Cell Context is the third axis of synergy for the combination of ATR inhibition and cisplatin in Ewing sarcoma.

PURPOSE: The importance of cellular context to the synergy of DNA Damage Response (DDR) targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR targeted combination therapy for Ewing Sarcoma (ES). EXPERIMENTAL DESIGN: We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for ES selectivity, and optimized for in vivo schedule. RESULTS: Berzosertib combined with cisplatin demonstrates profound synergy in multiple ES cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell cycle checkpoints, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of ES models leading to durable complete responses in 50% of animals bearing two different ES xenografts. CONCLUSION: These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule.

Primary ovarian insufficiency in RMND1 mitochondrial disease.

RMND1 (Required for Meiotic Nuclear Division 1 homolog) is a nuclear encoded mitochondrial protein. Biallelic variants inRMND1are described in patients with white matter encephalopathy, hearing loss and renal dysfunction. In addition to this phenotype, two independent families (3 patients) have been reported with ovarian failure. We report on a 17-year-old girl with RMND1 related mitochondrial disorder including white matter encephalopathy, hearing loss and renal insufficiency who presented primary ovarian insufficiency in whom a homozygous variant c.713 A > G (p.Asn238Ser) in the RMND1 gene was found. We report the fourth patient with RMND1 biallelic pathogenic variants and primary ovarian insufficiency.

Microplastics impede larval urchin selective feeding.

Microplastics are emergent threats to marine organisms as ingestion can cause a multitude of physiological problems. Suspension feeders, including marine invertebrate larvae, are particularly susceptible to ingesting microplastics due to similarities in physical appearance to algal cells. Larval feeding involves multiple stages: the capture and subsequent selection of particles followed by ingestion from the mouth to the stomach, digestion, and finally, egestion. Yet, little is known about which aspect of the feeding process is disrupted by microplastics. Here, we determine if prior exposure to microplastics alters the feeding behavior of the larval sea urchin Heliocidaris crassispina. We conducted two experiments: a food handling experiment studied larval survival, growth, and time required to fill and vacate the stomach; and a particle selection experiment analyzed changes in the ability of the larvae to selectively ingest algal cells over microplastics. In both experiments, larvae were pre-exposed to algae only (control), the addition of 10 µm polystyrene beads at 1 bead mL-1 or 1000 beads mL-1 until 3- or 7-days post-fertilization. Previous exposure to microplastics lengthened stomach filling time and impaired particle selection. While there was no significant change in survivorship and larval arm length, these sub-lethal impacts on larval feeding likely have more severe ramifications in vivo where food is limited, and thus, potentially threaten post-settlement success.

Expanding the clinical spectrum of biallelic ZNF335 variants.

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in 2 unrelated families. We describe, herein, 2 additional affected individuals with biallelic ZNF335 variants, 1 individual with a homozygous c.1399 T > C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A > G, p.(Glu1333Gly) variants with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra-axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335-associated microcephaly.

Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans.

BACKGROUND: A 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored. METHODS: Oxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle. RESULTS: OXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50-60%). CONCLUSIONS: This patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.

What can the CF registry tell us about rare CFTR-mutations? A Belgian study.

BACKGROUND: CFTR2 provides clinical and functional information of the most common CFTR-mutations. Rare mutations (RMs) occur in only a few patients with limited reported clinical data. Their role in CF-disease liability is hardly documented. METHODS: Belgian CF-Registry 2013 data were analyzed to identify CF with at least 1 RM (CF+RM). Clinical data and sweat chloride of CF+RM were compared to CF-controls, carrying 2 class 1 to 3 mutations (CFclassic). Disease severity was compared between both groups. To avoid bias in the comparison, transplanted patients were excluded from each group. RESULTS: Seventy-seven CF+RM were identified (77/1183 = 6.5%). Sixty-four different RM were detected, of which 21 had not been previously reported. All RMs, corresponding to HGVS (Human Genome Variation Society) nomenclature, were listed in supplementary data. Seven transplanted CF+RM were excluded for further analysis. CF+RM had higher age at diagnosis [median (IQR)] [3.7 y (0.3-18.3) vs. 0.3y (0.1-2,0) (p < 0.0001)], lower sweat chloride [96 mmol/L (64-107) vs. 104 mmol/L (97-115) (p < 0.0001)], higher FEV1%pred [77%pred (58-96) vs. 68%pred (48-86) (p = 0.017)], were less frequently pancreatic insufficient [56% vs. 98% (p < 0.0001)], Pseudomonas aeruginosa colonized [24% vs. 44% (p = 0.0093)] and needed fewer IV antibiotics [36% vs. 51% (p = 0.041)] than CFclassic. However, a wide spectrum of disease severity was seen amongst CF+RM. CONCLUSIONS: CF-patients with a RM cover 6.5% of the Belgian CF-population. Rare mutations can be found in severely ill patients, but more often in late diagnosed, pancreatic sufficient patients.

A new mutation in the calcium-sensing receptor gene causing hypocalcaemia: case report of a father and two sons.

BACKGROUND: Regulation of calcium is mediated by parathyroid hormone (PTH) and 1.25-dihydroxyvitamine D3. The calcium-sensing receptor (CaSR) regulates PTH release by a negative feedback system. Gain-of-function mutations in the CaSR gene reset the calcium-PTH axis, leading to hypocalcaemia. PATIENTS AND METHODS: We analysed a family with hypocalcaemia. The proband was a 47-year-old man (index, patient I1), who presented with paraesthesias in both limbs. He has two sons (patient II1 a nd I I2). The probands' lab results showed: serum calcium of 1.95 mmol/l, albumin 41 g/l, phosphate 0.81 mmol/l and PTH 6.6 ng/l (normal 15-65 ng/l). Based on this analysis, we suspected a hereditary form of hypocalcaemia and performed genetic testing by polymerase chain reaction and Sanger sequencing of the coding regions and intron boundaries of the CaSR gene. Genetic analysis revealed a new heterozygous mutation: c.2195A>G, p.(Asn732Ser) in exon 7. The lab results of patient II1 showed: serum calcium of 1.93 mmol/l, phosphate 1.31 mmol/l, albumin 41 g/l, and PTH 24.3 ng/l. His genotype revealed the same activating mutation and, like his father, he also lost his scalp hair at an early adolescent age. Patient II2 is asymptomatic, and has neither biochemical abnormalities, nor the familial CaSR gene mutation. He still has all his scalp hair. CONCLUSIONS: 1) The c.2195A>G, p.(Asn732Ser) mutation in exon 7 of the CaSR gene leads to hypocalcaemia, and has not been reported before in the medical literature. 2) Possibly, this mutation is linked to premature baldness.

Are AZFb deletions always incompatible with sperm production?

Deletions on the long arm of the Y chromosome are a well-known cause of male infertility and it is generally accepted that deletions involving the AZFb region are not compatible with sperm production. Here, we report on two patients for whom basic diagnostic tests showed a deletion of the AZFb region. Unexpectedly, both patients had some residual sperm production. Subsequently, extension and additional analyses of the AZFb region disclosed an aberrant deletion pattern. Therefore, these results emphasize the need for a detailed and powerful analysis of cases where first-line Yq deletion tests reveal an AZFb deletion. Moreover, our study clearly demonstrated that only a very careful selection of test markers will avoid the pitfall of a 'no further treatment possible' wrongful conclusion.

Detailed molecular characterization of a novel IDS exonic mutation associated with multiple pseudoexon activation.

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome.

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

TUBA1A mutations: from isolated lissencephaly to familial polymicrogyria.

BACKGROUND: Mutations in the TUBA1A gene have been reported in patients with lissencephaly and perisylvian pachygyria. METHODS: Twenty-five patients with malformations of cortical development ranging from lissencephaly to polymicrogyria were screened for mutations in TUBA1A. RESULTS: Two novel heterozygous missense mutations in TUBA1A were identified: c.629A>G (p.Tyr210Cys) occurring de novo in a boy with lissencephaly, and c.13A>C (p.Ile5Leu) affecting 2 sisters with polymicrogyria whose mother presented somatic mosaicism for the mutation. CONCLUSIONS: Mutations in TUBA1A have been described in patients with lissencephaly and pachygyria. We report a mutation in TUBA1A as a cause of polymicrogyria. So far, all mutations in TUBA1A have occurred de novo, resulting in isolated cases. This article describes familial recurrence of TUBA1A mutations due to somatic mosaicism in a parent. These findings broaden the phenotypic spectrum associated with TUBA1A mutations and have implications for genetic counseling.

Methylation of the CpG sites in the myotonic dystrophy locus does not correlate with CTG expansion size or with the congenital form of the disease.

We have studied the methylation status of the sequence 152 nucleotides upstream of the CTG repeat of the DM1 locus in patients' peripheral blood. We used the methylation-sensitive endonucleases SacII, HpaII and HhaI, followed by PCR. This allowed to correlate the methylation status of each CTG allele with its size. Contrary to previous findings, only the SacII site is often but not always differentially methylated among expanded CTG alleles. Importantly, this methylation was not restricted to congenital DM1, nor to large expansions, as it was also present in DM1 patients with a classical phenotype and various expansion sizes. On the other hand, we did not find any methylated alleles on the HhaI and HpaII sites, as was reported by Steinbach et al, which is in line with the results of Shaw and collaborators. The size range of the repeat expansions with methylation was from as small as 300 to as large as 2800 repeats.

Progressive myoclonic epilepsy as an adult-onset manifestation of Leigh syndrome due to m.14487T>C.

BACKGROUND: m.14487T>C, a missense mutation (p.M63V) affecting the ND6 subunit of complex I of the mitochondrial respiratory chain, has been reported in isolated childhood cases with Leigh syndrome (LS) and progressive dystonia. Adult-onset phenotypes have not been reported. OBJECTIVES: To determine the clinical-neurological spectrum and associated mutation loads in an extended m.14487T>C family. METHODS: A genotype-phenotype correlation study of a Belgian five-generation family with 12 affected family members segregating m.14487T>C was carried out. Clinical and mutation load data were available for nine family members. Biochemical analysis of the respiratory chain was performed in three muscle biopsies. RESULTS: Heteroplasmic m.14487T>C levels (36-52% in leucocytes, 97-99% in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100% in leucocytes, 100% in muscle). We found lower mutation loads (between 8 and 35% in blood) in adult patients with clinical features including migraine with aura, Leber hereditary optic neuropathy, sensorineural hearing loss and diabetes mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. INTERPRETATION: m.14487T>C resulted in a broad spectrum of phenotypes in our family. Depending on the mutation load, it caused severe encephalopathies ranging from infantile LS to adult-onset PME with dystonia. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.

Abdominal pain and vomiting as first sign of mitochondrial disease.

We describe a patient in whom abdominal pain and vomiting were the presenting symptoms of Mitochondrial Myopathy Encephalopathy, Lactic Acidosis with Stroke-like episodes syndrome (MELAS). Mitochondrial disorders usually present with neurological symptoms or with myopathic features at any age. Although many patients develop visceral symptoms at a certain moment during the course of the disease, only in a minority of patients these symptoms are the unique presenting ones. The proband was initially diagnosed as having gastro-oesophageal reflux and it was only after detailed clinical history that an underlying metabolic defect was suspected and the molecular defect identified.

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

BACKGROUND: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. AIMS: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. METHODS: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. RESULTS: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. CONCLUSION: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3' untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

Real and expected delivery rates of patients with myotonic dystrophy undergoing intracytoplasmic sperm injection and preimplantation genetic diagnosis.

BACKGROUND: This study aimed to analyse the reproductive outcome of a large cohort of myotonic dystrophy type 1 (DM1) patients undergoing ICSI and PGD. The secondary outcome parameter of this study was ovarian response as a way to express gonadal function in female DM1 patients. METHODS: Prospective cohort study. Real and expected cumulative delivery rates are descriptive. The reproductive outcome per cycle was compared with that of a control group of patients with X-linked recessive disorders. The comparative analysis of ovarian stimulation parameters in the study group versus the control group was carried out using both bivariate (crude) and multivariate (linear regression) analysis. RESULTS: Between 1995 and 2005, 205 cycles of ICSI and PGD were carried out for DM1 in 78 couples. The real cumulative delivery rate (max 6 cycles) overall was 46%. The expected overall cumulative delivery rate was 72%. Multivariate analysis did not show a significant difference in total dose of gonadotrophins used for ovarian stimulation between Group A (in which the female partner was affected) and a control group. CONCLUSIONS: This study shows that ICSI and PGD for DM1 offer good reproductive outcome, both in cumulative terms and per treatment cycle. There is no evidence of impaired gonadal function in female DM1 patients.

Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are important causes of human genetic disease, with mutations in tRNA genes particularly prevalent. In many patients, mutations are heteroplasmic, affecting a population of mtDNA molecules. Establishing the pathogenicity of homoplasmic mitochondrial tRNA (mt-tRNA) mutations, in which the mutation is present in every mtDNA molecule, is extremely difficult. These mutations must conform to specific pathogenic criteria, documenting unequivocally a functional defect of the mutant mt-tRNA. AIMS: To investigate the pathogenic nature of two homoplasmic mt-tRNA(Thr) deletions, m.15940delT (previously reported as pathogenic) and m.15937delA, by assessing the steady state levels of the mutant mt-tRNA in tissue and cell-line samples from six unrelated families, in which affected individuals were thoroughly investigated for mitochondrial DNA disease on the basis of clinical presentations. Rates of de novo mitochondrial protein synthesis were also examined in control and m.15937delA mutant fibroblasts. RESULTS: Our data strongly suggest that both single nucleotide deletions are neutral polymorphisms; no obvious defects were apparent in either steady state mt-tRNA(Thr) levels or rates of mitochondrial protein synthesis. CONCLUSIONS: These findings have important implications for the investigation of other families with suspected mtDNA disease, in particular the requirement to fulfil strict and established pathogenic criteria in order to avoid misattribution of pathogenicity to mt-tRNA variants.