The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line–activating mutations affect the thrombopoietin signaling axis. We have identified 2 ...families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3–human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.
•New germ-line mutations of JAK2 in the kinase domain were identified.•Specificity for MPL and resistance to JAK2 and HSP90 inhibitors was determined.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the ...selection of the patients likely to benefit from specific care.
MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G > T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing.
Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity.
Genetic testing of the proband's unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR.
We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.
Complex cortical malformations associated with mutations in tubulin genes are commonly referred to as "Tubulinopathies". To further characterize the mutation frequency and phenotypes associated with ...tubulin mutations, we studied a cohort of 60 foetal cases. Twenty-six tubulin mutations were identified, of which TUBA1A mutations were the most prevalent (19 cases), followed by TUBB2B (6 cases) and TUBB3 (one case). Three subtypes clearly emerged. The most frequent (n = 13) was microlissencephaly with corpus callosum agenesis, severely hypoplastic brainstem and cerebellum. The cortical plate was either absent (6/13), with a 2-3 layered pattern (5/13) or less frequently thickened (2/13), often associated with neuroglial overmigration (4/13). All cases had voluminous germinal zones and ganglionic eminences. The second subtype was lissencephaly (n = 7), either classical (4/7) or associated with cerebellar hypoplasia (3/7) with corpus callosum agenesis (6/7). All foetuses with lissencephaly and cerebellar hypoplasia carried distinct TUBA1A mutations, while those with classical lissencephaly harbored recurrent mutations in TUBA1A (3 cases) or TUBB2B (1 case). The third group was polymicrogyria-like cortical dysplasia (n = 6), consisting of asymmetric multifocal or generalized polymicrogyria with inconstant corpus callosum agenesis (4/6) and hypoplastic brainstem and cerebellum (3/6). Polymicrogyria was either unlayered or 4-layered with neuronal heterotopias (5/6) and occasional focal neuroglial overmigration (2/6). Three had TUBA1A mutations and 3 TUBB2B mutations. Foetal TUBA1A tubulinopathies most often consist in microlissencephaly or classical lissencephaly with corpus callosum agenesis, but polymicrogyria may also occur. Conversely, TUBB2B mutations are responsible for either polymicrogyria (4/6) or microlissencephaly (2/6).
The molecular basis underlying the clinical variability in symptomatic Duchenne muscular dystrophy (DMD) carriers are still to be precised. We report 26 cases of early symptomatic DMD carriers ...followed in the French neuromuscular network. Clinical presentation, muscular histological analysis and type of gene mutation, as well as X-chromosome inactivation (XCI) patterns using DNA extracted from peripheral blood or muscle are detailed. The initial symptoms were significant weakness (88%) or exercise intolerance (27%). Clinical severity varied from a Duchenne-like progression to a very mild Becker-like phenotype. Cardiac dysfunction was present in 19% of the cases. Cognitive impairment was worthy of notice, as 27% of the carriers are concerned. The muscular analysis was always contributive, revealing muscular dystrophy (83%), mosaic in immunostaining (81%) and dystrophin abnormalities in western blot analysis (84%). In all, 73% had exonic deletions or duplications and 27% had point mutations. XCI pattern was biased in 62% of the cases. In conclusion, we report the largest series of manifesting DMD carriers at pediatric age and show that exercise intolerance and cognitive impairment may reveal symptomatic DMD carriers. The complete histological and immunohistological study of the muscle is the key of the diagnosis leading to the dystrophin gene analysis. Our study shows also that cognitive impairment in symptomatic DMD carriers is associated with mutations in the distal part of the DMD gene. XCI study does not fully explain the mechanisms as well as the wide spectrum of clinical phenotype, though a clear correlation between the severity of the phenotype and inactivation bias was observed.
The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. ...In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs.
X-linked isolated lissencephaly sequence and subcortical band heterotopia are allelic human disorders associated with mutations of doublecortin (DCX), giving both familial and sporadic forms. DCX ...encodes a microtubule-associated protein involved in neuronal migration during brain development. Structural data show that mutations can fall either in surface residues, likely to impair partner interactions, or in buried residues, likely to impair protein stability. Despite the progress in understanding the molecular basis of these disorders, the prognosis value of the location and impact of individual DCX mutations has largely remained unclear. To clarify this point, we investigated a cohort of 180 patients who were referred with the agyria-pachygyria subcortical band heterotopia spectrum. DCX mutations were identified in 136 individuals. Analysis of the parents' DNA revealed the de novo occurrence of DCX mutations in 76 cases 62 of 70 females screened (88.5%) and 14 of 60 males screened (23%), whereas in the remaining cases, mutations were inherited from asymptomatic (n = 14) or symptomatic mothers (n = 11). This represents 100% of families screened. Female patients with DCX mutation demonstrated three degrees of clinical-radiological severity: a severe form with a thick band (n = 54), a milder form (n = 24) with either an anterior thin or an intermediate thickness band and asymptomatic carrier females (n = 14) with normal magnetic resonance imaging results. A higher proportion of nonsense and frameshift mutations were identified in patients with de novo mutations. An analysis of predicted effects of missense mutations showed that those destabilizing the structure of the protein were often associated with more severe phenotypes. We identified several severe- and mild-effect mutations affecting surface residues and observed that the substituted amino acid is also critical in determining severity. Recurrent mutations representing 34.5% of all DCX mutations often lead to similar phenotypes, for example, either severe in sporadic subcortical band heterotopia owing to Arg186 mutations or milder in familial cases owing to Arg196 mutations. Taken as a whole, these observations demonstrate that DCX-related disorders are clinically heterogeneous, with severe sporadic and milder familial subcortical band heterotopia, each associated with specific DCX mutations. There is a clear influence of the individual mutated residue and the substituted amino acid in determining phenotype severity.
Anionic boron‐bridged bisoxazolines (borabox ligands) have been synthesized and characterized in their protonated forms. The ligands are tuneable over a wide range, allowing either alkyl or aryl ...substituents at the oxazoline rings and the central bridging boron atom. The structural parameters of this new ligand type have been investigated by X‐ray analyses of palladium and copper complexes. Electronic properties have been studied by 13C NMR spectroscopy and by DFT calculations on palladium allyl complexes and compared to those of analogous bisoxazoline (box) complexes. Borabox complexes are more electron‐rich at the metal center than their neutral box congeners, and as a consequence of the longer bonds between the bridging atom and the oxazoline rings, their bite angles are larger. Palladium(II) complexes bearing an unsubstituted allyl ligand and homoleptic copper(II) complexes each possess an almost flat chelate ring. NMR analysis of a (1,3‐diphenylallyl)(borabox)palladium complex showed a 92:8 mixture of (syn,syn) and (anti,syn) allyl isomers, in contrast with a previously reported box analogue that existed exclusively in the (syn,syn) form. Comparison of the corresponding crystal structures revealed that the distance between the bisoxazoline and the allyl ligand in the borabox complex is shorter. In the copper‐catalyzed allylic oxidation of cyclohexene and cyclopentene with tert‐butyl perbenzoate, borabox ligands gave results similar—and in some cases superior—to those obtained with analogous box ligands.
Anionic versus neutral: The structural and electronic properties of allylpalladium(II) and ‐copper(II) complexes with borabox and box ligands (see scheme) were compared by NMR spectroscopy, X‐ray analysis and DFT calculations. The data showed borabox ligands to be stronger electron donors than analogous box ligands. (Borabox=boron‐bridged bisoxazoline; box=bisoxazoline.)
Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts ...with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.
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