Screening for genomic rearrangements and methylation abnormalities of the 15q11-q13 region in autism spectrum disorders.

International audienceBACKGROUND: Maternally derived duplications of the 15q11-q13 region are the most frequently reported chromosomal aberrations in autism spectrum disorders (ASD). Prader-Willi and Angelman syndromes, caused by 15q11-q13 deletions or abnormal methylation of imprinted genes, are also associated with ASD. However, the prevalence of these disorders in ASD is unknown. The aim of this study was to assess the frequency of 15q11-q13 rearrangements in a large sample of patients ascertained for ASD. METHODS: A total of 522 patients belonging to 430 families were screened for deletions, duplications, and methylation abnormalities involving 15q11-q13 with multiplex ligation-dependent probe amplification (MLPA). RESULTS: We identified four patients with 15q11-q13 abnormalities: a supernumerary chromosome 15, a paternal interstitial duplication, and two subjects with Angelman syndrome, one with a maternal deletion and the other with a paternal uniparental disomy. CONCLUSIONS: Our results show that abnormalities of the 15q11-q13 region are a significant cause of ASD, accounting for approximately 1% of cases. Maternal interstitial 15q11-q13 duplications, previously reported to be present in 1% of patients with ASD, were not detected in our sample. Although paternal duplications of chromosome 15 remain phenotypically silent in the majority of patients, they can give rise to developmental delay and ASD in some subjects, suggesting that paternally expressed genes in this region can contribute to ASD, albeit with reduced penetrance compared with maternal duplications. These findings indicate that patients with ASD should be routinely screened for 15q genomic imbalances and methylation abnormalities and that MLPA is a reliable, rapid, and cost-effective method to perform this screening

Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism

Evidence against haploinsuffiency of human ataxin 10 as a cause of spinocerebellar ataxia type 10

International audienc

Heterozygous and homozygous JAK2(V617F) states modeled by induced pluripotent stem cells from myeloproliferative neoplasm patients.

JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy

Clonal architecture of patient 1 CD34⁺ cells and origin of the iPS cell lines.

<p>(A) The allele burdens (by NGS) of the mutations and the status of chromosome 20 are indicated in CD3⁺, CD34⁺, iPSa and iPSb cell lines. (B) The <i>JAK2 </i>^V617F/V617F<i>ASXL1</i>^{<i>mut/wt</i>} subclone subsequently acquired new genetic abnormalities (<i>FBX015</i> and <i>MATN2</i>) mutations and an additional abnormal chromosome 20. During reprogramming or cell culture, 2 cell lines were generated: iPSa subclone <i>JAK2 </i>^V617F/V617F<i>ASXL1</i>^{<i>mut/wt</i>} and iPSb subclone <i>JAK2 </i>^V617F/V617F<i>ASXL1 </i>^{<i>mut/wt</i>}<i>FBX015 </i>^{<i>mut/wt</i>}<i>MATN2 </i>^{<i>mut/wt</i>} and <i>JAK2 </i>^V617F/V617F<i>ASXL1 </i>^{<i>mut/wt/wt</i>}<i>MATN2 </i>^{<i>mut/wt</i>} with an additional abnormal chromosome 20.</p

JAK2^V617F induces an increased sensitivity to EPO and TPO

<p>(A) GPA⁺CD41⁺ cells from P1(H), P2(h) or control were plated in methylcellulose in the presence of SCF (25 ng/mL) and increasing concentrations of EPO. EryP colonies were counted 12 days later. (B) The percentage of large EryP (>50 cells per colony) in total EryP was also calculated. Results are the mean ± SEM of 3 independent experiments. (C) Cloning efficiency of GPA⁺CD41⁺ cells for each genotype was calculated (D) GPA⁺ cells were cytokine-deprived overnight in serum-free medium and then seeded in IMDM alone for 4 hours. Cells were then stimulated with 10 U/mL EPO or not. (E) CD41⁺ cells from P1(H), P2(h) or control were plated in plasma clots without or with increasing concentrations of TPO. CFU-MK colonies were counted at day 10 after CD41a indirect staining. Results are the mean ± SEM of 3 independent experiments (*P<0.05). (F) Primary cells from one control and 4 patients (P1(H), P2(h), P3(H) and P4(h)) were grown either with SCF (25 ng/mL) ±EPO (1 U/mL) or with SCF (25 ng/mL) ± TPO (20 ng/mL), and cloned at one progenitor cell/well. The percentage of endogenous erythroid colonies (EEC) or endogenous CFU-MK was calculated for each condition.</p

Impact of different inhibitors on erythroid growth

<p>GPA⁺CD41⁺ cells were plated in methylcellulose with various inhibitors, in the presence of SCF (25 ng/mL), without EPO for P1(H), and with EPO (1 U/mL) for P1(H), P2(h) and control. EryP colonies were counted 12 days later. (A) Ruxolitinib ; (B) TG101348 ; (C) LY294002 ; (D) RAD001 ; (E) AUY922. Results are the mean ± SEM of 3 independent experiments (*P<0.05).</p

Hematopoietic differentiation of iPS cell lines

<p>(A) Cytological analysis showing EryP (red arrows), MK (black arrows), monocytes (green arrows) and granulocytes (blue arrows). (B) iPS cells from P1(H), P2(h) or control were induced for hematopoietic differentiation with EPO (1 U/mL), TPO (20 ng/mL), SCF (25 ng/mL) and IL-3 (10 ng/mL) till day 13. 1x10⁵ cells were plated in semi-solid conditions (both methylcellulose and plasma clots) and hematopoietic progenitors were counted in both conditions at day 12 and 10, respectively. (C) Proportions of each progenitor (CFU-MK, CFU-GM and EryP) was calculated for each iPS cell line. (D) Percentage of alpha and beta locus globins was calculated after qRT-PCR in iPS and ES-derived GPA⁺ cells or adult CD34⁺-derived erythroblasts. (E) Fold changes represent the amplification of CD41⁺ GPA⁺ cells at day 12 into CD41⁺ cells or GPA⁺ cells at day 18. Alternatively, fold changes represent the amplification of CD14⁺ cells between days 15 and 21. (F) Hematopoietic differentiation potential of iPS cells was quantified by plating one TRA-1-81⁺ cell per well in a 96-well plate coated with OP9 stromal cells. The absolute number of CD41⁺, GPA⁺ and CD14⁺ cells in each clone was measured by flow cytometry at day 18 (mean ± SEM, n>10; 2 independent experiments) (Mann Whitney test, two-tailed, * P<0.05, ***P<0.001).</p

Detection of 9 different point mutations of <i>PCDH19</i> in 11 female patients by direct sequencing.

<p>A) Sequence electropherograms of the mutations and the missense variant (c.3319C>G/p.Arg1107Gly) identified in association with the c.859G>T/p.Glu287X nonsense mutation. The mutation nomenclature is based on the <i>PCDH19</i> transcript reference EF676096. Nucleotides are numbered according to the cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to the journal guidelines (<a href="http://www.hgvs.org/mutnomen" target="_blank">www.hgvs.org/mutnomen</a>). B) Alignment of the regions surrounding the mutations (indicated by an arrow) in orthologous and paralogous proteins, showing the high conservation of each affected amino-acid in vertebrates and in the delta protocadherin paralogous genes.</p

Clinical characteristics of patients with <i>PCDH19</i> mutations.

<p>2*: patient N 06 1258 is the sister of patient N 06 1257 (index case); PMD = psychomotor development, Nl = normal, F = febrile, unit = unilateral, GTC = generalized tonic-clonic, W-S = words-sentences, abs = absent, AED = anti epileptic drugs.</p