DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores ...the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed ...that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2A294V ). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2A294G ) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2A294V alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2A294V channel alone. Although the total Kv7.2A294V expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2A294V /Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2A294V , the current density of homomeric Kv7.2A294G was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2A294G /Kv7.2/Kv7.3 and Kv7.2A294G /Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant.
Although 22q11.2 deletion syndrome (22q11.2DS) is the most recurrent human microdeletion syndrome associated with a highly variable phenotype, little is known about the condition's true incidence and ...the phenotype at diagnosis. We performed a multicenter, retrospective analysis of postnatally diagnosed patients recruited by members of the Association des Cytogénéticiens de Langue Française (the French-Speaking Cytogeneticists Association). Clinical and cytogenetic data on 749 cases diagnosed between 1995 and 2013 were collected by 31 French cytogenetics laboratories. The most frequent reasons for referral of postnatally diagnosed cases were a congenital heart defect (CHD, 48.6%), facial dysmorphism (49.7%) and developmental delay (40.7%). Since 2007 (the year in which array comparative genomic hybridization (aCGH) was introduced for the routine screening of patients with intellectual disability), almost all cases have been diagnosed using FISH (96.1%). Only 15 cases (all with an atypical phenotype) were diagnosed with aCGH; the deletion size ranged from 745 to 2904 kb. The deletion was inherited in 15.0% of cases and was of maternal origin in 85.5% of the latter. This is the largest yet documented cohort of patients with 22q11.2DS (the most commonly diagnosed microdeletion) from the same population. French cytogenetics laboratories diagnosed at least 108 affected patients (including fetuses) per year from among a national population of ∼66 million. As observed for prenatal diagnoses, CHDs were the most frequently detected malformation in postnatal diagnoses. The most common CHD in postnatal diagnoses was an isolated septal defect.
Letter to the Editor SMARCE1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1) is a constant component of the SWI/SNF (SWItch/Sucrose ...Non-Fermentable) chromatin remodeling complexes. The biallelic hit resulting in the loss of protein expression 5 further argues in favor of a driver role of SMARCE1 in our case. ...the tumor we report enlarges the spectrum of SMARCE1-related brain tumors to a so far undescribed high-grade neoplasm. ...a CCM could be formally ruled out because the tumor was not dura matter based, microscopic findings are not consistent with clear cell meningioma, immunohistochemical features were those of a tumor without SSTR2A expression and methylation profiling was clearly in disfavor of this diagnosis.
Lynch syndrome accounts for 3–5% of colorectal cancers and is due to a germline mutation in one of the mismatch repair genes
MLH1
,
MSH2
,
MSH6
, and
PMS2
. Somatic hypermethylation of the
MLH1
...promoter is commonly associated to sporadic cases. Strategies have been developed to identify patients with Lynch Syndrome based on clinical findings, tumoral phenotype, family history and immunohistochemistry analysis. However, there still are some pitfalls in this strategy, possibly responsible for an underdiagnosis of Lynch syndrome. Here we report the case of a 37 years-old man presenting with two concomitant tumors located in the rectosigmoid and in the ileocecal angle. Both tumors were microsatellites instability-high (MSI-H) and showed a loss of
MLH1
and
PMS2
protein expression, but only one had
MLH1
promoter hypermethylation. Constitutional analysis of mismatch repair genes could not be performed from a blood sample, because of the early death of the patient. However, tumoral tissue analyses revealed in both tumors a pathogenic variant in the
MLH1
gene. Further analysis of the surrounding tumor-free tissue also showed the presence of this alteration of the
MHL1
gene. Finally, the same pathogenic variant was present constitutionally in one of the siblings of the patient, confirming its hereditary nature. This new case of concomitant presence of
MLH1
promoter hypermethylation and
MLH1
germline mutation demonstrates that the presence of
MLH1
promoter hypermethylation should not rule out the diagnosis of Lynch Syndrome.
Intellectual Disability (ID) is characterized by deficits in intellectual functions such as reasoning, problem-solving, planning, abstract thinking, judgment, and learning. As new avenues are ...emerging for treatment of genetically determined ID (such as Down's syndrome or Fragile X syndrome), it is necessary to identify objective reliable and sensitive outcome measures for use in clinical trials.
We developed a novel visual analogical reasoning paradigm, inspired by the Progressive Raven's Matrices, but appropriate for Intellectually Disabled patients. This new paradigm assesses reasoning and inhibition abilities in ID patients.
We performed behavioural analyses for this task (with a reaction time and error rate analysis, Study 1) in 96 healthy controls (adults and typically developed children older than 4) and 41 genetically determined ID patients (Fragile X syndrome, Down syndrome and ARX mutated patients). In order to establish and quantify the cognitive strategies used to solve the task, we also performed an eye-tracking analysis (Study 2).
Down syndrome, ARX and Fragile X patients were significantly slower and made significantly more errors than chronological age-matched healthy controls. The effect of inhibition on error rate was greater than the matrix complexity effect in ID patients, opposite to findings in adult healthy controls. Interestingly, ID patients were more impaired by inhibition than mental age-matched healthy controls, but not by the matrix complexity. Eye-tracking analysis made it possible to identify the strategy used by the participants to solve the task. Adult healthy controls used a matrix-based strategy, whereas ID patients used a response-based strategy. Furthermore, etiologic-specific reasoning differences were evidenced between ID patients groups.
We suggest that this paradigm, appropriate for ID patients and developmental populations as well as adult healthy controls, provides an objective and quantitative assessment of visual analogical reasoning and cognitive inhibition, enabling testing for the effect of pharmacological or behavioural intervention in these specific populations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A population of patients with unexplained neurological symptoms from six major French university hospitals was screened over a 28-month period for primary creatine disorder (PCD). Urine ...guanidinoacetate (GAA) and creatine:creatinine ratios were measured in a cohort of 6,353 subjects to identify PCD patients and compile their clinical, 1H-MRS, biochemical and molecular data. Six GAMT N-guanidinoacetatemethyltransferase (EC 2.1.1.2) and 10 X-linked creatine transporter (SLC6A8) but no AGAT (GATM) L-arginine/glycine amidinotransferase (EC 2.1.4.1) deficient patients were identified in this manner. Three additional affected sibs were further identified after familial inquiry (1 brother with GAMT deficiency and 2 brothers with SLC6A8 deficiency in two different families). The prevalence of PCD in this population was 0.25% (0.09% and 0.16% for GAMT and SLC6A8 deficiencies, respectively). Seven new PCD-causing mutations were discovered (2 nonsense c.577C > T and c.289C > T and 1 splicing c.391 + 15G > T mutations for the GAMT gene and, 2 missense c.1208C > A and c.926C > A, 1 frameshift c.930delG and 1 splicing c.1393-1G > A mutations for the SLC6A8 gene). No hot spot mutations were observed in these genes, as all the mutations were distributed throughout the entire gene sequences and were essentially patient/family specific. Approximately one fifth of the mutations of SLC6A8, but not GAMT, were attributed to neo-mutation, germinal or somatic mosaicism events. The only SLC6A8-deficient female patient in our series presented with the severe phenotype usually characterizing affected male patients, an observation in agreement with recent evidence that is in support of the fact that this X-linked disorder might be more frequent than expected in the female population with intellectual disability.
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