A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

Epigenetic clock as a correlate of anxiety.

DNA methylation changes consistently throughout life and age-dependent alterations in DNA methylation can be used to estimate one's epigenetic age. Post-mortem studies revealed higher epigenetic age in brains of patients with major depressive disorder, as compared with controls. Since MDD is highly correlated with anxiety, we hypothesized that symptoms of anxiety, as well as lower volume of grey matter (GM) in depression-related cortical regions, will be associated with faster epigenetic clock in a community-based sample of young adults. Participants included 88 young adults (53% men; 23-24 years of age) from the European Longitudinal Study of Pregnancy and Childhood (ELSPAC) who participated in its neuroimaging follow-up and provided saliva samples for epigenetic analysis. Epigenetic age was calculated according to Horvath (Horvath, 2013). Women had slower epigenetic clock than men (Cohen's d = 0.48). In women (but not men), slower epigenetic clock was associated with less symptoms of anxiety. In the brain, women (but not men) with slower epigenetic clock had greater GM volume in the cerebral cortex (brain size-corrected; R2 = 0.07). Lobe-specific analyses showed that in women (but not men), slower epigenetic clock was associated with greater GM volume in frontal lobe (R2 = 0.16), and that GM volume in frontal lobe mediated the relationship between the speed of epigenetic clock and anxiety trait (ab = 0.15, SE = 0.15, 95% CI [0.007; 0.369]). These findings were not replicated, however, in a community-based sample of adolescents (n = 129; 49% men; 12-19 years of age), possibly due to the different method of tissue collection (blood vs. saliva) or additional sources of variability in the cohort of adolescents (puberty stages, socioeconomic status, prenatal exposure to maternal smoking during pregnancy).

A novel germline mutation of the SFTPA1 gene in familial interstitial pneumonia.

Different genes related to alveolar stability have been associated with familial interstitial pneumonia (FIP). Here, we report a novel, rare SFTPA1 variant in a family with idiopathic interstitial pneumonia (IIP). We performed whole-exome sequencing on germline DNA samples from four members of one family; three of them showed signs of pulmonary fibrosis (idiopathic interstitial pneumonia) with autosomal-dominant inheritance. A heterozygous single nucleotide variant c.532 G > A in the SFTPA1 gene has been identified. This variant encodes the substitution p.(Val178Met), localized within the carbohydrate recognition domain of surfactant protein A and segregates with the genes causing idiopathic interstitial pneumonia. This rare variant has not been previously reported. We also analyzed the detected sequence variant in the protein structure in silico. The replacement of valine by the larger methionine inside the protein may cause a disruption in the protein structure. The c.532 G > A variant was further validated using Sanger sequencing of the amplicons, confirming the diagnosis in all symptomatic family members. Moreover, this variant was also found by Sanger sequencing in one other symptomatic family member and one young asymptomatic family member. The autosomal-dominant inheritance, the family history of IIP, and the evidence of a mutation occurring in part of the SFTPA1 gene all suggest a novel variant that causes FIP.

C-terminal RUNX1 mutation in familial platelet disorder with predisposition to myeloid malignancies.

Here we report a C-terminal RUNX1 mutation in a family with platelet disorder and predisposition to myeloid malignancies. We identified the mutation c.866delG:p.Gly289Aspfs*22 (NM_001754) (RUNX1 b-isoform NM_001001890; c.785delG:p.Gly262Aspfs*22) using exome sequencing of samples obtained from eight members of a single family. The mutation found in our pedigree is within exon eight and the transactivation domain of RUNX1. One of the affected individuals developed myelodysplastic syndrome (MDS), which progressed to acute myelogenous leukemia (AML). A search for the second hit which led to the development of MDS and later AML in this individual revealed the PHF6 gene variant (exon9:c.872G > A:p.G291E; NM_001015877), BCORL1 (exon3:c.1111A > C:p.T371P; NM_001184772) and BCOR gene variant (exon4:c.2076dupT:p.P693fs; NM_001123383), which appear to be very likely second hits participating in the progression to myeloid malignancy.

Complex analysis of the p53 tumor suppressor in lung carcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.

The origin of deletion 22q11 in chronic lymphocytic leukemia is related to the rearrangement of immunoglobulin lambda light chain locus.

The technology of array comparative genomic hybridization (array-CGH/aCGH) enabled the identification of novel genomic aberrations in chronic lymphocytic leukemia (CLL) including the monoallelic and biallelic deletions affecting 22q11 locus. In contrast to previous publications, we hypothesized that the described 22q11 deletions are a consequence of the rearrangement of immunoglobulin lambda light chain locus (IGL) segments surrounding several protein-coding genes located in this region. Indeed, using array-CGH and PCR analysis we show that all deletions (n=7) affecting the 22q11 locus in our cohort (n=40) are based on the physiological mechanism of IGL rearrangement. This demonstrates that this loss of genetic material is likely not pathogenic and in fact is merely a marker of IGL rearrangement.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia.

MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.

Gene expression profiling in follicular lymphoma and its implication for clinical practice.

Follicular lymphoma (FL) is characterized by an indolent and relapsing course. Recently, the clinical outcome of FL has been distinguished by immune microenvironment-associated gene signatures. In our study, gene expression profiling (GEP) was performed in 31 non-selected patients with follicular lymphoma (FL), 12 of whom were in relapse and the remaining 19 newly diagnosed. A custom oligonucleotide microarray (Agilent 8 × 15K) was used which contained probes for about 3500 genes, including those that had been previously published as demonstrating significant prognostic value. An unsupervised approach was not able to recognize clinically different FLs. As the previously published prognostically relevant gene signatures could not be properly verified, probably due to microarray platform differences, template matching was therefore used in order to define two gene sets with differential gene expression among our samples. These gene sets shared an overrepresentation of genes with similar biological functions and were termed 'T-CELL' and 'PROLIFERATION' profiles. The 'poor profile' was then defined by a high PROLIFERATION score (upper tertile) and/or low T-CELL score (lower tertile). The 'poor profile' cohort contained a significantly higher proportion of relapsed cases (p < 0.05, Fisher's exact test). Additionally, a comparison of samples from initial diagnosis and from relapse showed significant differences mainly in the T-CELL profile (p = 0.036; χ(2)). This supports the hypothesis that the number of T-cells and their expression pattern play a major role in FL development.

Inactivation of p53 and amplification of MYCN gene in a terminal lymphoblastic relapse in a chronic lymphocytic leukemia patient.

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. A proportion of patients eventually progress to a higher stage of malignancy. A recent association has been observed between the presence of aberrant somatic hypermutations in leukemic cells (hypermutations occurring outside of the immunoglobulin locus) and the transformation to a diffuse large B-cell lymphoma or prolymphocytic leukemia. In this study, we report on the rarely observed blastic transformation in a CLL patient who had previously been shown to harbor aberrant somatic hypermutations in the TP53 tumor-suppressor gene (Mol Immunol 2008;45:1525-29). The enzyme responsible, the activation-induced cytidine deaminase, was still active within the transformation, as evidenced by the ongoing class-switch recombination of cytoplasmic immunoglobulins. The transformation was accompanied by a complete p53 inactivation, as well as complex karyotype changes including prominent amplification of MYCN oncogene. Our case-study supports the view that the aberrant somatic hypermutation is associated with transformation of CLL to a more aggressive malignancy.