International audienceGermline loss-of-function mutations in the gene have recently been identified in patients fulfilling the National Institutes of Health (NIH) diagnostic criteria for neurofibromatosis type 1 (NF1) but with no (neurofibromin 1) mutation found, suggesting a neurofibromatosis type 1-like syndrome

Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of B150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with B90h for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

Constitutional dominant loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisted of multiple café-au-lait macules, axillary freckling, learning disabilities and macrocephaly. SPRED1 is a negative regulator of the RAS MAPK pathway and can interact with neurofibromin, the NF1 gene product. Individuals with NF1 have a higher risk of haematological malignancies. SPRED1 is highly expressed in haematopoietic cells and negatively regulates haematopoiesis. SPRED1 seemed to be a good candidate for leukaemia predisposition or transformation. We performed SPRED1 mutation screening and expression status in 230 paediatric lymphoblastic and acute myeloblastic leukaemias (AMLs). We found a loss-of-function frameshift SPRED1 mutation in a patient with Legius syndrome. In this patient, the leukaemia blasts karyotype showed a SPRED1 loss of heterozygosity, confirming SPRED1 as a tumour suppressor. Our observation confirmed that acute leukaemias are rare complications of the Legius syndrome. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority of AMLs at diagnosis compared with normal or paired complete remission bone marrows. SPRED1 decreased expression correlated with genetic features of AML. Our study reveals a new mechanism which contributes to deregulate RAS MAPK pathway in the vast majority of paediatric AMLs.

The wide clinical spectrum of the ABCB4 gene (ATP-binding cassette subfamily B member 4) deficiency syndromes in humans includes low phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptivesinduced cholestasis (CIC), and progressive familial intrahepatic cholestasis type 3 (PFIC3). No ABCB4 mutations are found in a significant proportion of patients with these syndromes. In the present study, 102 unrelated adult patients with LPAC (43 patients) or CIC/ICP (59 patients) were screened for ABCB4 mutations using DNA sequencing. Heterozygous ABCB4 point or short insertion/deletion mutations were found in 37% (16/43) of the LPAC patients and in 27% (16/59) of the ICP/CIC patients. High-resolution gene dosage methodologies were used in the 70 negative patients. Here, we describe for the first time ABCB4 partial or complete heterozygous deletions in 7% (3/43) of the LPAC patients, and in 2% (1/59) of the ICP/CIC patients. Our observations urge to systematically test patients with LPAC, ICP/CIC, and also children with PFIC3 for the presence of ABCB4 deletions using molecular tools allowing detection of gross rearrangements. In clinical practice, a comprehensive ABCB4 alteration-screening algorithm will permit the use of ABCB4 genotyping to confirm the diagnosis of LPAC or ICP/CIC, and allow familial testing. An early diagnosis of these biliary diseases may be beneficial because of the preventive effect of ursodeoxycholic acid on biliary complications. Further comparative studies of patients with well-characterized genotypes (including deletions) and phenotypes will help determine whether ABCB4 mutation types influence clinical outcomes.

Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies.

Dihydropteroate synthase (DHPS) gene mutations have raised concerns about emerging sulfonamide resistance in Pneumocystis jirovecii. DHPS and dihydrofolate reductase (DHFR) gene products were amplified in clinical specimens from South African patients. One of 53 DHPS genes sequenced contained the double mutation Thr55Ala Pro57Ser. DHFR gene mutations detected were Ala67Val and the new mutations Arg59Gly and C278T

Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

BACKGROUND: Children under 1 year of age with hypoxic pneumonia regularly have concurrent cytomegalovirus (CMV) viremia. In these children, the diagnosis of CMV-associated pneumonia and the prediction of an outcome are difficult. It is unclear whether quantification of blood CMV viral load (CMV-VL) can predict outcomes in these children.METHODS: This was a retrospective study including children (1–12 months of age), with detectable CMV-VL and hypoxic pneumonia admitted to the paediatric intensive care unit of Tygerberg Hospital, Cape Town, South Africa between 1 January 2014 and 31 December 2015. Clinical, radiological and biochemical data were collected.RESULTS: Of the 87 participants included (median age: 3.9 months, IQR 2.2–4.8), 35 were (40%) born prematurely. The median weight-for-age Z-score was –2.68 (IQR –3.0 to –0.83); 37 (43%) were severely underweight for age; 27 (31%) were HIV-positive, 3 were on ART. The median CMV-VL was log 4.0 (IQR 3.3–4.79); CMVhigh was defined as CMV-VL > median; CMV-VL < median was classified as CMVlow. Overall survival was 90%; 12 (15.4%) remained oxygen-dependent at Day 28 post-admission. There was no difference in survival, 24-h post-admission ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2), oxygen dependence or ventilation duration between CMVlow and CMVhigh. High-frequency oscillation ventilation duration was longer (P = 0.005) and Pneumocystis jirovecii (PJP) co-infection more frequent (P = 0.018) in CMVhigh.CONCLUSION: CMV-VL is unable to predict the clinical outcome in children with hypoxic pneumonia. Specific treatment for CMV should be considered in all children at risk of CMV-associated pneumonia with detectable CMV-VL.