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Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by a period of stagnation followed by regression in the development of young girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with features suggestive, but not diagnostic of RTT. Long distance PCR coupled with long-read direct sequencing was employed to sequence the entire MECP2 gene coding region in all cases. Mutations were identified in 44/55 (80%) unrelated classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Twenty-one different mutations were identified (12 missense, four nonsense and five frame-shift mutations); 14 of these were novel. All missense mutations were located either in the methyl-CpG-binding domain or in the transcription repression domain. Nine recurrent mutations were characterized in a total of 33 unrelated cases (73% of all cases with MECP2 mutations). Significantly milder disease was noted in patients carrying missense mutations as compared with those with truncating mutations ( P = 0. 0023), and milder disease was associated with late as compared with early truncating mutations ( P = 0.0190).

The frequency of breakthrough invasive fungal diseases (IFDs) during echinocandin therapy is unclear. We retrospectively analyzed 534 hematologic patients treated with echinocandin (caspofungin, N = 55; micafungin, N = 306; anidulafungin, N = 173). Four proven IFDs were found, caused by Candida parapsilosis (N = 2), C. parapsilosis and Candida glabrata (N = 1), and Fusarium species (N = 1). Four cases of possible IFDs were observed, all showing pulmonary infection. One case showed features suggestive of hepatosplenic candidiasis. Six of these eight cases had previously received the purine analog clofarabine. Breakthrough IFD during echinocandin treatment occurred infrequently (1.5 %), caused predominantly by Candida species. Clofarabine usage was an important risk factor.

Clinical outcome and mutations of 96 core-binding factor acute myeloid leukemia (AML) patients 18–60 years old were examined. Complete remission (CR) after induction was 94.6%. There was no significant difference in CR, leukemia-free-survival (LFS) and overall survival (OS) between t(8;21) (N=67) and inv(16) patients (N=29). Univariate analysis showed hematopoietic stem cell transplantation at CR1 as the only clinical parameter associated with superior LFS. Next-generation sequencing based on a myeloid gene panel was performed in 72 patients. Mutations in genes involved in cell signaling were associated with inferior LFS and OS, whereas those in genes involved in DNA methylation were associated with inferior LFS. KIT activation loop (AL) mutations occurred in 25 patients, and were associated with inferior LFS (P=0.003) and OS (P=0.001). TET2 mutations occurred in 8 patients, and were associated with significantly shorter LFS (P=0.015) but not OS. Patients negative for KIT-AL and TET2 mutations (N=41) had significantly better LFS (PP=0.012) than those positive for both or either mutation. Multivariate analysis showed that KIT-AL and TET2 mutations were associated with inferior LFS, whereas age ⩾40 years and marrow blast ⩾70% were associated with inferior OS. These observations provide new insights that may guide better treatment for this AML subtype.

We report a 32 year-old Chinese lady with history of tetralogy of Fallot, presented to us with chest pain due to hypocalcemia secondary to hypoparathyroidism. With her dysmorphic facial features and intellectual disability 22q11.2 deletion was suspected and confirmed by genetic study. Clinicians should consider the diagnosis of DiGeorge syndrome in adult patient with past medical history of congenital heart disease, facial dysmorphism, intellectual disability and primary hypoparathyroidism.

Familial occurrence of distal foregut atresia (DFA) (Type 1) is rare. Diagnosis is based upon the clinical symptomatology and confirmed by radiological studies, surgery and histology. A number of reports have described families in which several family members have been involved and suggested an autosomal recessive mode of inheritance. Little is known about the underlying genetic causes or indeed the likely pathogenic mechanism. We report a family in which there are five affected cases including three siblings where the DFA appears to be inherited in an autosomal dominant inheritance pattern with reduced penetrance.

A 55-year-old woman with myelodysplastic syndrome was admitted because of pyrexia and bilateral lower limb muscle pain. The creatine kinase (CK) was 1152 U/l (reference range: 40–161 U/l). Urine for myoglobin was positive. Blood culture grew methicillin-resistant Staphylococcus aureus (MRSA), sensitive to vancomycin, fusidic acid, clindamycin and erythromycin. She was treated with intravenous vancomycin (1 g every 12 h). Computed tomography (CT) of pelvis and lower limbs showed inflammatory changes in the thighs. She was discharged after 7 days of vancomycin treatment, with the CK normal and blood culture negative. She was re-admitted a week later because of recurrent pyrexia. There were multiple indurated lesions over her right lateral thigh, right forearm, left calf and the …

Introduction and aim. Relapse following allogeneic hematopoietic stem cell transplantation (HSCT) is a major cause of treatment failure and is associated with a poor prognosis. Overall survivals are around 50% at 5 years following allogeneic HSCT in intermediate and high risk AML. Survivals remain less than 20% in poor-risk and very poor-risk patients based on the cytogenetic profile. Thus, prevention of relapse following allogeneic HSCT remains an unmet clinical need. Low-dose azacitidine maintenance post-HSCT has been shown to augment graft-versus-leukemia effect and may prolong survivals. We aim to prospectively evaluate the effect of azacitidine maintenance following allogeneic HSCT in high risk AML and MDS. Method. Consecutive patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in remission following first allogeneic HSCT or second allogeneic HSCT (from the original donor) were recruited. High risk AML in this study comprised patients with poor risk karyotype, secondary AML transformed from underlying MDS, presence of fms-like tyrosine kinase 3-internal tandem duplication (FLT3 -ITD) and non-remission before HSCT. Azacitidine was administered at 100mg daily for 3 days per cycle every 28 days until progression or a maximum of 8 cycles. The clinicopathologic and treatment characteristics were determined. The occurrence of graft-versus-host disease (GVHD) was determined. DNA chimerism was determined in the bone marrow before the initiation of azacitidine, after 4th and 8th cycles of azacitidine and at 1 year. DNA chimerism was determined by quantification of polymorphic short tandem repeat sequences. The progression-free survival (PFS) and overall survival (OS) were determined by Kaplan-Meier analysis. Results. Thirty-four patients with high-risk AML (N=31) and MDS (N=3) were recruited. The median duration of follow-up was 14 months (range: 2 - 44 months). Twenty-two patients received azacitidine maintenance after first allogeneic HSCT, whereas 12 patients received azacitidine maintenance after a second allogeneic HSCT from the same donor following relapse from a first allogeneic HSCT For patients receiving azacitidine after first HSCT, at a median follow-up of 18.5 months (range: 5- 36 months), the median PFS was not reached, and the median OS was 32 months (95% confidence interval [C.I.]: 24.85-39.15). The 24-month PFS and OS were 66.1% and 73.2% respectively. Acute and chronic GVHD occurred in 7 (31.8%) and 17 patients (77%). For patients receiving azacitidine after second HSCT, at a median follow-up of 14 months (range: 9 - 46 months), the median PFS and OS were 9 months (95% C.I.:6.94-11.04) and 14 months (range: 11.77 - 16.23 months). The 24-month PFS and OS were 25% and 14% respectively. Acute and chronic GVHD occurred in 1 (8.3%) and 5 (41.7%) patients respectively. In both groups, 100% donor chimerism was achieved during azacitidine maintenance. Conclusion. Azacitidine maintenance following first allogeneic HSCT resulted in favorable 2-year survivals in selected patients with high-risk AML and MDS. Nevertheless, survivals were poor despite azacitidine maintenance after second allogeneic HSCT from the same donor. Full donor chimerism was maintained during azacitidine maintenance. Disclosures No relevant conflicts of interest to declare.

Introduction Mutations in the family of SCN genes encoding sodium channels are responsible for several disorders affecting the central and peripheral nervous systems and muscle. Disease arising from sodium channel mutants range from the relatively benign (e.g. mild myotonia) to the fatal (e.g. long-QT syndrome), with a wide variety of disorders spanning the spectrum of severity. Identified SCN4a mutations to date have been consistently autosomal dominant and associated with paramyotonia congenita, potassium-mediated periodic paralysis or aggravated myotonia due to defects altering the biophysical properties of sodium channels that mediate membrane hyper- or hypo-excitability. Here we describe a newly recognised autosomal-recessive syndrome comprising severe congenital hypotonia with respiratory failure in a family of Punjabi descent, with 2 of 3 children affected. Methods and results Using whole exome sequencing we identified two new mutations (g. 62025363 C >T, D1069N and g. 62025425 T >G, splice site) in the SCN4A gene, confirmed via Sanger sequencing. Reverse transcriptase polymerase chain reaction shows that the splice-site mutation in SCN4A leads to altered RNA. To investigate the impact of the missense mutation, c.3205G >A, Chinese hamster ovary (CHOk1) cells transfected with either a WT or D1069N SCN4A were examined for their biophysical properties. A set of depolarizing test pulses was used to measure the voltage dependence of activation and indicated biophysical changes in the encoded voltage-gated sodium channel (NaV1.4). Conclusions Together, our findings characterise the first reported evidence of an autosomal recessive SCN4a sodium channelopathy comprising severe congenital neuromuscular hypotonia and respiratory failure with biophysical dysfunction of NaV1.4 attributable to SCN4a compound heterozygous gene mutation.

Npm1 +/− heterozygous mice develop a haematological disorder with features resembling human myelodysplastic syndrome (MDS). Promoter hypermethylation of the NPM1 gene may lead to suppressed gene transcription and hence functional haploinsufficiency, which contributes to the development of MDS. Thirty-one patients with MDS and eight normal individuals were studied for promoter methylation and mRNA expression of NPM1 . Methylation-specific PCR (MSP), COBRA and bisulfite sequencing were used to examine the NPM1 methylation status. Quantitative PCR was used to assess the expression of NPM1 . NPM1 DNA methylation was rare, occurring in one of 31 cases as determined by MSP. There was no significant difference in NPM1 mRNA expression between MDS and normal blood samples. In conclusion, the finding suggests that NPM1 methylation is rare in MDS and does not play a major role in its pathogenesis.

Internal tandem duplication (ITD) of FLT3 (fms-like tyrosine kinase 3) in acute myeloid leukemia (AML) is associated with inferior clinical outcome. Sorafenib is effective in targeting chemo-refractory FLT3-ITD+ AML. However, leukemia progresses invariably. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), which activate a sodium/hydrogen exchanger 1 (NHE1) and known to be up-regulated at leukemia progression during continuous sorafenib treatment, may underlie TKI resistance. TESC was over-expressed in FLT3-ITD+ AML lines MOLM-13 and MV4-11. Knocking down TESC by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene) amiloride (HMA). Sorafenib resistant MOLM-13 cell line (M13-RE) was generated by long term culture of sorafenib. M13-RE significantly increased its sensitivity to HMA. HMA also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. Treating MOLM-13, MV4-11 and primary FLT3-ITD+ AML cells with HMA significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of the TESC-NHE1-pHi axis in mediating sorafenib resistance in AML. Disclosures: No relevant conflicts of interest to declare.

Approximately 20 percent of patients with acute promyelocytic leukemia (APL) relapse after initial remission. More than 80 percent of these patients achieve a second complete remission (CR2). Autologous hematopoietic stem cell transplantation (HSCT) at CR2 is a widely accepted post-remission strategy. However, only a few prospective trials have examined the optimal post-remission therapy for relapsed APL. In this study, we prospectively evaluated the outcome of 65 patients with APL in their first relapse (R1) treated with oral arsenic trioxide (As2O3) and chemotherapy-based re-induction followed by oral arsenic trioxide-based maintenance. Sixty-five patients (men=37; women=28) at a median age of 39 (3-76) years were treated with oral As2O3, all-trans retinoic acid (ATRA), and ascorbic acid combined with idarubicin (6mg/m2/day for 5 days) for re-induction. On achieving CR2, 2 cycles of idarubicin consolidation (6mg/m2/day for 2 days each cycle) were given. That was followed by oral As2O3-based maintenance (2 weeks every 2 months) for 2 years. The median duration of follow-up was 90 (21–378) months. All evaluable patients achieved CR2. Twenty-one patients (32.8%) subsequently had a second relapse (R2). On multivariate analysis, male gender was associated with an increased risk of second relapse (P=0.01). The age, white blood cell (WBC) count, platelet count, peak WBC during reinduction and the occurrence of differentiation syndrome did not impact on the risk of second relapse. The median overall survival (OS) was not reached. The 5-year and 10-year OS were 82.2% and 74.4% respectively. Significantly inferior OS was associated with male gender (P=0.02), prior oral As2O3-based maintenance at CR1 (P=0.01) and central nervous system (CNS) involvement at relapse (P In conclusion, our study showed that durable remissions and long-term survival were achieved with oral As2O3-based maintenance without the need of autologous HSCT at CR2. Disclosures: No relevant conflicts of interest to declare.

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoproliferative disorder, and relapsed/refractory disease has a poor prognosis. In patients with relapsed / refractory MCL, optimal treatment strategy remains undefined. Oral arsenic trioxide (As2O3) was initially developed for the treatment of relapsed acute promyelocytic leukemia (APL). As2O3 inhibits neoplastic cellular proliferation by a wide array of mechanisms, including induction of apoptosis, targeting of signaling pathways, and down-regulation of BCL-2. Evidence in vitro also suggested that As2O3 might be effective in lymphoma, but clinical data are hitherto not available. In this study, we investigated the use of an oral-As2O3-based regimen for the treatment of patients with relapsed / refractory MCL. Thirty-nine patients (men=34, women=5) at 64 (41–82) years of age with relapsed/refractory MCL, who had received 2 (1–5) prior regimens and were ineligible for high-dose chemotherapy, were treated with a continuous oral regimen, comprising oral arsenic trioxide (oral-As2O3), chlorambucil and ascorbic acid. The overall response rate was 49% (complete response: 28%; partial response: 21%). Only grade 1/2 toxicities were observed (hematologic: 56%, hepatic: 8%). Independent prognostic factors for response were increased lactate dehydrogenase (P=0.04) and unfavorable MCL international prognostic index (P=0.04). At a median follow up of 21(range:1-118) months, the median progression-free-survival (PFS) was 16 months, and overall-survival (OS) 38 months. The 2-year and 5-year PFS were 41% and 29% respectively. The 2-year and 5-year OS were 56% and 43% respectively. Independent prognostic factors for PFS were female gender (P=0.002), Eastern Cooperative Oncology Group (ECOG) performance score of 2 (P=0.009), and non-response to treatment (P Disclosures: No relevant conflicts of interest to declare.

Seckel syndrome (MIM 210600) is an autosomal recessive disorder of low birth weight, severe microcephaly, and dysmorphic facial appearance with receding forehead, prominent nose, and micrognathia. We have performed a genomic screen in two consanguineous families of Pakistani origin and found that the disorder segregates with markers between loci D3S1316 and D3S3710, which map to chromosome 3q22.1-q24. Analysis using HOMOZ/MAPMAKER gave a maximum LOD score of 8.72. All five affected individuals were homozygous for the same allele, for two adjacent polymorphic markers within the region segregating with the disease, narrowing the region to 12 cM.

A 70-year-old woman presented with a 4-month history of right leg ulcers (upper left panel). Biopsy showed infiltration by abnormal medium to large size lymphoid cells (upper left middle panel, haematoxylin eosin). There was no obvious angiocentricity or angioinvasion. The abnormal lymphoid cells were positive for cytoplasmic CD3 epsilon (upper right middle panel, immunoperoxidase), CD56 (upper right panel), and T-cell intracellular antigen-1. In-situ hybridization for EpsteinBarr virus encoded RNA was positive. Overall features were consistent with extranodal natural killer (NK)/T-cell lymphoma, nasal type. She was treated with chemotherapy. After the second course, blistering skin eruptions occurred over the trunk, buttocks and legs (lower left panel). A skin biopsy revealed erythema multiforme-like areas with scattered cytoid bodies (white arrow) throughout all levels of the epidermis, and areas with pemphigus vulgaris-like changes showing early suprabasal acantholysis (black arrow) (lower right panel). Direct immunofluorescence studies showed weak intercellular and basement membrane staining with both C3 and IgG, confirming the diagnosis of paraneoplastic pemphigus (PNP). She deteriorated progressively and died 3 months after presentation despite immunosuppression and further chemotherapy. Paraneoplastic pemphigus characteristically presents as painful oral and mucosal ulcerations, with concomitant skin involvement simulating pemphigus vulgaris or erythema multiforme. Bronchiolitis obliterans may occur and can be fatal. PNP is caused by serum autoantibodies against epithelial proteins, particularly the plakin family, leading to epithelial injury in the skin, and mucosal and bronchial linings. Therefore, the underlying neoplasms are almost exclusively of B-cell lineage. However, T-cells and CD56+ NK cells have also been postulated to be effectors of PNP. This case illustrated the rare association between NK cell lymphoma and PNP, suggesting therefore that NK cells might be involved in the pathogenesis of PNP. Clinicians should also be alert to the possibility of PNP in lymphomas not of B-cell lineage.

Individuals with severe, undiagnosed developmental disorders (DDs) are enriched for damaging de novo mutations (DNMs) in developmentally important genes. We exome sequenced 4,293 families with individuals with DDs, and meta-analysed these data with published data on 3,287 individuals with similar disorders. We show that the most significant factors influencing the diagnostic yield of de novo mutations are the sex of the affected individual, the relatedness of their parents and the age of both father and mother. We identified 94 genes enriched for damaging de novo mutation at genome-wide significance (P < 7 × 10−7), including 14 genes for which compelling data for causation was previously lacking. We have characterised the phenotypic diversity among these genetic disorders. We demonstrate that, at current cost differentials, exome sequencing has much greater power than genome sequencing for novel gene discovery in genetically heterogeneous disorders. We estimate that 42% of our cohort carry pathogenic DNMs (single nucleotide variants and indels) in coding sequences, with approximately half operating by a loss-of-function mechanism, and the remainder resulting in altered-function (e.g. activating, dominant negative). We established that most haplo insufficient developmental disorders have already been identified, but that many altered-function disorders remain to be discovered. Extrapolating from the DDD cohort to the general population, we estimate that developmental disorders caused by DNMs have an average birth prevalence of 1 in 213 to 1 in 448 (0.22-0.47% of live births), depending on parental age.AbbreviationsPTVProtein-Truncating VariantDNMDe Novo MutationDDDevelopmental DisorderDDDDeciphering Developmental Disorders study

published_or_final_version, Springer Open Choice, 21 Feb 2012