The position of nonsense mutations can predict the phenotype severity: A survey on the DMD gene.

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.

Copy Number Variants Account for a Tiny Fraction of Undiagnosed Myopathic Patients.

Next-generation sequencing (NGS) technologies have led to an increase in the diagnosis of heterogeneous genetic conditions. However, over 50% of patients with a genetically inherited disease are still without a diagnosis. In these cases, different hypotheses are usually postulated, including variants in novel genes or elusive mutations. Although the impact of copy number variants (CNVs) in neuromuscular disorders has been largely ignored to date, missed CNVs are predicted to have a major role in disease causation as some very large genes, such as the dystrophin gene, have prone-to-deletion regions. Since muscle tissues express several large disease genes, the presence of elusive CNVs needs to be comprehensively assessed following an accurate and systematic approach. In this multicenter cohort study, we analyzed 234 undiagnosed myopathy patients using a custom array comparative genomic hybridization (CGH) that covers all muscle disease genes at high resolution. Twenty-two patients (9.4%) showed non-polymorphic CNVs. In 12 patients (5.1%), the identified CNVs were considered responsible for the observed phenotype. An additional ten patients (4.3%) presented candidate CNVs not yet proven to be causative. Our study indicates that deletions and duplications may account for 5⁻9% of genetically unsolved patients. This strongly suggests that other mechanisms of disease are yet to be discovered.

Targeted gene panel screening is an effective tool to identify undiagnosed late onset Pompe disease.

Mutations in the GAA gene may cause a late onset Pompe disease presenting with proximal weakness without the characteristic muscle pathology, and therefore a test for GAA activity is the first tier analysis in all undiagnosed patients with hyperCKemia and/or limb-girdle muscular weakness. By using MotorPlex, a targeted gene panel for next generation sequencing, we analyzed GAA and other muscle disease-genes in a large cohort of undiagnosed patients with suspected inherited skeletal muscle disorders (n = 504). In this cohort, 275 patients presented with limb-girdle phenotype and/or an isolated hyperCKemia. Mutational analysis identified GAA mutations in ten patients. Further seven affected relatives were identified by segregation studies. All the patients carried the common GAA mutation c.-32-13T >G and a second, previously reported mutation. In the subcohort of 275 patients with proximal muscle weakness and/or hyperCKemia, we identified late-onset Pompe disease in 10 patients. The clinical overlap between Pompe disease and LGMDs or other skeletal muscle disorders suggests that GAA and the genes causing a metabolic myopathy should be analyzed in all the gene panels used for testing neuromuscular patients. However, enzymatic tests are essential for the interpretation and validation of genetic results.

Functional Antagonism between OTX2 and NANOG Specifies a Spectrum of Heterogeneous Identities in Embryonic Stem Cells.

Embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF) plus fetal bovine serum (FBS) exhibit heterogeneity in the expression of naive and primed transcription factors. This heterogeneity reflects the dynamic condition of ESCs and their versatility to promptly respond to signaling effectors promoting naive or primed pluripotency. Here, we report that ESCs lacking Nanog or overexpressing Otx2 exhibit an early primed identity in LIF + FBS and fail to convert into 2i-induced naive state. Conversely, Otx2-null ESCs possess naive identity features in LIF + FBS similar to Nanog-overexpressing ESCs and convert poorly into FGF-induced early primed state. When both Nanog and Otx2 are inactivated, ESCs cultured in LIF + FBS exhibit primed identity and weakened ability to convert into naive state. These data suggest that, through mutual antagonism, NANOG and OTX2 specify the heterogeneous identity of ESCs cultured in LIF + FBS and individually predispose them for optimal response to naive or primed inducing factors.

The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients.

OBJECTIVE: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. METHODS: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. RESULTS: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. CONCLUSIONS: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.

Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast.

Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.

Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?

Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.

Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F.

Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-ß super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.