefined selection criteria for clinical trials on patients with cystic fibrosis receiving viral and non-viral lung delivery systems of the CFTR gene. Several features in the reviewed studies were examined, including clinical phase (1–3), sample size, delivery target cells/vector, and reported adverse effects. A quantitative estimate of treatment intervention success was evaluated using a meta-analysis approach.Results A total of 20 studies with 549 patients were included in the review. The studies involved the delivery of the defective gene to the lung, nasal mucosa, and sinuses, and were mainly phase 1–2, randomized controlled trials; there were no phase three studies. The vector for gene transfer was liposome or viral. % predicted FEV1 was statistically significant between intervention and control patients in two trials. Gene transfer was detected to a higher degree in intervention patients than control; this outcome measure was assessed using bronchoscopy assessment of vector-specific DNA and mRNA expression in lung and nasal mucosa. These effects, however, were temporary. The safety of the gene therapy approach was confirmed. ConclusionReportedly, the gene therapy approach is safe but has limited and temporary efficacy. Newer approaches should thus be engineered to deliver the necessary genetic material with the desired, full-scale efficacy.

Is there an unmet medical need for improved hearing restoration?

Hearing impairment, the most prevalent sensory deficit, affects more than 466 million people worldwide (WHO). We presently lack causative treatment for the most common form, sensorineural hearing impairment; hearing aids and cochlear implants (CI) remain the only means of hearing restoration. We engaged with CI users to learn about their expectations and their willingness to collaborate with health care professionals on establishing novel therapies. We summarize upcoming CI innovations, gene therapies, and regenerative approaches and evaluate the chances for clinical translation of these novel strategies. We conclude that there remains an unmet medical need for improving hearing restoration and that we are likely to witness the clinical translation of gene therapy and major CI innovations within this decade.

O-114 Preimplantation genetic testing for monogenic diseases without necessary familial members

Abstract Preimplantation genetic testing for monogenic diseases (PGT-M) is available for any single gene defects theoretically, as long as the disease-causing locus has been unequivocally identified. However, PGT-M has faced great challenges for couples with more than one genetic mutation, de novo mutations or without essential family members for linkage analysis. In order to overcome these challenges, we developed a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) and chromosomal structural rearrangements (PGT-SR) study, as well as monogenic diagnosis study for couples bearing de novo mutations or without necessary familial members. The innovation of our strategy is to use the gamete (polar body or single sperm) or embryo as proband for linkage analyses (GEPLA) to detect an embryo’s carrier status. GEPLA strategy uses affected embryo as proband or gametes carrying mutant alleles as proband for SNP linkage analyses, that were suitable for all types of patients mentioned above. Using this novel developed method, nine autosomal dominant polycystic kidney disease (ADPKD) couples with either de novo mutation or without a positive family history were recruited. Moreover, other two couples with single gene disorders (SMA and NF2) which lack of essential family members for linkage analysis including de novo mutation, and also combined with reciprocal translocation were recruited too. For nine ADPKD couples, a total of 34 embryos from 13 PGT-M cycles were examined and two couples were successfully delivered healthy babies. For the other two couples, 15 embryos were screened, and two embryos were determined as free of the monogenic disease and specific chromosomal abnormalities created by reciprocal translocations. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients lacking of essential family members for linkage analysis, and even for reciprocal chromosome translocation concurrently.

O-044 ICSI in the lab: from vintage to AI

Abstract With the advent of in vitro fertilization (IVF) by Patrick Steptoe and Bob Edwards in the late 70s, the first conception outside of the human body resulted in the birth of Louise Brown. Although a terrific success, limitations of IVF surfaced, represented by the unexpected complete fertilization failure with suboptimal or dysfunctional spermatozoa. This prompted curiosity toward individual spermatozoa for a deeper understanding of its role aimed at enhancing the interaction between complementary gametes. Techniques were designed to manipulate the oocytes, such as stripping, partially digesting, or cracking the zona pellucida (ZP). These methods were palliative solutions to overcome fertilization failure and were often plagued by polyspermy. As a result, more direct approaches were implemented to overcome the ZP, such as subzonal injection (SUZI) that although more consistent, was still unable to overcome the shortcomings of dysfunctional spermatozoa. This laid the foundation for the utilization of ICSI that, whilst attempted by some investigators, became popular when Gianpiero Palermo serendipitously inserted one spermatozoon into the ooplasm during SUZI. Consistent fertilization then followed by injecting a cohort of oocytes by ICSI in SUZI cycles, and replacement of these embryos led to 4 pregnancies described in the first clinical ICSI report. To minimize oocyte damage, the procedure was further refined by inducing a deep invagination of the oolemma toward the 9 o’clock position, granting higher chances of post-injection survival. What set apart ICSI from other forms of ART was that any sperm sample, regardless of quality/quantity, would yield fertilization. Indeed, ICSI is the sole insemination method used with epididymal and testicular spermatozoa and has therefore revolutionized fertility treatment of azoospermic men. Indeed, even immotile testicular spermatozoa can still fertilize and yield successful pregnancies, albeit at a lower rate than their motile counterpart. Also, for these semen sources, aggressive sperm immobilization was introduced to enhance sperm membrane permeabilization and grant optimal fertilization results. Furthermore, ICSI has transformed the field of reproductive medicine by assisting other reproductive techniques, such as testing embryos for single gene defects to reduce the occurence of sperm DNA contamination, or overcoming the cryostress-induced changes of the ZP during cryopreservation allowing the oocyte to be fertilized at a higher rate. Oocyte cryopreservation now empowers women in their reproductive age to ordain their childbearing future. ICSI has proven to be the ultimate technique to overcome male infertility and has broadened its indication by yielding consistent fertilization and successful pregnancies in most circumstances, ensuring that men have the chance of fathering their own progeny. To date, ICSI is applied in several countries, and in some, is performed as the preferred/sole insemination method contributing to the birth of millions of babies worldwide. Thus far, no concerning differences have been seen in the health of ICSI versus standard IVF offspring, or even naturally conceived. In fact, it has been currently established that young adults of both genders born through ICSI retain their reproductive health. Despite its growing popularity, ICSI does not always succeed but still provides an invaluable platform to deepen our knowledge of gamete biology and helps to investigate/overcome some of the most severe and persistent forms of infertility. For example, combined with assisted gamete treatment, ICSI allows couples plagued by sperm-bound oocyte-activation-deficiency to achieve pregnancy. The need to increase access and curtail costs of reproductive care has led to the testing of automation in ART. This is also occurring with ICSI and to date, different automated modules have been proposed for oocyte denudation, sperm tracking, and robotic ICSI. Concurrently, there has been an interest in experimenting with artificial intelligence in the IVF laboratory to minimize human shortcomings and ensure that the best spermatozoon is chosen.

Very-Early Onset Chronic Active Colitis with Heterozygous Variants in LRBA1 and CARD11, a Case of “Immune TOR-Opathies”

Background A small subset of cases of inflammatory bowel disease (IBD) occurs as a result of single gene defects, and typically occurs in young or very young pediatric patients, referred to as “monogenic very-early onset IBD (VEO-IBD)”. The gene variants leading to monogenic VEO-IBD are often associated with primary immunodeficiency syndromes. Case report A six year-old girl presented to our gastroenterology clinic with LRBA deficiency with a heterozygous mutation at c.1399 A > G, p Met467Val, histopathologic chronic active colitis without granulomas and clinical chronic colitis. Her gastrointestinal symptoms began at age 5 with bloody diarrhea, abdominal pain and weight loss. Whole exome sequencing revealed a CARD11 heterozygous de novo mutation (c.220 + 1G > A). She was in clinical remission on only abatacept. Discussion We present a case of monogenic VEO-IBD associated with two heterozygous variants in LRBA1 and CARD11, both considered as key players in the newly proposed “immune TOR-opathies”.

Use of preimplantation genetic testing for monogenic disorders and subsequent prenatal care and diagnostic testing.

The goal of preimplantation genetic testing for monogenic or single gene defects (PGT-M) is to identify inherited pathogenic variants in the embryo prior to embryo transfer, increasing the likelihood of an unaffected child. Prenatal diagnostic testing is recommended to confirm the results of PGT-M. The purpose of this study was to characterize the population undergoing PGT-M over time. This retrospective study examined patients who had a positive pregnancy test after PGT-M from 2012 to 2019. A query of the internal assisted reproductive technology database and chart review were used. One hundred and 42 patients completed IVF cycles for PGT-M during this time period and progressed past 10weeks gestation. There were more PGT-M cycles over time with 46 cycles between 2012 and 2015 and 96 cycles between 2016 and 2019. Patients varied on the decision to pursue prenatal diagnostic testing after PGT-M. For those with known follow-up (130/142), 16 patients underwent diagnostic testing (12%) and 114 did not. As PGT-M is increasingly utilized prior to pregnancy, it is important for genetic counselors and OB/GYNs to understand the characteristics and outcomes of the population of patients undergoing PGT-M, including how to counsel about the residual risk of an affected pregnancy after PGT-M.

Diagnosis, monitoring, and management of pyruvate kinase deficiency in children.

Pyruvate kinase (PK) deficiency is a rare, congenital red blood cell disorder caused by a single gene defect. The spectrum of genotypes, variants, and phenotypes are broad, commonly requiring a multimodal approach including enzyme and genetic testing for accurate and reliable diagnosis. Similarly, management of primary and secondary sequelae of PK deficiency varies, mainly including supportive care with transfusions and surgical interventions to improve symptoms and quality of life. Given the risk of acute and long-term complications of PK deficiency and its treatment, regular monitoring and management of iron burden and organ dysfunction is critical. Therefore, all children and adolescents with PK deficiency should receive regular hematology care with visits at least every 6months regardless of transfusion status. We continue to learn more about the spectrum of symptoms and complications of PK deficiency and best practice for monitoring and management through registry efforts (NCT03481738). The treatment of PK deficiency has made strides over the last few years with newer disease-modifying therapies being developed and studied, with the potential to change the course of disease in childhood and beyond.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

SummaryEmbryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Inborn errors of metabolism and their impact in paediatric dentistry.

The management of paediatric patients with inborn errors of metabolism (IEM) presents an unparalleled challenge for paediatric dentists owing to the multiplex of interrelated dental manifestations and metabolic management necessitating modifications to dental care. Inborn errors of metabolism describe a largely heterogenous group of genetic disorders namely attributable to a single gene defect essential for a specific metabolic pathway. Approximately 400 disorders have been described with an overall incidence of 1 in 5000 live births worldwide. Clinical presentation is classically inconspicuous and insidious in the neonatal period with pathophysiology attributable to accumulation of toxic by-products which interfere with normal function, or insufficient synthesis of essential compounds. This paper aims to discuss the primary oral and maxillofacial manifestations across the scope of inborn errors of metabolism, whilst also considering how metabolic treatment has the propensity to complicate dental management.

EP146: Application of exome sequencing in patients of congenital anomalies with or without intellectual disability

Congenital anomalies (CA) and intellectual disability (ID) both comprise a heterogeneous spectrum of diseases. They affect about 3% of live birth worldwide. The causes of CA can be divided into genetic and nongenetic. About a quarter of CA has been estimated to have a genetic cause previously, with the two most common causes as single gene defects and chromosome abnormalities. ID has highly variable severity and frequently combines with multiple congenital anomalies (MCA). Exome sequencing (ES) is not only massive parallel but also high throughput sequencing.

Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs

The bleeding disorder hemophilia A (HA) is caused by a single-gene (F8) defect and its clinical symptom can be substantially improved by a small increase in the plasma coagulation factor VIII (FVIII) level. In this study, we used F8-defective human induced pluripotent stem cells from an HA patient (F8d-HA hiPSCs) and F8-corrected (F8c) HA hiPSCs produced by CRISPR/Cas9 genome engineering of F8d–HA hiPSCs. We obtained a highly enriched population of CD157+ cells from CRISPR/Cas9-edited F8c-HA hiPSCs. These cells exhibited multiple cellular and functional phenotypes of endothelial cells (ECs) with significant levels of FVIII activity, which was not observed in F8d-HA hiPSC-ECs. After transplantation, the engineered F8c–HA hiPSC-ECs dramatically changed bleeding episodes in HA animals and restored plasma FVIII activity. Notably, grafting a high dose of ECs substantially reduced the bleeding time during multiple consecutive bleeding challenges in HA mice, demonstrating a robust hemostatic effect (90% survival). Furthermore, the engrafted ECs survived more than 3 months in HA mice and reversed bleeding phenotypes against lethal wounding challenges. We also produced F8c-HA hiPSC-derived 3D liver organoids by assembling three different cell types in microwell devices and confirmed its therapeutic effect in HA animals. Our data demonstrate that the combination of genome-engineering and iPSC technologies represents a novel modality that allows autologous cell-mediated gene therapy for treating HA.

Genetic diagnosis in Sudanese and Tunisian families with syndromic intellectual disability through exome sequencing.

Intellectual disability is a form of neurodevelopmental disorders that begin in childhood and is characterized by substantial intellectual difficulties as well as difficulties in conceptual, social, and practical areas of living. Several genetic and nongenetic factors contribute to its development; however, its most severe forms are generally attributed to single-gene defects. High-throughput technologies and data sharing contributed to the diagnosis of hundreds of single-gene intellectual disability subtypes. We applied exome sequencing to identify potential variants causing syndromic intellectual disability in six Sudanese patients from four unrelated families. Data sharing through the Varsome portal corroborated the diagnosis of one of these patients and a Tunisian patient investigated through exome sequencing. Sanger sequencing validated the identified variants and their segregation with the phenotypes in the five studied families. We identified three pathogenic/likely pathogenic variants in CCDC82, ADAT3, and HUWE1 and variants of uncertain significance in HERC2 and ATP2B3. The patients with the CCDC82 variants had microcephaly and spasticity, two signs absent in the two previously reported families with CCDC82-related intellectual disability. In conclusion, we report new patients with pathogenic mutations in the genes CCDC82, ADAT3, and HUWE1. We also highlight the possibility of extending the CCDC82-linked phenotype to include spastic paraplegia and microcephaly.