Monogenetic Diseases Research Articles

CRISPR CLIP: Comprehensive Reviews on Interventional Studies using Precision Recombinant Technologies: Clinical Landmarks, Implications, and Prospects

Abstract Title CRISPR CLIP: Comprehensive Reviews on Interventional Studies using Precision Recombinant Technologies: Clinical Landmarks, Implications, and Prospects. Objectives To consolidate clinical trials that utilized the CRISPR technology to synthesise cures for various genetic diseases as a means to provide a window into the progress made so far while paving the way forward for future research and practices. Design Systematic review. (PROSPERO CRD42023479511) Methods Trials from 7 databases’ (ClinicalTrials.gov, European Union Clinical Trials Registry, ISRCTN registry, ICTRP/trialsearch.who.int, ChiCTR.org.cn, Clinical Trial Registry India, and Cochrane Library/Trials) inception to March 9, 2024, were considered. Exclusion criteria were unrelated, duplicated, non-English, unavailable full texts, diagnostic studies, correlational studies, observational studies, abstract-only papers, reviews or conference papers. Included studies were appraised using the ten-item CASP tool to assess methodological quality. Results The review identified 82 RCTs utilizing CRISPR and revealed four main themes: Diseases targeted, Countries of Clinical trials, Type of interventions, and Trial trends over the years. Geographically, the United States and China lead in the number of CRISPR clinical trials, followed by the European Union. However, Africa, Asia, and South America have very few trials. Among disease classes, cancer is the most prevalent focus with 39 studies, followed by monogenetic blood diseases, like Thalassemia and sickle cell anaemia. The biological agent CTX001 and Cyclophosphamide each feature in 11 studies. The peak year for clinical trials was 2018, marked by a significant increase with 16 studies conducted. Discussion Despite conducting a comprehensive search, the majority of trials were concentrated in the United States and China. Additionally, potential oversights due to vague titles, English-only studies, and indexing issues may have occurred. Nonetheless, by incorporating data from seven distinct databases, this review significantly contributes to understanding CRISPR's utilization in therapeutic clinical trials, paving the way for future research directions. The review underscores the burgeoning interest in CRISPR-based interventions. Current trials barely tap CRISPR's potential for treating genetic diseases. Funding The study received no external funding.

Expanded carrier screening for 224 monogenic disease genes in 1,499 Chinese couples: asingle-center study.

Expanded carrier screening (ECS) is a preventive genetic test that enables couples to know their risk of having a child affected by certain monogenetic diseases. This study aimed to evaluate the carrier frequency for rare monogenic diseases in the general Chinese population and the impacts of ECS on their reproductive decisions and pregnancy outcomes. This single-center study was conducted between September 2022 and April 2023. An ECS panel containing 224 recessive genes was offered to 1,499 Chinese couples from the general population who were at early gestational ages or planned to conceive. Overall, 55.0 % of the individuals carried for at least one recessive condition. There were 16 autosomal recessive (AR) genes with a carrier frequency of≥1/100 and 22 AR genes with a carrier frequency of <1/100 to ≥1/200. The most common AR and X-linked diseases were GJB2-related non-syndromic hearing loss, and hemolytic anemia, respectively. Fifty-five couples (3.67 %; 1 in 27.3) were at increased risk of having an affected child with 19 pregnant at the time of testing. Of these, 10 opted for amniocentesis, and four affected pregnancies were identified, with three of them being terminated. This study not only provides valuable information about the recessive genetic landscape, but also establishes a solid foundation for couple-based ECS in a real clinical setting.

Preclinical ex vivo IL2RG gene therapy using autologous hematopoietic stem cells as an effective and safe treatment for X-linked severe combined immunodeficiency disease

X-linked severe combined immunodeficiency disease (X-SCID) is a rare inherited disease caused by mutations in the interleukin 2 receptor subunit gamma gene (IL2RG), which encodes the common γ chain protein, a subunit of the receptor for lymphocytes. X-SCID is characterized by profound defects in T-cell, B-cell, and natural killer cell function. Here, we report a Chinese cohort of nine X-SCID patients with six novel IL2RG mutations. Among those, the two adolescent patients carrying missense mutation with an atypical immunotype were confirmed by further analyzing IL-2-JAK-STAT5 signaling, T cell proliferation, and T cell receptor excision circles (Trecs). Interestingly, Bacillus Calmette-Guérin (BCG) disease occurred commonly in this cohort. Although allogeneic hematopoietic stem-cell transplantation is curative for the disease, it is not available to all patients due to the lack of suitable matched donors. Autologous gene therapy using a self-inactivating lentiviral vector (SIN-LV) technology, developed in recent decades has provided an alternative therapy for treating such mono-genetic diseases. Here, we performed the pre-clinical studies to assess our SIN-LV carrying IL2RG on human ED7R cells deficient in IL2RG and CD34+ stem cells derived from the bone marrow of a healthy donor and a patient with X-SCID. This work is done complied with the established “Good Manufacturing Practice” (GMP) used in the clinical trials. In addition, a safety study is performed using the transduced CD34+ cells implanted into the axilla of nude mice in vivo. Overall, our studies have demonstrated the efficiency and safety of SIN-IL2RG-LV, which paves the way for conducting X-SCID gene therapy clinical trials in China in the near future.

Family-based whole-exome sequencing implicates a variant in lysyl oxidase like 4 in atypical femur fractures.

Atypical femur fractures (AFFs) are rare adverse events associated with bisphosphonate use, having unclear pathophysiology. AFFs also cluster in families and have occurred in patients with monogenetic bone diseases sometimes without bisphosphonate use, suggesting an underlying genetic susceptibility. Our aim was to identify a genetic cause for AFF in a Caucasian family with seven members affected by osteoporosis, including three siblings with bisphosphonate-associated AFFs. Using whole-exome sequencing, we identified a rare pathogenic variant c.G1063A (p.Gly355Ser) in lysyl oxidase like 4 (LOXL4) among 64 heterozygous rare, protein-altering variants shared by the three siblings with AFFs. The same variant was also found in a fourth sibling with a low-trauma femur fracture above the knee, not fulfilling all the ASBMR criteria of AFF and in one of 73 unrelated European AFF patients. LOXL4 is involved in collagen cross-linking and may be relevant for microcrack formation and bone repair mechanisms. Preliminary functional analysis showed that skin fibroblast-derived osteoblasts from the unrelated patient with the LOXL4 variant expressed less collagen type I and elastin, while osteogenic differentiation and mineralization were enhanced compared with two controls. In conclusion, this LOXL4 variant may underlie AFF susceptibility possibly due to abnormal collagen metabolism leading to increased formation of microdamage or compromised healing of microcracks in the femur.

Different diseases, different needs: Patient preferences for gene therapy in lysosomal storage disorders, a probabilistic threshold technique survey

BackgroundGene therapy is currently in development for several monogenetic diseases including lysosomal storage disorders. Limited evidence is available on patient preferences for gene therapy in this population. In this study, we compare gene therapy-related risk tolerance between people affected by three lysosomal storage diseases currently faced with different therapeutic options and prognoses.MethodsA survey including the probabilistic threshold technique was developed in which respondents were asked to choose between gene therapy and the current standard of care. The attributes included to establish participants’ risk tolerance were previously identified in focus groups of affected people or their representatives, namely: risk of mild side effects, severe side effects, the need for additional medication, and the likelihood of long-term effectiveness. The survey was distributed among people receiving outpatient care for type 1 Gaucher disease (good prognosis with current treatment options), Fabry disease (varying prognosis with current treatment options, XY-genotype on average more severely affected than XX), and parents representing people with severe forms of mucopolysaccharidosis type III A/B (poor prognosis, no disease-specific therapy available).ResultsA total of 85 surveys were completed (15 Gaucher disease respondents, 62 Fabry disease respondents (17 self-identifying male), eight parents of ten people with mucopolysaccharidosis type III). Disease groups with higher disease severity trended towards higher risk tolerance: Gaucher disease respondents were most cautious and predominantly preferred the current standard of care as opposed to MPS III representatives who were more risk tolerant. Respondents with Fabry disease were most heterogeneous in their risk tolerance, with male participants being more risk tolerant than female participants. Long-term effectiveness was the attribute in which respondents tolerated the least risk.ConclusionsPeople affected by a lysosomal storage disease associated with a poorer prognosis and less effective current treatment options trended towards more risk tolerance when choosing between gene therapy and the current standard of care. This study shows the importance of involvement of patient preferences before and during the development process of new treatment modalities such as gene therapy for rare diseases, to ensure that innovative therapies align with the wishes and needs of people affected by these diseases.

The High Diagnostic Yield of Prenatal Exome Sequencing Followed by 3400 Gene Panel Analysis in 629 Ongoing Pregnancies With Ultrasound Anomalies.

The aim of this study was to evaluate the diagnostic yield of routine exome sequencing (ES) in fetuses with ultrasound anomalies. We performed a retrospective analysis of the ES results of 629 fetuses with isolated or multiple anomalies referred in 2019-2022. Variants in a gene panel consisting of approximately 3400 genes associated with multiple congenital anomalies and/or intellectual disability were analyzed. We used trio analysis and filtering for de novo variants, compound heterozygous variants, homozygous variants, X-linked variants, variants in imprinted genes, and known pathogenic variants. Pathogenic and likely pathogenic variants (class five and four, respectively) were identified in 14.0% (88/629, 95% CI 11.5%-16.9%) of cases. In the current cohort, the probability of detecting a monogenetic disorder was ∼1:7 (88/629, 95% CI 1:8.7-1:5.9), ranging from 1:9 (49/424) in cases with one major anomaly to 1:5 (32/147) in cases with multiple system anomalies. Our results indicate that a notable number of fetuses (1:7) with ultrasound anomalies and a normal chromosomal microarray have a (likely) pathogenic variant that can be detected through prenatal ES. These results warrant implementation of exome sequencing in selected cases, including those with an isolated anomaly on prenatal ultrasound.

Lower limb maltorsion and acetabular deformity in children and adolescents with X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a rare monogenetic skeletal disorder. Lower limb deformities contribute substantially to impaired gait quality and burden of disease in patients with XLH. Standardized data regarding onset and severity of lower limb maltorsion are unavailable. This study aimed to evaluate lower limb maltorsion using rotational magnetic resonance imaging (MRI) and computed tomography (CT). Rotational MRI and CT of children and adolescents with verified XLH were evaluated retrospectively. Femoral and tibial torsion, acetabular anteversion, and axial acetabular coverage were measured and compared with published age-adapted radiographic, clinical measurements and MRI data, respectively. Fifteen patients (mean age, 10.7 years) were included in the study. Decreased femoral torsion was observed in 47% (14/30 femora) and femoral retrotorsion in 17% (5/30 femora). Ten of 13 hips with coxa vara deformity presented with decreased femoral antetorsion. Reduced external tibial torsion manifested in 64% (18/28 tibiae). Abnormal axial femoral head coverage was present in 67% (20/30 hips), whereas 53% (16/30 hips) showed increased acetabular anteversion. Femoral and tibial torsional pathologies were found in children and adolescents with XLH. The occurrence of severe femoral retrotorsion in a 2-year-old child prior to ambulation raises questions regarding the biomechanical impact of gait on the development of torsional deformities in XLH.

Survival in myotonic dystrophy type 1: a long time follow up-study with special reference to gastrointestinal symptoms.

Myotonic dystrophy type 1 (DM1) is a monogenetic disease affecting many organs. Gastrointestinal symptoms are prevalent and of considerable consequences for affected individuals. The life expectancy is shortened and the objective of the study is to evaluate if gastrointestinal symptoms can predict the outcome of the disease. Fifty-one patients with DM1 were interviewed regarding symptoms from the gastrointestinal tract in the mid-1990s. Survival of all patients was evaluated in 2023 and the impact of symptoms on survival was assessed. At the beginning of the study, the mean age was 35.9 years, (median 37.0, 9-63). At the end of the study 47 out of the 51 patients were deceased at a mean age of 53.7 years (median 55.7, 32.5-79.0). Patients with the congenital form of DM1 (n = 6) died at an age of 46.0 years (median 45.2, 40.0-53.6). There was no correlation between the gastrointestinal symptoms and survival. Albeit prevalent and of considerable clinical consequence, gastrointestinal symptoms are not correlated to survival in myotonic dystrophy type 1.

Prevalence of hemoglobinopathies detected by high performance liquid chromatography in tertiary care centre, Kota, Rajasthan

Background: Hemoglobinopathies are the most common heterogeneous group of monogenetic disorder in the world. Their prevalence varies with geographical regions. India is developing country and many studies have shown a significant burden of hemoglobinopathies in India. Our aim is to estimate prevalence of various hemoglobinopathies in our region. Methods: The present study was conducted at the department of pathology, Government Medical College, Kota on patients referred for antenatal or voluntary premarital checkup, patients with clinical history and complete blood count (CBC) suggestive of hemolytic anemia, and family members of known cases of hemoglobinopathies. Transfusion-dependent patients were also included. However, patients with history of blood transfusion within the last 1 month were excluded. EDTA samples were used for CBC using 6-part differential cell counter and high-performance liquid chromatography (HPLC) using BIORAD D10 analyzer. The results were tabulated and analyzed. Results: Of the 226 cases studied, 139 (61.5%) were females and 87 (38.5%) were males. Most common age group was 21 to 30 years. The number of thalassemia traits were 26 (11.5%), sickle cell traits were 7 (3.1%), sickle cell homozygous was 1 (0.4%), compound heterozygous for sickle cell and thalassemia were 2 (0.9%), while the remaining 190 patients (84.07%) were found to have normal HPLC, with presence or absence of nutritional deficiency. Conclusions: In our study, we found a high prevalence of hemoglobinopathies among patients. The most common disorder detected was beta thalassemia trait. Most of the hemoglobinopathies found in our study could be accurately quantified by HPLC which is a rapid, sensitive, and reproducible method for the detection of different hemoglobinopathies.

Assessment the carrier frequency of monogenic diseases in populations requiring assisted reproductive technology

PurposeThe objective of this study is to assess the carrier frequency and pathogenic variation of monogenetic diseases in a population of 114 subjects in Han Chinese from Hebei province who are undergoing assisted reproductive technology through the utilization of Expanded Carrier Screening (ECS).MethodsThe study utilized a panel consisting of 155 severe monogenic recessive genetic diseases for ECS. Next-generation sequencing technology was employed to identify specific variants associated with ECS in a cohort of 114 subjects from 97 couples, comprising 97 females and 17 male spouses.ResultsA total of 114 individuals received ECS. The carrier rate of pathogenic genes in the enrolled population was 44.74% (51/114). Among the 97 females, the carrier rate of pathogenic genes was higher in those without assisted reproduction indicators than in those with assisted reproduction indicators (59.09% vs. 41.33%). However, the carrier rate of pathogenic genes in males without assisted reproductive technology was slightly lower than that with assisted reproductive technology (40% vs. 41.67%). Among both female and male participants, the carrier rate of pathogenic genes between individuals without indicators of assisted reproduction and those with such indicators was 55.55% vs. 41.38%. In 51 carriers, 72.55% (37/51) carried one genetic variant, 25.49% (13/51) carried two genetic variants, and 1.96% (1/51) carried three genetic variants. A total of 38 pathogenic genes were detected in this study, and GJB2 and MMACHC were most common. The carrier rates of the two genes were both 5.26% (6/114). A total of 55 variations were detected, and c.235delC was most frequently found. The carrier rate was 3.51% (4/114). The incidence of couples carrying the same pathogenic genes was 1.03% (1/97).ConclusionsThe findings elucidate the carrier rate of pathogenic genes among 155 severe monogenic recessive genetic diseases and underscore the significance of ECS as a preventive measure against congenital anomalies. When both partners carry the same genetic mutation for a monogenic disease, preventive strategies can be taken in offspring through preimplantation genetic testing (PGT), prenatal genetic testing, or the utilization of donor gametes. ECS is instrumental in assessing reproductive risk, guiding fertility-related decisions, and reducing the prevalence of monogenic recessive genetic disorders in subsequent generations.

Efficacy of an AAV vector encoding a thermostable form of glucocerebrosidase in alleviating symptoms in a Gaucher disease mouse model

Almost all attempts to date at gene therapy approaches for monogenetic disease have used the amino acid sequences of the natural protein. In the current study, we use a designed, thermostable form of glucocerebrosidase (GCase), the enzyme defective in Gaucher disease (GD), to attempt to alleviate neurological symptoms in a GD mouse that models type 3 disease, i.e. the chronic neuronopathic juvenile subtype. Upon injection of an AAVrh10 (adeno-associated virus, serotype rh10) vector containing the designed GCase (dGCase) into the left lateral ventricle of Gba−/−;Gbatg mice, a significant improvement in body weight and life-span was observed, compared to injection of the same mouse with the wild type enzyme (wtGCase). Moreover, a reduction in levels of glucosylceramide (GlcCer), and an increase in levels of GCase activity were seen in the right hemisphere of Gba−/−;Gbatg mice, concomitantly with a significant improvement in motor function, reduction of neuroinflammation and a reduction in mRNA levels of various genes shown previously to be elevated in the brain of mouse models of neurological forms of GD. Together, these data pave the way for the possible use of modified proteins in gene therapy for lysosomal storage diseases and other monogenetic disorders.

Inhibition of GSK3α,β rescues cognitive phenotypes in a preclinical mouse model of CTNNB1 syndrome

CTNNB1 syndrome is a rare monogenetic disorder caused by CTNNB1 de novo pathogenic heterozygous loss-of-function variants that result in cognitive and motor disabilities. Treatment is currently lacking; our study addresses this critical need. CTNNB1 encodes β-catenin which is essential for normal brain function via its dual roles in cadherin-based synaptic adhesion complexes and canonical Wnt signal transduction. We have generated a Ctnnb1 germline heterozygous mouse line that displays cognitive and motor deficits, resembling key features of CTNNB1 syndrome in humans. Compared with wild-type littermates, Ctnnb1 heterozygous mice also exhibit decreases in brain β-catenin, β-catenin association with N-cadherin, Wnt target gene expression, and Na/K ATPases, key regulators of changes in ion gradients during high activity. Consistently, hippocampal neuron functional properties and excitability are altered. Most important, we identify a highly selective inhibitor of glycogen synthase kinase (GSK)3α,β that significantly normalizes the phenotypes to closely meet wild-type littermate levels. Our data provide new insights into brain molecular and functional changes, and the first evidence for an efficacious treatment with therapeutic potential for individuals with CTNNB1 syndrome.

P-551 Application of simultaneous PGT-M/PGT-A using whole genome sequencing and haplotyping for monogenetic disorders, multiple variants, low penetrance, and de novo disorders

Abstract Study question Is the concurrent use of high-resolution PGT-A and genome-wide haplotyping with disease-specific assays for PGT-M effective in an unselected patient population at-risk for monogenetic disorders? Summary answer PGT-A/PGT-M is an effective treatment for couples/patients burdened with genetic diseases, that provides optimal family planning, prevents vertical transmission, and reduces personal and societal encumbrance What is known already Societal awareness of genetic testing expanded the use of carrier screening, diagnostic genetic tests, and higher in vitro fertilization (IVF) utilization has increased demand for preimplantation genetic testing for monogenetic disorders (PGT-M), as an alternative to invasive prenatal tests. The scope of PGT-M testing expands beyond common severe childhood disorders, to include serious and mild late onset disorders, cancer predisposition, variants of unknown significance, de novo mutations, and variants in multiple genes. This highlights novel ethical dilemmas, necessitating adjustment of generic genome wide approaches to complement, or eventually replace, targeted disease-specific PGT-M assays. Study design, size, duration This is a retrospective study of 359 couples at-risk for monogenetic disorders referred to the CReATe Fertility Centre, Toronto, Canada. Our objective was to evaluate the efficiency of concurrent use of genome wide haplotyping and disease specific assays for PGT-M and high resolution PGT-A in an unselected patient population at risk for monogenetic disorders, multiple and de novo variants, low penetrance and variants of unknown significance. Participants/materials, setting, methods All patients received comprehensive genetic counselling. For each couple/patient, DNA from gamete providers, related family member/s and whole-genome amplified (WGA)DNA from a single trophectoderm(TE) biopsy were analyzed. Whole-genome SNP-array (Karyomapping, Illumina, CA) and STR-analysis were used to obtain parental haplotypes and Sanger-sequencing was used for direct variant analysis. PGT-A was performed using Illumina platform(NextSeq 550). NxClinical (BioDiscovery) and BlueFuse Multi(Illumina) software were used for data analysis. Main results and the role of chance 1826 embryos, from 235 IVF cycles were analyzed for 88 unique genes (14 cancer predisposing, 42 autosomal recessive and 32 dominant genes) and a total of 282 unique variants. The most frequent genes tested were BRCA1/2 -21% of all cases, followed by HBB-8.7%, CFTR-8.7% and CYP21A2-8.7%. PGT-M for 11 cases was done for a de novo variant detected in a sibling and in 2 cases for a parental mosaic variant. Multiple genes and VUS were analyzed in 12 and 6 cases, respectively. PGT-A analysis showed 31.6% aneuploidy, 9.6% mosaicism and 58.8% euploidy. Genome wide haplotyping was used for euploid and mosaic embryos with an average case call rate of 95%, and average case Allele Drop Out (ADO) rate of 2.7%. Recombination events in 3% of cases prevented assignment of haplotype. Direct mutation testing was obtained for all samples and was 100% concordant with the predicted haplotypes. In cases with no predicted haplotype, direct testing established the PGT-M diagnosis. The distribution for both recessive and dominant disorders was in Mendelian order. To date 66 live births confirmed the results from PGT-M diagnosis. Limitations, reasons for caution Ethical considerations and extensive genetic counselling are prerequisites for PGT-A/M testing implementation. Simultaneous screening for embryo aneuploidy and monogenetic disorders optimizes patient treatment and family planning, but may not be available in all jurisdictions. Although the data was collected prospectively, retrospective design is a limitation. Wider implications of the findings Simultaneous PGT-A and PGT-M with direct variant testing provides optimal embryo prioritization for transfer and significantly reduces time to a healthy pregnancy in couples at risk for inherited disorders. Karyomapping is effective for generic haplotyping, with a low risk of undetected recombination events in the genomic region of interest. Trial registration number not applicable

A retrospective survey on follow-up of splenectomy patients due to β-thalassemia and Sickle cell Anemia in Karbala, Iraq during 2010-2023

Background: Hemoglobinopathy is considered a common monogenetic genetic disorder worldwide. Splenectomy is considered a therapeutic strategy in patients with hemoglobinopathy. The aim of current study was to provide a survey on the splenectomy and 5 years follow-up in different clinical forms of β-thalassemia (intermedia, Major) and Sickle cell Anemia (SCA) patients who referred to Hereditary Blood Disease Center in the Karbala Teaching Hospital for Children in Karbala, Iraq. Materials and Methods: In this retrospective study we tried to evaluate 126 hemoglobinopathy and thalassemia patients from Karbala City, Iraq. All cases of splenectomy due to hemoglobinopathy and thalassemia during 2010-2023 who referred to the Hereditary Blood Disease Center in the Karbala Teaching Hospital for Children in Karbala, Iraq were included. Patient data was collected at three-time points. The first was after the splenectomy, the second during 1-5 years, and the third step after 5 years. Clinical and laboratory data were retrieved from the patient’s file. Results: The mean age of splenectomy of included patients was 14.1±7.5 years. From 126 cases, 103 (81.74%) were β-Thalassemia, 13 (10.32%) were SCA, and 10 (7.94%) were Sickle cell beta-thalassemia. The mean age in SCA was significantly less than two other groups (mean age in β-Thalassemia, SCA and Sickle cell beta thalassemia were 18.2±8.7, 24.2±12.7 and 25.2±9.5, respectively) (p=0.008). Platelet and WBC count represents a significant increase during 1-5 years after splenectomy in comparison with 1 year after splenectomy (for Platelet and WBC p=0.03 and 0.001, respectively). Conclusion: splenectomy is considered the last therapeutic option in hemoglobinopathy patients. All Hemoglobinopathy patients represented significant improvement after splenectomy. Because there was no suitable treatment in the past, splenectomy was considered a therapeutic solution. It should be said that periodic follow-up of splenectomy patients in hemoglobinopathy plays an important role in improving the management of these diseases.

Steps on the way to a new nomenclature of liver diseases

The term «steatotic liver disease (SLD)» is proposed to cover all patients with hepatosteatosis. Patients with SLD with one or more of the five cardiometabolic risk factors (CMRF) are further divided into «metabolic dysfunction‑associated steatotic liver disease» (MASLD) or «metabolic‑alcohol‑associated liver disease» (MetALD)/other combination of etiologies. СMRF includes: 1) body mass index ≥25 kg/m2 (≥23 kg/m2 for people of Asian nationality) or waist circumference &gt;94 cm for men and &gt;80 cm for women or taking into account ethnicity; 2) fasting serum glucose level ≥5.6 mmol/l (≥100 mg/dL) or glucose level 2 hours after exercise ≥7.8 mmol/l (≥140 mg/dL) or glycated hemoglobin ≥5.7% (≥39 mmol/L) or type 2 diabetes mellitus, or type 2 diabetes treatment; 3) blood pressure ≥130/85 mm Hg or antihypertensive treatment; 4) plasma triglycerides ≥1.70 mmol/l (≥150 mg/dL) or lipid‑lowering therapy; and 5) plasma high‑density lipoprotein cholesterol ≤1.0 mmol/L (Ј 40 mg/dL) for men and ≤1.3 mmol/L (Ј 50 mg/dL) for women or the patient’s receipt of lipid‑lowering treatment. Patients with SLD and at least one of the СMRF belong to the category of MASLD, if they have no other causes of steatosis. Patients in the new category of MetALD have characteristics corresponding to MASLD and consume alcohol in the amount of 20 to 50 g/day for women and 30 to 60 g/day for men. In the absence of any СMRF, cryptogenic SLD or SLD with another specific etiology (alcoholic liver disease, drug‑induced liver damage, and monogenetic diseases) are distinguished. In the new nomenclature, the stages of steatosis and liver fibrosis have not been changed.

Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production

Successful cell and gene therapy clinical trials have resulted in the United States Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of Major Histocompatibility Complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB which facilitates expansion, reduces handling, and increases titers by 2-fold compared to previous methods. SJ293TS-DPB stably produced lentiviral vectors for over four months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

Modeling the cell biology of monogenetic intestinal epithelial disorders.

Monogenetic variants are responsible for a range of congenital human diseases. Variants in genes that are important for intestinal epithelial function cause a group of disorders characterized by severe diarrhea and loss of nutrient absorption called congenital diarrheas and enteropathies (CODEs). CODE-causing genes include nutrient transporters, enzymes, structural proteins, and vesicular trafficking proteins in intestinal epithelial cells. Several severe CODE disorders result from the loss-of-function in key regulators of polarized endocytic trafficking such as the motor protein, Myosin VB (MYO5B), as well as STX3, STXBP2, and UNC45A. Investigations of the cell biology and pathophysiology following loss-of-function in these genes have led to an increased understanding of both homeostatic and pathological vesicular trafficking in intestinal epithelial cells. Modeling different CODEs through investigation of changes in patient tissues, coupled with the development of animal models and patient-derived enteroids, has provided critical insights into the enterocyte differentiation and function. Linking basic knowledge of cell biology with the phenotype of specific patient variants is a key step in developing effective treatments for rare monogenetic diseases. This knowledge can also be applied more broadly to our understanding of common epithelial disorders.

PURA syndrome-causing mutations impair PUR-domain integrity and affect P-body association.

Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

PURA syndrome-causing mutations impair PUR-domain integrity and affect P-body association

Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

Ligand-based virtual-screening identified a novel CFTR ligand which improves the defective cell surface expression of misfolded ABC transporters.

Cystic fibrosis (CF) is a monogenetic disease caused by the mutation of CFTR, a cAMP-regulated Cl- channel expressing at the apical plasma membrane (PM) of epithelia. ∆F508-CFTR, the most common mutant in CF, fails to reach the PM due to its misfolding and premature degradation at the endoplasmic reticulum (ER). Recently, CFTR modulators have been developed to correct CFTR abnormalities, with some being used as therapeutic agents for CF treatment. One notable example is Trikafta, a triple combination of CFTR modulators (TEZ/ELX/IVA), which significantly enhances the functionality of ΔF508-CFTR on the PM. However, there's room for improvement in its therapeutic effectiveness since TEZ/ELX/IVA doesn't fully stabilize ΔF508-CFTR on the PM. To discover new CFTR modulators, we conducted a virtual screening of approximately 4.3 million compounds based on the chemical structures of existing CFTR modulators. This effort led us to identify a novel CFTR ligand named FR3. Unlike clinically available CFTR modulators, FR3 appears to operate through a distinct mechanism of action. FR3 enhances the functional expression of ΔF508-CFTR on the apical PM in airway epithelial cell lines by stabilizing NBD1. Notably, FR3 counteracted the degradation of mature ΔF508-CFTR, which still occurs despite the presence of TEZ/ELX/IVA. Furthermore, FR3 corrected the defective PM expression of a misfolded ABCB1 mutant. Therefore, FR3 may be a potential lead compound for addressing diseases resulting from the misfolding of ABC transporters.