High incidence of Noonan syndrome features including short stature and pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P<0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients

Congenital thrombocytopenia associated with a heterozygous variant in the MEIS1 gene encoding a transcription factor essential for megakaryopoiesis

The transcription factor MEIS1 (myeloid ectotrophic insertion site 1) is crucial for the maintenance of hematopoietic stem cells and for megakaryopoiesis. Germline variants in MEIS1 are associated with restless-leg syndrome, but were not previously shown to cause cytopenias. This is the first report of a patient with congenital thrombocytopenia associated with a sequence variant in MEIS1, presenting with early onset severe thrombocytopenia and mild signs of bone marrow stress. Whole exome sequencing revealed a de novo monoallelic splice site variant in MEIS1, NM_002398.3:exon4:c.432 + 5 G > C, leading to a premature stop codon. We propose that heterozygous mutations in MEIS1 may cause congenital thrombocytopenia

Pattern of NKLR-ligands on primary melanoma cells.

<p>Figure shows the median fluorescence intensity (MFI) of the binding of various NKLR-Ig fusion proteins to primary melanoma cells, as indicated in the figure. Figure shows a representative experiment.</p

Correlation between NKLR expression prior to and after rapid expansion.

<p>Each NKLR was tested on gated NK cells and is presented in a different panel, as indicated. NK cultures from different donors (N = 7) were tested at day 14 (black squares) and at day 21 (X's) of rapid expansion protocol (REP). The NKLR expression prior to rapid expansion was tested on un-manipulated peripheral blood NK cells. Each dot represents the average of at least three staining experiments. FAB - fold above background.</p

HLA haplotype of melanoma and NK donors.

<p>Table shows the full high resolution HLA typing of Mel008 cells, as well as the healthy NK donors (NK#1 and NK#2) and melanoma NK donors (NKmel#1 and NKmel#2).</p

NKLR profile analysis on gated NK cells.

<p>Figure shows the NKLR analysis of 3 different donors, as indicated in the left. NK cells were defined as CD56⁺CD3⁻ cells, as depicted in the left vertical panel. Gated NK cells were further co-stained for NKG2D and CD16 (central vertical panel) or for NKp46 and NKp30 (right vertical panel). Circles highlight various subpopulations.</p

NK NKLR expression profile in healthy donors vs. melanoma patients.

<p>The expression profile of each NKLR is presented in an independent panel, as indicated. Each dot represents either a healthy donor or a melanoma patient. Blood specimens were collected from 30 healthy donors and 40 stage IV melanoma patients with clinical evidence of disease. Y-axis denotes the percent of NKLR-positive cells out of gated NK cell. Horizontal lines represent median values. P value under each panel was calculated with an aparametric t-test and represents the statistical significance of the difference between median values.</p

Expression of NKLRs on NK cells during activation and expansion.

<p>(A) Ex vivo expansion of peripheral blood NK cells. Figure shows the average results of NK cultures derived from different donors (N = 7). % NK purity was determined by double staining for CD3 and CD56 in flow cytometry. CD3-depleted cells (Day 0) were either activated overnight with 500 IU/ml IL-2 (Day 1) or underwent rapid expansion for 14 (Day 14) or 21 days (Day 21). Fold expansion was calculated relatively to the amount of NK cells in Day 0. (B) NKLR expression during <i>ex vivo</i> expansion. All NK cultures were independently stained for the indicated NKLRs. Expression of NKLRs in each donor was normalized according to the corresponding expression on gated NK cells by the un-manipulated peripheral blood mononuclear cells (PBMC). Figure shows the average of normalized results on all healthy donors tested (N = 7). Error bars represent standard error. * denotes a statistical significance of P value<0.05. ** denotes a statistical significance of P value<0.01.</p

Enhanced expression of NKLRs correlates with improved cytotoxicity against melanoma cells.

<p>(A) KIR expression during ex vivo expansion. All NK cultures were independently stained for the indicated KIRs. Expression of KIRs in each donor was normalized according to the corresponding expression on gated NK cells by the un-manipulated peripheral blood mononuclear cells (PBMC). Figure shows the average of normalized results on all healthy donors tested (N = 7). Error bars represent standard error. * denotes a statistical significance of P value<0.05. (B) The expression of each NKLR was tested on two representative bulk NK cultures on day 1 and day 14 of ex vivo expansion process, as indicated in the figure. Expression is presented as median fluorescence intensity (MFI). (C) NK#1 and (D) NK#2 cultures were tested in killing assays against Mel008 cells in various effector-to-target (E∶T) ratios. NKG2D or/and NKp30 on NK cultures #1 and #2 at day 14 were blocked by pre-incubation with mAb at a concentration of 2 µg/ml or 4 µg/ml, respectively. Figure shows a representative experiment. (E) NK cells with low expression of NKp30 were derived from melanoma patients (NKmel#1 and NKmel#2) and tested in killing assays against Mel008 cells in an E∶T of 20∶1. NKG2D or/and NKp30 were blocked as described above. Figure shows a representative experiment.</p