Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

A state-of-the-art review of tamoxifen as a potential therapeutic for duchenne muscular dystrophy.

Introduction: This systematic review analyzes the state-of-art repurposing of the drug tamoxifen (TAM) in the treatment of Duchenne Muscular Dystrophy (DMD), including its mechanism of action, toxicological findings, and past and ongoing clinical trials. A parallel aim of this work was to explore whether evidence exists to support further funding of investigation on TAM treatment for DMD patients with a pivotal trial in young patients. Bringing evidence and answering the scientific question of whether this treatment could improve the quality-of-life of DMD patients is needed to establish guidelines and accelerate access to promising therapies for DMD patients. Methods: The search was conducted in January 2022 utilizing PubMed. All MeSH terms for "Duchenne Muscular Dystrophy" and "tamoxifen" were used. The inclusion and exclusion criteria were defined according to the PICOS framework. Results: The included publications all explored the use of TAM with promising outcomes in muscular strength recovery and a decrease in pathology biomarkers. Two reviews recognize TAM as a potential treatment for DMD patients and state that drug repurposing plays a crucial role in the quest for a drug candidate to treat this rare disease. Conclusion: According to available data, TAM shows promise as a treatment for DMD, both pharmacologically and clinically. However, published data to date are insufficient to definitively conclude the beneficial effect of TAM on quality-of-life and ultimately survival, particularly in the youngest patients diagnosed with DMD.

Biosafety in ex vivo gene therapy and conditional ablation of lentivirally transduced hepatocytes in nonhuman primates.

Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand.

Knobloch syndrome: novel mutations in COL18A1, evidence for genetic heterogeneity, and a functionally impaired polymorphism in endostatin.

Knobloch syndrome (KNO) is an autosomal recessive disorder characterized by high myopia, vitreoretinal degeneration with retinal detachment, and congenital encephalocele. Pathogenic mutations in the COL18A1 gene on 21q22.3 were recently identified in KNO families. Analysis of two unrelated KNO families from Hungary and New Zealand allowed us to confirm the involvement of COL18A1 in the pathogenesis of KNO and to demonstrate the existence of genetic heterogeneity. Two COL18A1 mutations were identified in the Hungarian family: a 1-bp insertion causing a frameshift and a premature in-frame stop codon and an amino acid substitution. This missense variant is located in a conserved amino acid of endostatin, a cleavage product of the carboxy-terminal domain of collagen alpha 1 XVIII. D1437N (D104N in endostatin) likely represents a pathogenic mutation, as we show that the endostatin N104 mutant is impaired in its affinity towards laminin. Linkage to the COL18A1 locus was excluded in the New Zealand family, providing evidence for the existence of a second KNO locus. We named the second unmapped locus for Knobloch syndrome KNO2. Mutation analysis excluded COL15A1, a member of the multiplexin collagen subfamily similar to COL18A1, as being responsible for KNO2.

A step toward liver gene therapy: efficient correction of the genetic defect of hepatocytes isolated from a patient with Crigler-Najjar syndrome type 1 with lentiviral vectors.

BACKGROUND: Ex vivo liver gene therapy may be a future alternative to orthotopic liver transplantation for the treatment of some liver diseases. We previously described the transduction in suspension with lentiviral vectors and immediate hepatocyte transplantation (SLIT) protocol and its high transduction rate with normal human hepatocytes. We also reported SLIT efficiency in the animal model of Crigler-Najjar type 1 syndrome (CN-1), the Gunn rat. Here, we evaluated SLIT efficiency with diseased human hepatocytes. METHODS: Hepatocytes of the liver from a 4-year-old patient presenting CN-1 were isolated. They were transduced with liver-specific lentiviral vectors expressing uridine-diphosphate-glucuronosyltransferase (hUGT1A1) or green fluorescent protein, and then analyzed in vitro for transduction efficiency and hUGT1A1 expression, or transplanted in nonobese diabetic/severe combined immunodeficiency (SCID) mice to evaluate long-term survival of transplanted cells. RESULTS: More than 90% of CN-1 hepatocytes were transduced. Hepatocytes produced hUGT1A1 protein after lentiviral transduction. After having been subjected to the SLIT, lentivirally transduced CN-1 hepatocytes engrafted long term (up to 26 weeks posttransplantation) in recipient livers and expressed green fluorescent protein or hUGT1A1 vector. CONCLUSION: The SLIT protocol allowed for a high transduction of CN-1 hepatocytes and restoration of the expression of the deficient protein. Furthermore, long-term survival of lentivirally transduced CN-1 hepatocytes in the liver of immunodeficient mice was demonstrated. This study is therefore an important step toward human application of lentiviral gene therapy.

Proteomic and transcriptomic analysis of human CD8(+) T lymphocytes over-expressing telomerase.

Human T lymphocytes have a finite life span resulting from progressive telomere shortening that occurs at each cell division, eventually leading to chromosomal instability. It has been shown that ectopic expression of the human telomerase reverse transcriptase (hTERT) gene into various human cells results in the extension of their replicative life span, without inducing changes associated with transformation. However, it is still unclear whether cells that over-express telomerase are physiologically and biochemically indistinguishable from normal cells. To address this question, we compared the proteome of young and aged human CD8(+) T lymphocytes with that of T cells transduced with hTERT. Interestingly, we found no global changes in the protein pattern in young T cells, irrespective of telomerase expression. In contrast, several relevant proteins with differential expression patterns were observed in hTERT-transduced T cells with extended life span upon long-term culture. Altogether, our data revealed that T lymphocytes over-expressing telomerase displayed an intermediate protein pattern, sharing a similar protein expression not only with young T cells, but also with aged T cells. Finally, the results obtained from this global proteomic approach are in agreement with the overall gene transcription profiling performed on the same T-cell derived clones.

Mechanisms regulating the proliferative potential of human CD8+ T lymphocytes overexpressing telomerase.

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.

Mechanisms that limit the in vitro proliferative potential of human CD8+ T lymphocytes.

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16(INK4a) is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16(INK4a) and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16(INK4a) expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16(INK4a) and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16(INK4a) limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.

The Caenorhabditis elegans ortholog of C21orf80, a potential new protein O-fucosyltransferase, is required for normal development.

Down syndrome (DS), as a phenotypic result of trisomy 21, is the most frequent aneuploidy at birth and the most common known genetic cause of mental retardation. DS is also characterized by other phenotypes affecting many organs, including brain, muscle, heart, limbs, gastrointestinal tract, skeleton, and blood. Any of the human chromosome 21 (Hsa21) genes may contribute to some of the DS phenotypes. To determine which of the Hsa21 genes are involved in DS, the effects of disrupting and overexpressing individual human gene orthologs in model organisms, such as the nematode Caenorhabditis elegans, can be analyzed. Here, we isolated and characterized C21orf80 (human chromosome 21 open reading frame 80), a potential novel protein O-fucosyltransferase gene that encodes three alternatively spliced transcripts. Transient expression of tagged C21orf80 proteins suggests a primary intracellular localization in the Golgi apparatus. To gain insight into the biological role of C21orf80 and its potential role in DS, we isolated its C. elegans ortholog, pad-2, and performed RNA interference (RNAi) and overexpression experiments. pad-2(RNAi) embryos showed failure to undergo normal morphogenesis. Transgenic worms with elevated dosage of pad-2 displayed severe body malformations and abnormal neuronal development. These results show that pad-2 is required for normal development and suggest potential roles for C21orf80 in the pathogenesis of DS.