Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific ...knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.
The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in ...exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.
A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used the ...DMD gene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, the DMD gene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, because DMD mutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.
This paper describes the clinical practice and performance of cell-free DNA sequencing-based non-invasive prenatal testing (NIPT) as a screening method for fetal trisomy 21, 18, and 13 (T21, T18, and ...T13) and sex chromosome aneuploidies (SCA) in a general Italian pregnancy population.
The AMES-accredited laboratory offers NIPT in maternal blood as a screening test for fetal T21, T18, T13 and SCA. Samples were sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay.
A retrospective analysis was performed on 36,456 consecutive maternal blood samples, including 35,650 singleton pregnancies, 800 twin pregnancies, and 6 triplet pregnancies. Samples were tested between April 2017 and September 2019. The cohort included 46% elevated-risk and 54% low-risk patients. A result indicative of a classic trisomy was found in 356 (1%) of singleton or twin samples: 254 T21, 69 T18, and 33 T13. In addition, 145 results (0.4%) were indicative of a SCA. Of the combined 501 screen-positive cases, 484 had confirmatory diagnostic testing. NIPT results were confirmed in 99.2% (247/249) of T21 cases, 91.2% (62/68) of T18 cases, 84.4% (27/32) of T13 cases, and 86.7% (117/135) of SCA cases. In the 35,955 cases reported as unaffected by a classic trisomy or SCA, no false negative cases were reported. Assuming that false negative results would be reported, the sensitivity of NIPT was 100.00% for T21 (95% Cl 98.47-100.0), T18 (95% Cl 94.17-100.0), and T13 (95% Cl 87.54-100.0). The specificities were 99.99% (95% Cl 99.98-100.0), 99.98% (95% Cl 99.96-100.0), 99.99% (95% Cl 99.97-100.0), and 99.95% (95% Cl 99.92-99.97) for T21, T18, T13, and SCA, respectively.
This retrospective analysis of a large cohort of consecutive patients who had whole-genome sequencing-based NIPT for classic trisomies and SCA shows excellent detection rates and low false positive rates.
The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible ...for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.
Disrupting variants in the
gene are associated with Duchenne or Becker muscular dystrophy (DMD/BMD) or with hyperCKemia, all of which present very different degrees of clinical severity. The clinical ...phenotypes of these disorders could not be distinguished in infancy or early childhood. Accurate phenotype prediction based on DNA variants may therefore be required in addition to invasive tests, such as muscle biopsy. Transposon insertion is one of the rarest mutation types. Depending on their position and characteristics, transposon insertions may affect the quality and/or quantity of dystrophin mRNA, leading to unpredictable alterations in gene products. Here, we report the case of a three-year-old boy showing initial skeletal muscle involvement in whom we characterized a transposon insertion (Alu sequence) in exon 15 of the
gene. In similar cases, the generation of a null allele is predicted, resulting in a DMD phenotype. However, mRNA analysis of muscle biopsy tissue revealed skipping of exon 15, which restored the reading frame, thus predicting a milder phenotype. This case is similar to very few others already described in the literature. This case further enriches our knowledge of the mechanisms perturbing splicing and causing exon skipping in
, helping to properly guide clinical diagnosis.
De novo DDX3X variants account for 1-3% of syndromic intellectual disability (ID) in females and have been occasionally reported in males. Furthermore, somatic DDX3X variants occur in several ...aggressive cancers, including medulloblastoma. We report three unrelated females with severe ID, dysmorphic features, and a common brain malformative pattern characterized by malformations of cortical development, callosal dysgenesis, basal ganglia anomalies, and midbrain-hindbrain malformations. A pilocytic astrocytoma was incidentally diagnosed in Patient 1 and trigonocephaly was found in Patient 2. With the use of family based whole exome sequencing (WES), we identified three distinct de novo variants in DDX3X. These findings expand the phenotypic spectrum of DDX3X-related disorders, demonstrating unique neuroradiological features resembling those of the tubulinopathies, and support a role for DDX3X in neuronal development. Our observations further suggest a possible link between germline DDX3X variants and cancer development.
Inherited retinal diseases (IRDs) are the leading cause of vision loss in the working-age population. We performed a retrospective epidemiological study to determine the genetic basis of IRDs in a ...large Italian cohort (n = 2790) followed at a single referral center. We provided, mainly by next generation sequencing, potentially conclusive molecular diagnosis for 2036 patients (from 1683 unrelated families). We identified a total of 1319 causative sequence variations in 132 genes, including 353 novel variants, and 866 possibly actionable genotypes for therapeutic approaches. ABCA4 was the most frequently mutated gene (n = 535; 26.3% of solved cases), followed by USH2A (n = 228; 11.2%) and RPGR (n = 102; 5.01%). The other 129 genes had a lower contribution to IRD pathogenesis (e.g. CHM 3.5%, RHO 3.5%; MYO7A 3.4%; CRB1 2.7%; RPE65 2%, RP1 1.8%; GUCY2D 1.7%). Seventy-eight genes were mutated in five patients or less. Mitochondrial DNA variants were responsible for 2.1% of cases. Our analysis confirms the complex genetic etiology of IRDs and reveals the high prevalence of ABCA4 and USH2A mutations. This study also uncovers genetic associations with a spectrum of clinical subgroups and highlights a valuable number of cases potentially eligible for clinical trials and, ultimately, for molecular therapies.
High throughput sequencing analysis has facilitated the rapid analysis of the entire titin (TTN) coding sequence. This has resulted in the identification of a growing number of recessive titinopathy ...patients. The aim of this study was to (1) characterize the causative genetic variants and clinical features of the largest cohort of recessive titinopathy patients reported to date and (2) to evaluate genotype-phenotype correlations in this cohort.
We analyzed clinical and genetic data in a cohort of patients with biallelic pathogenic or likely pathogenic TTN variants. The cohort included both previously reported cases (100 patients from 81 unrelated families) and unreported cases (23 patients from 20 unrelated families).
Overall, 132 causative variants were identified in cohort members. More than half of the cases had hypotonia at birth or muscle weakness and a delayed motor development within the first 12 months of life (congenital myopathy) with causative variants located along the entire gene. The remaining patients had a distal or proximal phenotype and a childhood or later (noncongenital) onset. All noncongenital cases had at least one pathogenic variant in one of the final three TTN exons (362-364).
Our findings suggest a novel association between the location of nonsense variants and the clinical severity of the disease.
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