Congenital neutropenia: From lab bench to clinic bedside and back.

Neutropenia is a hematological condition characterized by a decrease in absolute neutrophil count (ANC) in peripheral blood, typically classified in adults as mild (1-1.5 × 109/L), moderate (0.5-1 × 109/L), or severe (< 0.5 × 109/L). It can be categorized into two types: congenital and acquired. Congenital severe chronic neutropenia (SCN) arises from mutations in various genes, with different inheritance patterns, including autosomal recessive, autosomal dominant, and X-linked forms, often linked to mitochondrial diseases. The most common genetic cause is alterations in the ELANE gene. Some cases exist as non-syndromic neutropenia within the SCN spectrum, where genetic origins remain unidentified. The clinical consequences of congenital neutropenia depend on granulocyte levels and dysfunction. Infants with this condition often experience recurrent bacterial infections, with approximately half facing severe infections within their first six months of life. These infections commonly affect the respiratory system, digestive tract, and skin, resulting in symptoms like fever, abscesses, and even sepsis. The severity of these symptoms varies, and the specific organs and systems affected depend on the genetic defect. Congenital neutropenia elevates the risk of developing acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS), particularly with certain genetic variants. SCN patients may acquire CSF3R and RUNX1 mutations, which can predict the development of leukemia. It is important to note that high-dose granulocyte colony-stimulating factor (G-CSF) treatment may have the potential to promote leukemogenesis. Treatment for neutropenia involves antibiotics, drugs that boost neutrophil production, or bone marrow transplants. Immediate treatment is essential due to the heightened risk of severe infections. In severe congenital or cyclic neutropenia (CyN), the primary therapy is G-CSF, often combined with antibiotics. The G-CSF dosage is gradually increased to normalize neutrophil counts. Hematopoietic stem cell transplants are considered for non-responders or those at risk of AML/MDS. In cases of WHIM syndrome, CXCR4 inhibitors can be effective. Future treatments may involve gene editing and the use of the diabetes drug empagliflozin to alleviate neutropenia symptoms.

A multistep approach to the genotype-phenotype analysis of Polish patients with tuberous sclerosis complex.

The aim of this study was to evaluate a cost-effective diagnostic strategy for identification of casual variants for tuberous sclerosis complex (TSC) in the Polish population and to correlate the genetic results with selected phenotypic features. Fifty-five patients, aged 3-44 years, with a clinical diagnosis of TSC were enrolled into the study. All patients received a three-step analysis: next generation sequencing screening (NGS), multiplex ligation-dependent probe amplification (MLPA) and deep sequencing. This multistep approach obtained positive results in 51/55 (93%) patients: of the 51 positives TSC1 variants were observed in 16 (31%) and TSC2 variants in 35 (69%); these included 13 novel variants and two patients with mosaicism. Four patients (7%) had no mutation identified (NMI). Among the TSC1 gene variants, there were five nonsense, four frameshift, three large deletions, two missense and two splicing variants. For the TSC2 gene, 11 were missense, eight splicing, six frameshift, four large deletions, two in-frame deletions and four nonsense variants. The patients with TSC2 changes had their clinical diagnosis of TSC at a younger age than those with TSC1 changes (one year vs three years, p = 0.041). The TSC2 group demonstrated a higher number of major symptoms per patient (p = 0.04). Subependymal giant cell astrocytoma with concomitance of other brain lesions was more common in patients with missense mutations in either gene (23% vs 0%, p = 0.02). Such a multistep molecular diagnostic strategy could increase the possibility of detecting causal variants for TSC and may allow detection of mosaicism at low levels. Missense pathogenic variants in TSC1 or TSC2 gene might be associated with a higher risk of brain lesions.

Clinical and molecular characterization of craniofrontonasal syndrome: new symptoms and novel pathogenic variants in the EFNB1 gene.

BACKGROUND: Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder that results from pathogenic variants in the EFNB1 gene. The syndrome paradoxically presents with greater severity of the symptoms in heterozygous females than hemizygous males. RESULTS: We have recruited and screened a female cohort affected with CFNS. Our primary finding was the description of monozygotic twins, i.e., patients 5 and 6, discordant for the CFNS phenotype. Intriguingly, patient 5 presented classical CFNS gestalt, whereas patient 6 manifested only very subtle craniofacial features, not resembling CFNS. Besides, we have expanded the mutational spectrum of the EFNB1 gene through reporting four novel pathogenic variants-p.(Trp12*), p.(Cys64Phe), p.(Tyr73Metfs*86), p.(Glu210*). All those alterations were found applying either targeted NGS of a custom gene panel or PCR followed by Sanger sequencing and evaluated using in silico predictors. Lastly, we have also expanded the CFNS phenotypic spectrum by describing in patient 3 several novel features of the syndrome, such as bifid hallux, bicornuate uterus, and abnormal right ovary segmented into six parts. CONCLUSIONS: We have described the unreported so far differences of the clinical phenotype in the monozygotic twin patients 5 and 6 harboring an identical p.(Glu210*) variant located in the EFNB1 gene. With our finding, we have pointed to an unusual phenomenon of mildly affected females with CFNS, who may not manifest features suggestive of the syndrome. Consequently, this study may be valuable for geneticists consulting patients with craniofacial disorders.

Surface expression of Cytokine Receptor-Like Factor 2 increases risk of relapse in pediatric acute lymphoblastic leukemia patients harboring IKZF1 deletions.

We prospectively examined whether surface expression of Cytokine Receptor-Like Factor 2 (CRLF2) on leukemic blasts is associated with survival and induction treatment response in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Flow cytometric analysis of bone marrow-derived leukemia cells revealed that 7.51% (29/286) of 386 pediatric BCP-ALL patients were CRLF2-positive (CRLF2pos) at diagnosis. The median minimal residual disease (MRD) was lower in CRLF2pos than CRLF2-negative (CRLF2neg) patients on day 15 (MRD15) after induction therapy [0.01% (0.001-0.42%) vs. 0.45% (0.05-3.50%); p=0.001]. By contrast, the MRD15 was higher in Ikaros family Zinc Finger Protein 1 (IKZF1)-deleted BCP-ALL patients than in BCP-ALL patients without IKZF1 deletions [1.18% (0.06-12.0%) vs 0.33% (0.03-2.6%); p=0.003]. Subgroup analysis showed that MRD15 levels were lower in IKZF1Δ/CRLF2pos patients than in IKZF1Δ/CRLF2neg patients [0.1% (0.02-5.06%) vs. 2.9% (0.25-12%); p=0.005]. Furthermore, MRD15 levels were higher in IKZF1WT/CRLF2neg patients than in IKZF1WT/CRLF2pos patients [0.40% (0.04-2.7%) vs. 0.001% (0.001-0.01%)]. Despite the low MRD15 levels, IKZF1Δ/CRLF2pos patients showed poorer relapse-free survival (RFS) than other patient groups (p=0.003). These findings demonstrate that surface CRLF2 expression is associated with increased risk of relapse in pediatric BCP-ALL patients harboring IKZF1 deletions.

Polymorphism in IKZF1 gene affects clinical outcome in diffuse large B-cell lymphoma.

IKZF1 encodes a transcription factor involved in B-cell maturation and differentiation. We genotyped 218 diffuse large B-cell lymphoma (DLBCL) patients and 715 unrelated controls using a TaqMan allelic discrimination assay. No statistical difference was observed in the genotype distribution of the IKZF1 rs4132601 polymorphism between DLBCL patients and controls. However, the 2-year PFS rate of patients with the IKZF1 TT genotype was 54.3% compared to 68.6% in those with the IKZF1 G+ genotypes. Moreover, the IKZF1 rs4132601 polymorphism retained its independent prognostic impact on PFS. A more pronounced effect of the IKZF1 TT genotype on PFS was detected in patients with low/intermediate low IPI-risk group. When analysis was restricted to patients with GCB-type pattern, those with the IKZF1 TT genotype achieved a lower 5-year OS rate than the patients with the IKZF1 G+ genotypes (19.6 vs. 56%). This study provides the first evidence for the association of IKZF1 variants with DLBCL outcome.

Biallelic loss of CDKN2A is associated with poor response to treatment in pediatric acute lymphoblastic leukemia.

The inactivation of tumor suppressor genes located within 9p21 locus (CDKN2A, CDKN2B) occurs in up to 30% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but its independent prognostic significance remains controversial. In order to investigate the prognostic impact of deletions and promoter methylation within 9p21, 641 children with newly diagnosed BCP-ALL using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) were investigated. A total of 169 (26.4%) microdeletions in 9p21 were detected, of which 71 were homozygous. Patients with CDKN2A homozygous deletions were older at diagnosis (p < .001), more frequently steroid resistant (p = .049), had higher WBC count (p < .001), higher MRD at Day 15 (p = .013) and lower relapse-free survival [p = .028, hazard ratio: 2.28 (95% confidence interval: 1.09-4.76)] than patients without these alterations. CDKN2A homozygous deletions coexisted with IKZF1 and PAX5 deletions (p < .001). In conclusion, CDKN2A homozygous deletions, but not promoter methylation, are associated with poor response to treatment and increased relapse risk of pediatric BCP-ALL.

Human leukocyte antigen-G polymorphisms influence the clinical outcome in diffuse large B-cell lymphoma.

The role of HLA-G is extensively studied in cancer due to its inhibition of the immune response. Several polymorphisms in the HLA-G gene have been reported to significantly affect its expression. We, therefore, investigated whether functionally relevant HLA-G polymorphisms, HLA-G-725C/G/T, and HLA-G 14-base pair, have any influence on the susceptibility to diffuse large B-cell lymphoma (DLBCL) and its clinical course. The polymorphisms were genotyped in 207 previously untreated patients with DLBCL and 150 unrelated controls. A significant difference in genotype distribution of HLA-G polymorphic genotypes between the patients and controls was found. The frequencies of the HLA-G-725GG or the HLA-G-725GC genotype were lower, and those of the HLA-G ins/ins genotype were higher in the patients compared with the controls. Patients carrying the HLA-G-725CC genotype presented a higher probability of overall survival (OS) than subjects with other genotype combinations of HLA-G-725C/G/T (P = 0.003). The homozygous HLA-G del/del had a lower probability of OS than subjects carrying the HLA-G deletion/insertion (del/ins) or the HLA-G ins/ins genotype (P = 0.009). Two HLA-G genotype-based risk groups were defined according to the genotype distribution. The high-risk (HR) group presented a shorter OS than low-risk (LR) patients (P = 0.001). In a multivariate analysis adjusted for International Prognostic Index (IPI) factors, both the intermediate high/high IPI-risk group (P < 0.0001) and the HR genotype group (P = 0.004) independently increased the risk of death. This is the first study indicating an important role of HLA-G polymorphisms for the clinical course of DLBCL. The potential influence of HLA-G polymorphisms on the susceptibility to DLBCL thus deserves further study.