•A consensus was reached on an updated definition of LGMD, and current sub-types were evaluated by application of the updated definition.•Consensus was reached on the most useful LGMD classification ...system that also allowed space for further discoveries of new sub-types.•Potential ramifications of the new definition and classification of LGMD for patients were discussed and several action points around how to disseminate this proposal were identified.
α-thalassemia is characterized in about 80% of cases by deletions generated by the presence of duplications and interspersed repeated sequences in the α-globin gene cluster. In a project on the ...molecular basis of α-thalassemia in Southern Italy, we identified six families, showing an absence of the most common deletions, and normal α-globin gene sequences. Multiplex Ligation-dependent Probe Amplification (MLPA), qRT-PCR, and the sequencing of long-range PCR amplicon have been used for the identification and characterization of new deletions. MLPA analysis for the identification of α- and β-globin rearrangement revealed the presence of five new α-thalassemia deletions. The set-up of qRT-PCR allowed us to delimit the extent of the deletions ranging from about 10 kb to more than 250 kb, two of them being of the telomeric type. The long-range PCR generated a specific anomalous fragment in three deletions, and only several unspecific bands in the other two deletions. The sequencing of the anomalous amplicons revealed the breakpoints of two deletions: the --PA, 34 kb long, identified in two families, and the telomeric --AG, 274 kb long. The anomalous fragment containing the breakpoint of the deletion --FG was partially sequenced, and it was not possible to identify the breakpoints due to the presence of several repetitive Alu sequences. The analysis of the breakpoint regions of the --Sciacca and --Puglia, respectively, are about 10 and 165 kb long, and revealed the presence of repeats that most likely impaired the amplification of a specific fragment for the identification of the breakpoint. MLPA, in association with qRT-PCR and long-range PCR, is a good approach for the identification and molecular characterization of rare or new deletions. Breakpoint analysis confirms that Alu sequences play an important role in favoring unequal crossing-over. Southern Italy shows considerable genetic heterogeneity, as expected with its central position in the Mediterranean basin, favoring migratory flows.
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.
Mutations in the human desmin gene (DES) may cause both autosomal dominant and recessive cardiomyopathies leading to heart failure, arrhythmias and atrio-ventricular blocks, or progressive ...myopathies. Cardiac conduction disorders, arrhythmias and cardiomyopathies usually associated with progressive myopathy are the main manifestations of autosomal dominant desminopathies, due to mono-allelic pathogenic variants. The recessive forms, due to bi-allelic variants, are very rare and exhibit variable phenotypes in which premature sudden cardiac death could also occur in the first or second decade of life. We describe a further case of autosomal recessive desminopathy in an Italian boy born of consanguineous parents, who developed progressive myopathy at age 12, and dilated cardiomyopathy four years later and died of intractable heart failure at age 17. Next Generation Sequencing (NGS) analysis identified the homozygous loss-of-function variant c.634C>T; p.Arg212*, which was likely inherited from both parents. Furthermore, we performed a comparison of clinical and genetic results observed in our patient with those of cases so far reported in the literature.
MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established ...the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes ...in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (
carried the c.1240C>T (p.Arg414Cys) homozygous variant in
;
carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in
); two patients with
-related myopathy (
carried the c.1325G>A (p.Arg442His) variant in
;
carried the c.1357C>T (p.Arg453Cys) variant in
); one patient with desminopathy (
carried the c.46C>T (p.Arg16Cys) variant in
); two patients with mitochondrial myopathy (
carried the m.3243A>G variant in
;
carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in
). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management.
We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three ...new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neurofibromatosis type 1 (NF1) is an autosomal dominant condition, with a birth incidence of approximately 1:2000-3000, caused by germline pathogenic variants in
, a tumor suppressor gene encoding ...neurofibromin, a negative regulator of the RAS/MAPK pathway. This explains why NF1 is included in the group of RASopathies and shares several clinical features with Noonan syndrome. Here, we describe the main clinical characteristics and complications associated with NF1, particularly those occurring in pediatric age. NF1 has complete penetrance and shows wide inter- and intrafamilial phenotypic variability and age-dependent appearance of manifestations. Clinical presentation and history of NF1 are multisystemic and highly unpredictable, especially in the first years of life when penetrance is still incomplete. In this scenario of extreme phenotypic variability, some genotype-phenotype associations need to be taken into consideration, as they strongly impact on genetic counseling and prognostication of the disease. We provide a synthetic review, based on the most recent literature data, of all known genotype-phenotype correlations from a genetic and clinical perspective. Molecular diagnosis is fundamental for the confirmation of doubtful clinical diagnoses, especially in the light of recently revised diagnostic criteria, and for the early identification of genotypes, albeit few, that correlate with specific phenotypes.
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.
PDF
