Frameshift mutation hotspot identified in Smith-Magenis syndrome: case report and review of literature

Smith-Magenis syndrome (SMS) is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1) gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC) in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other), resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels) and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident

Establishing and validating noninvasive prenatal testing procedure for fetal aneuploidies in Vietnam

Noninvasive prenatal testing (NIPT) for fetal aneuploidies has been widely adopted in developed countries. Despite the sharp decrease in the cost of massively parallel sequencing, the technical know-how and skilled personnel are still one of the major limiting factors for applying this technology to NIPT in low-income settings. Here, we present the establishment and validation of our NIPT procedure called triSure for detection of fetal aneuploidies.We established the triSure algorithm based on the difference in proportion of fetal and maternal fragments from the target chromosome to all chromosomes. Our algorithm was validated using a published data set and an in-house data set obtained from high-risk pregnant women in Vietnam who have undergone amniotic testing. Several other aneuploidy calling methods were also applied to the same data set to benchmark triSure performance.The triSure algorithm showed similar accuracy to size-based method when comparing them using published data set. Using our in-house data set from 130 consecutive samples, we showed that triSure correctly identified the most samples (overall sensitivity and specificity of 0.983 and 0.986, respectively) compared to other methods tested including count-based, sized-based, RAPIDR and NIPTeR.We have demonstrated that our triSure NIPT procedure can be applied to pregnant women in low-income settings such as Vietnam, providing low-risk screening option to reduce the need for invasive diagnostic tests

8-chloro-adenosine activity in FLT3-ITD acute myeloid leukemia

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD (+) MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.Peer reviewe

White-to-brown metabolic conversion of human adipocytes by JAK inhibition

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of novel therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus Kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a novel role for the JAK/STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity

Sequence-Based Genotyping for Marker Discovery and Co-Dominant Scoring in Germplasm and Populations

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike

Molecular Characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and Impact of T-Cell Epitope Mutations on HLA Recognition (ANRS 12159)

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score). with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area

An Outbreak of Severe Infections with Community-Acquired MRSA Carrying the Panton-Valentine Leukocidin Following Vaccination

Background: Infections with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging worldwide. We investigated an outbreak of severe CA-MRSA infections in children following out-patient vaccination. Methods and Findings: We carried out a field investigation after adverse events following immunization (AEFI) were reported. We reviewed the clinical data from all cases. S. aureus recovered from skin infections and from nasal and throat swabs were analyzed by pulse-field gel electrophoresis, multi locus sequence typing, PCR and microarray. In May 2006, nine children presented with AEFI, ranging from fatal toxic shock syndrome, necrotizing soft tissue infection, purulent abscesses, to fever with rash. All had received a vaccination injection in different health centres in one District of Ho Chi Minh City. Eight children had been vaccinated by the same health care worker (HCW). Deficiencies in vaccine quality, storage practices, or preparation and delivery were not found. Infection control practices were insufficient. CA-MRSA was cultured in four children and from nasal and throat swabs from the HCW. Strains from children and HCW were indistinguishable. All carried the Panton-Valentine leukocidine (PVL), the staphylococcal enterotoxin B gene, the gene complex for staphylococcal-cassette-chromosome mec type V, and were sequence type 59. Strain HCM3A is epidemiologically unrelated to a strain of ST59 prevalent in the USA, althoughthey belong to the same lineage. Conclusions. We describe an outbreak of infections with CA-MRSA in children, transmitted by an asymptomatic colonized HCW during immunization injection. Consistent adherence to injection practice guidelines is needed to prevent CA-MRSA transmission in both in- and outpatient settings