In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.
BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.
- MeSH
- adenosindeaminasa genetika metabolismus MeSH
- alely MeSH
- dítě MeSH
- dvouvláknová RNA metabolismus MeSH
- editace RNA MeSH
- epilepsie enzymologie genetika MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutace MeSH
- nemoci mozku enzymologie genetika metabolismus MeSH
- neurovývojové poruchy enzymologie genetika MeSH
- pokrevní příbuzenství MeSH
- předškolní dítě MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- rodokmen MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
PURPOSE OF THE STUDY In patients older than 40 years of age, treatment of chondral lesions of the knee employing microfractures does not provide satisfactory outcomes. One of the contributing factors may be the age-related lack of sufficient mesenchymal stromal cells able to efficiently migrate into the desired site of the lesion. Concomitant application of mononuclear cells (MNCs) or cultured mesenchymal stem cells (MSCs) isolated from bone marrow and seeded on a 3D scaffold could provide an alternative enhancing therapeutic efficacy in these patients. The aim of our study was to assess two different sources of bone marrow for isolation of progenitor cells. To be specific, material obtained from proximal tibia and iliac crest (the commonly used bone marrow source) was compared in terms of quantity and quality of cells and their suitability for cellbased treatment of chondral lesions. MATERIAL AND METHODS Bone marrow was collected during a total knee replacement surgery from the iliac crest and the proximal metaphysis of the tibia from ten volunteers older than 40 years of age, using aspiration biopsy needles. The MNCs from the obtained material were isolated, cultured and analyzed for their phenotypic features. Both sources were compared as to the yield and viability of MNCs and MSCs as well as the ability of MSCs of chondrogenic differentiation. RESULTS The MNCs concentration/yield was significantly higher in samples from the iliac crests. Similar results were obtained with cultured MSCs after the first passage when the MSCs/MNCs ratio was compared. Nevertheless, the qualitative analysis that included MSCs immuno-phenotyping, viability and population doubling time showed no difference between the two tested bone marrow sources. DISCUSSION Particularly in older patients, therapies of chondral defects employing bone marrow-stimulating techniques result in unsatisfactory outcomes. Therefore, orthopedic surgeons have turned their attention to cell-based treatments. Bone marrow located in pelvic bone and metaphysis of long bones (distal femur, proximal tibia) contains mononuclear cells that possess features of MSCs. In accordance with other authors, quantitative differences between the cells from two anatomical locations were found in our cohort of patients. Qualitatively, however, there was no significant variability observed. We also confirmed that the metaphysis of proximal tibia is a suitable source for cultured MSCs. Moreover, in contrast to several reports, the quality of these cells does not appear to decrease with the patients age. CONCLUSIONS The iliac crest represents a superior bone marrow source for the MNCs and MSCs yield when compared with tibia. However, there was no qualitative difference between the isolated and cultured cells. The population doubling time analysis showed that the tibia is a good alternative source of MSCs which can be obtained at therapeutically relevant numbers for example for the treatment of chondral lesions of the knee. Key words: bone marrow, mesenchymal stem cells, MSC, mononuclear cells, chondral defects, osteoarthritis.
- MeSH
- buněčná diferenciace MeSH
- buňky kostní dřeně MeSH
- kmenové buňky MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky * MeSH
- os ilium MeSH
- senioři MeSH
- Check Tag
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
Bi-allelic loss-of-function variants in genes that encode subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of childhood-onset and complex hereditary spastic paraplegia: SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). Here, we report a detailed cross-sectional analysis of clinical, imaging and molecular data of 156 patients from 101 families. Enrolled patients were of diverse ethnic backgrounds and covered a wide age range (1.0-49.3 years). While the mean age at symptom onset was 0.8 ± 0.6 years [standard deviation (SD), range 0.2-5.0], the mean age at diagnosis was 10.2 ± 8.5 years (SD, range 0.1-46.3). We define a set of core features: early-onset developmental delay with delayed motor milestones and significant speech delay (50% non-verbal); intellectual disability in the moderate to severe range; mild hypotonia in infancy followed by spastic diplegia (mean age: 8.4 ± 5.1 years, SD) and later tetraplegia (mean age: 16.1 ± 9.8 years, SD); postnatal microcephaly (83%); foot deformities (69%); and epilepsy (66%) that is intractable in a subset. At last follow-up, 36% ambulated with assistance (mean age: 8.9 ± 6.4 years, SD) and 54% were wheelchair-dependent (mean age: 13.4 ± 9.8 years, SD). Episodes of stereotypic laughing, possibly consistent with a pseudobulbar affect, were found in 56% of patients. Key features on neuroimaging include a thin corpus callosum (90%), ventriculomegaly (65%) often with colpocephaly, and periventricular white-matter signal abnormalities (68%). Iron deposition and polymicrogyria were found in a subset of patients. AP4B1-associated SPG47 and AP4M1-associated SPG50 accounted for the majority of cases. About two-thirds of patients were born to consanguineous parents, and 82% carried homozygous variants. Over 70 unique variants were present, the majority of which are frameshift or nonsense mutations. To track disease progression across the age spectrum, we defined the relationship between disease severity as measured by several rating scales and disease duration. We found that the presence of epilepsy, which manifested before the age of 3 years in the majority of patients, was associated with worse motor outcomes. Exploring genotype-phenotype correlations, we found that disease severity and major phenotypes were equally distributed among the four subtypes, establishing that SPG47, SPG50, SPG51 and SPG52 share a common phenotype, an 'AP-4 deficiency syndrome'. By delineating the core clinical, imaging, and molecular features of AP-4-associated hereditary spastic paraplegia across the age spectrum our results will facilitate early diagnosis, enable counselling and anticipatory guidance of affected families and help define endpoints for future interventional trials.
- MeSH
- adaptorový proteinový komplex 4 genetika MeSH
- corpus callosum diagnostické zobrazování MeSH
- dítě MeSH
- dospělí MeSH
- kohortové studie MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- magnetická rezonanční tomografie metody trendy MeSH
- mladiství MeSH
- mladý dospělý MeSH
- předškolní dítě MeSH
- průřezové studie MeSH
- registrace MeSH
- spastická paraplegie dědičná diagnostické zobrazování genetika MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
We report on a family with spinocerebellar ataxia type 1 (SCA1), in which the age at onset and the severity of the disease do not correlate with the number of CAG repeat units. Although a marked anticipation was observed in the proband, it was not a consequence of an expansion of the CAG tract. None of the expanded alleles contained CAT interruptions. The pathologic expansion in this family was stable during the paternal but not maternal transmission, where it expanded by one trinucleotide and unexpectedly did not lead to anticipation. Our observations suggest that factors other than the length of the CAG repeat play a considerable role in determination of the disease course.
- MeSH
- alely MeSH
- dospělí MeSH
- expanze trinukleotidových repetic MeSH
- fenotyp MeSH
- finanční podpora výzkumu jako téma MeSH
- genotyp MeSH
- lidé středního věku MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- rodokmen MeSH
- spinocerebelární ataxie genetika patologie MeSH
- věk při počátku nemoci MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
Expansion of CAG trinucleotide repeats has been shown to cause a number of autosomal dominant spinocerebellar ataxias such as SCA1, SCA2, SCA3/MJD, SCA6 and SCA7. These disorders are characterized by a wide inter- and intrafamiliar variation in clinical features. The same mutation can result in different phenotypes and the very similar phenotypes can be caused by different mutations. Therefore it is necessary to investigate more SCA genes (according to prevalence) to identify the causal elongation. We developed a fast and efficient screening method based on touchdown multiplex PCR with fluorescent labelled primers for the most common types of SCAs (SCA 1, 2, 3 and 7). It has been reliable in 113 probands tested. Fragment analysis was performed by using 6% denaturing polyacrylamide gel and employing the automated DNA sequencer. This method considerably shortens the process of molecular genetic screening of SCAs and might be used as a tip for designing other SCA screening sets.
Although spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease is the most common type of SCA worldwide, we did not identify any cases of the disease amongst SCA patients in the Czech population. It has been proposed that the prevalence of large normal alleles correlates with the frequency of various types of SCA. We have therefore attempted to resolve the absence of SCA3 in our population by investigating, within 204 normal chromosomes, the frequency and nature of CAG repeats as well as two intragenic polymorphisms. We found that large normal alleles with more than 33 CAG repeats were observed at a frequency of only 0.49%. Whereas most of the expanded alleles worldwide have the CA haplotype, this was the least common (5.4%) variant observed in our study, although it was associated with a larger mean CAG repeat length (26.9). We postulate that the absence of SCA3 in the Czech population might be explained by the lack of large normal alleles and consequently a relatively small reservoir for aberrant CAG expansions at the SCA3 locus.
- MeSH
- alely MeSH
- financování organizované MeSH
- frekvence genu MeSH
- lidé MeSH
- Machado-Josephova nemoc genetika MeSH
- mutace MeSH
- mutační analýza DNA MeSH
- polymorfismus genetický MeSH
- proteiny nervové tkáně genetika MeSH
- spinocerebelární ataxie genetika MeSH
- trinukleotidové repetice MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
ADHD (attention hyperactivity disorder) is a polygenetic disorder with various candidate genes. At this time, more than thirty dopaminergic, noradrenergic, serotonergic and GABA-ergic genes are known. The research of only some candidate genes (DRD4, DAT, DRD5, DBH, 5HTT, HTR1B and SNAP25) brought relatively consistent results confirming the heredity of ADHD syndromes. The results of research of other genes (DRD2, DRD3, MAO, ADR2A, GABA A3, GABA B3) are not clear yet. This paper summarizes the most important genetic data in correlations with biochemical periphery parameters (especially for DBH, HVA, MHPG, serotonin). Hypothetically, certain subgroups of ADHD may be identified by correlation of biochemical characteristics and some candidate genes. The paper discusses some implications for future research. Review.
