Background
Myofibrillar myopathies (MFM) are a subgroup of protein aggregate myopathies (PAM) characterized by a common histological picture of myofibrillar dissolution, Z-disk disintegration, and ...accumulation of degradation products into inclusions. Mutations in genes encoding components of the Z-disk or Z-disk-associated proteins occur in some patients whereas in most of the cases, the causative gene defect is still unknown. We aimed to search for pathogenic mutations in genes not previously associated with MFM phenotype.
Methods
We performed whole-exome sequencing in four patients from three unrelated families who were diagnosed with PAM without aberrations in causative genes for MFM.
Results
In the first patient and her affected daughter, we identified a heterozygous p.(Arg89Cys) missense mutation in
LMNA
gene which has not been linked with PAM pathology before. In the second patient, a heterozygous p.(Asn4807Phe) mutation in
RYR1
not previously described in PAM represents a novel, candidate gene with a possible causative role in the disease. Finally, in the third patient and his symptomatic daughter, we found a previously reported heterozygous p.(Cys30071Arg) mutation in
TTN
gene that was clinically associated with cardiac involvement.
Conclusions
Our study identifies a new genetic background in PAM pathology and expands the clinical phenotype of known pathogenic mutations.
Background:
Neuromuscular disorders (NMDs) are a heterogeneous group of genetic diseases, caused by mutations in genes involved in spinal cord, peripheral nerve, neuromuscular junction, and muscle ...functions. To advance the knowledge of the pathological mechanisms underlying NMDs and to eventually identify new potential drugs paving the way for personalized medicine, limitations regarding the availability of neuromuscular disease-related biological samples, rarely accessible from patients, are a major challenge.
Aim:
We characterized urinary stem cells (USCs) by in-depth transcriptome and protein profiling to evaluate whether this easily accessible source of patient-derived cells is suitable to study neuromuscular genetic diseases, focusing especially on those currently involved in clinical trials.
Methods:
The global transcriptomics of either native or MyoD transformed USCs obtained from control individuals was performed by RNA-seq. The expression of 610 genes belonging to 16 groups of disorders (
http://www.musclegenetable.fr/
) whose mutations cause neuromuscular diseases, was investigated on the RNA-seq output. In addition, protein expression of 11 genes related to NMDs including
COL6A
,
EMD
,
LMNA
,
SMN
,
UBA1
,
DYNC1H1
,
SOD1
,
C9orf72
,
DYSF
,
DAG1
, and
HTT
was analyzed in native USCs by immunofluorescence and/or Western blot (WB).
Results:
RNA-seq profile of control USCs shows that 571 out of 610 genes known to be involved in NMDs, are expressed in USCs. Interestingly, the expression levels of the majority of NMD genes remain unmodified following USCs MyoD transformation. Most genes involved in the pathogenesis of all 16 groups of NMDs are well represented except for channelopathies and malignant hyperthermia related genes. All tested proteins showed high expression values, suggesting consistency between transcription and protein representation in USCs.
Conclusion:
Our data suggest that USCs are human cells, obtainable by non-invasive means, which might be used as a patient-specific cell model to study neuromuscular disease-causing genes and that they can be likely adopted for a variety of
in vitro
functional studies such as mutation characterization, pathway identification, and drug screening.
Hypertrophic cardiomyopathy is an autosomal dominant disease. The main feature of this disorder is its occurrence in patients who present a left ventricular hypertrophy, unexplained by the loading ...conditions, usually asymmetric with greatest involvement most commonly of the interventricular septum.
Case presentation During a sports medicine control, a ultrasound scan in a 17 years old patient has shown a concentric left ventricular parietal hypertrophy associated with a 23 mm mid- basal interventricular septum thickness. After genetic counselling, a positive family history for hypertrophic cardiac disease and parents’ consanguineity was found. The genetic basis of the hypertrophic cardiomyopathy was investigated through a dedicated gene panel. The genetic test has revealed the presence of the variant c.3424G>A (p.Glu1142Lys) in the MYH7 gene in a homozygous state. Genotyping of the parents and of the two brothers revealed the presence of the MYH7 variant in heterozygosity in both parents and in the younger brother. In all of them, variable signs of hypertrophic cardiomyopathy were found.
Our findings report the presence of a homozygous variant in a sarcomeric gene (MYH7) which gave rise to early HCM, whereas the variant in a heterozygous state was associated to much milder cardiac phenotypes in the affected relatives. The onset and the progression of the hypertrophic cardiomyopathy in the reported family is to be referred to the presence of the variant in hetero- or homo-zygosity in a gene dosage manner.
Hypertrophic cardiomyopathy; Imaging; Genetic analysis.
The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and ...other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Brugada syndrome (BrS) is an inherited cardiac channelopathy first diagnosed in 1992 but still considered a challenging disease in terms of diagnosis, arrhythmia risk prediction, pathophysiology and ...management. Despite about 20% of individuals carrying pathogenic variants in the SCN5A gene, the identification of a polygenic origin for BrS and the potential role of common genetic variants provide the basis for applying polygenic risk scores for individual risk prediction. The pathophysiological mechanisms are still unclear, and the initial thinking of this syndrome as a primary electrical disease is evolving towards a partly structural disease. This review focuses on the main scientific advancements in the identification of biomarkers for diagnosis, risk stratification, pathophysiology and therapy of BrS. A comprehensive model that integrates clinical and genetic factors, comorbidities, age and gender, and perhaps environmental influences may provide the opportunity to enhance patients’ quality of life and improve the therapeutic approach.
We studied the incidence of acute graft versus host disease (GvHD) and its outcome in three consecutive time frames (year <2000; 2000-2010; >2010), in 3,120 patients allografted in two transplant ...Centers between 1976 and 2020. The median age increased over the three periods from 32 to 42 to 54 years (
< 0.00001). The median day of onset of GvHD in the three periods was day +14, day +16, and day +30, respectively (
< 0.0001). The cumulative incidence (CI) of GvHD grades II-IV in the three periods was 47, 24, and 16%, respectively (
< 0.00001). The CI of GvHD grades III-IV was 13, 5, and 4% (
< 0.001). In multivariate analysis, significant predictive factors for GvHD II-IV, on top of year of transplant, were anti-thymocyte globulin (ATG) (RR 0.67,
> 0.001); post-transplant cyclophosphamide (PTCY) (RR 0.41,
< 0.001), a family mismatched donor (RR 1.31,
= 0.03) a matched unrelated donor (RR 2.1,
< 0.001), an unrelated mismatched donor (RR1.8,
= 0.001), donor age above 40 years (RR 1.27,
< 0.001), hematological malignancy-as compared to aplastic anemia (RR 2.3,
< 0.001). When selecting only GvHD grade II, in a multivariate analysis, there was a significant reduction of transplant-related mortality (TRM) for patients grafted in 2001-2010 (RR 0.62,
< 0.0001) and for patients grafted in 2011-2020 (RR 0.35,
< 0.0001) as compared to grafts before the year 2000. A similar reduction in time was seen for patients with GvHD grades III-IV. The overall TRM in the three periods was 30, 22, and 16% (
< 0.0001) and survival was 47, 51, and 58% (
< 0.0001). Relapse risk was unchanged. In conclusion, we showed improved prevention of acute GvHD with time, together with a significant delay in the onset of the disease. Treatment of GvHD has also improved over time, as suggested by both reduced TRM and improved survival in more recent transplant periods.
ObjectivesTo describe a cohort of patients with arrhythmogenic left ventricular cardiomyopathy (ALVC), focusing on the spectrum of the clinical presentations.MethodsPatients were retrospectively ...evaluated between January 2012 and June 2020. Diagnosis was based on (1) ≥3 contiguous segments with subepicardial/midwall late gadolinium enhancement in the left ventricle (LV) at cardiac magnetic resonance plus a likely pathogenic/pathogenic arrhythmogenic cardiomyopathy (AC) associated genetic mutation and/or familial history of AC and/or red flags for ALVC (ie, negative T waves in V4-6/aVL, low voltages in limb leads, right bundle branch block like ventricular tachycardia) or (2) pathology examination of explanted hearts or autoptic cases suffering sudden cardiac death (SCD). Significant right ventricular involvement was an exclusion criterion.ResultsFifty-two patients (63% males, age 45 years (31–53)) composed the study cohort. Twenty-one (41%) had normal echocardiogram, 13 (25%) a hypokinetic non-dilated cardiomyopathy (HNDC) and 17 (33%) a dilated cardiomyopathy (DCM). Of 47 tested patients, 29 (62%) were carriers of a pathogenic/likely pathogenic DNA variant. Clinical contexts leading to diagnosis were SCD in 3 (6%), ventricular arrhythmias in 15 (29%), chest pain in 8 (15%), heart failure in 6 (12%) and familial screening in 20 (38%). Thirty patients (57%) had previously received a diagnosis other than ALVC with a diagnostic delay of 6 years (IQR 1–7).ConclusionsALVC is hidden in different clinical scenarios with a phenotypic spectrum ranging from normal LV to HNDC and DCM. Ventricular arrhythmias, chest pain, heart failure and SCD are the main clinical presentations, being familial screening essential for the affected relatives’ identification.
The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining ...mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene.
We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis.
This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), ...since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.
We defined urinary stem cell (USC) RNA-seq signatures and provided evidence that they are a non-invasive, affordable cell source to study Duchenne muscular dystrophy (DMD), an X-linked disease, due to dystrophin gene mutations. Based on USCs’ RNA profiling and whole-genome sequencing (WGS), we identified three undetected atypical DMD mutations.
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