Vimentin regulates nuclear segmentation in neutrophils.

Granulocytes are indispensable for various immune responses. Unlike other cell types in the body, the nuclei of granulocytes, particularly neutrophils, are heavily segmented into multiple lobes. Although this distinct morphological feature has long been observed, the underlying mechanism remains incompletely characterized. In this study, we utilize cryo-electron tomography to examine the nuclei of mouse neutrophils, revealing the cytoplasmic enrichment of intermediate filaments on the concave regions of the nuclear envelope. Aided by expression profiling and immuno-electron microscopy, we then elucidate that the intermediate-filament protein vimentin is responsible for such perinuclear structures. Of importance, exogenously expressed vimentin in nonimmune cells is sufficient to form cytoplasmic filaments wrapping on the concave nuclear surface. Moreover, genetic deletion of the protein causes a significant reduction of the number of nuclear lobes in neutrophils and eosinophils, mimicking the hematological condition of the Pelger-Huët anomaly. These results have uncovered a new component establishing the nuclear segmentation of granulocytes.

Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly.

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Protean Regulation of Leukocyte Function by Nuclear Lamins.

The leukocyte nucleus must be sufficiently elastic to squeeze through tissue barriers during migration, but not so collapsible as to risk damaging chromatin. The proper balance is struck in part by the composition of the nuclear lamina, a flexible meshwork composed mainly of intermediate filaments woven from type A and type B lamin proteins, that is located subjacent to the inner nuclear membrane. There is now increasing evidence that, in addition to influencing nuclear shape and stiffness and cell migration, lamins and lamin-interacting proteins may also interact functionally with chromatin to influence leukocyte gene expression, differentiation, and effector function, including T cell differentiation, B cell somatic hypermutation, and the formation of neutrophil extracellular traps (NETosis).

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Antenatal diagnostic dilemma in a pseudodominant pedigree with lamin-B receptor (LBR)-related regressive spondylometaphyseal dysplasia.

The lamin-B receptor (LBR) encodes a dual-functioning inner nuclear membrane protein essential for cholesterol biosynthesis and chromatin organization. LBR pathogenic variants cause distinct phenotypes due to the dual function of LBR, including Pelger-Huët anomaly (PHA), PHA with mild skeletal anomalies (PHASK; MIM# 618019), LBR-related regressive type of spondylometaphyseal dysplasia (LBR-R-SMD), Greenberg dysplasia (MIM# 215140). We here report the first case with radiological manifestations of LBR-R-SMD in the fetal period, and milder skeletal findings in the similarly affected father. Direct sequencing of LBR revealed homozygous c.1534C>T (p.Arg512Trp) in exon 12 in both affected individuals. Our report further refines the early phenotype in LBR-R-SMD, and demonstrates that the p.Arg512Trp mutation is associated with PHA. We propose that LBR-R-SMD should be considered as a differential diagnosis in pregnancies with sonographic evidence of short and bowed tubular bones with narrow thorax. Evaluating peripheral blood smears of expectant parents for the presence of PHA may lead to a clinical diagnosis, allowing for comprehensive prenatal genetic counseling.

[Rare diseases recognizable from blood smears]. / Seltene Erkrankungen am Blutbild erkennen.

The examination of peripheral blood smears is not only essential for the differential diagnostics of hematological diseases but can also provide important indications for general internal diseases, infections, hereditary diseases and poisoning. By the systematic analysis of a blood smear for alterations to thrombocytes, erythrocytes and leukocytes, a blood smear investigation can make a decisive contribution to the formulation of a diagnosis. In this way evidence of rare diseases can also be gained when taking the corresponding clinical findings into consideration.

Transient Deformation of Neutrophils in Kawasaki Disease.

In the treatment of Kawasaki disease, resistance to high-dose immunoglobulin intravenous (IGIV) can occur. The neutrophil morphology analyses in 17 patients revealed that transient pseudo-Pelger-Huët anomaly was more frequently detected in the IGIV-resistant group. This finding may aid the prediction of IGIV resistance.

An anadysplasia-like, spontaneously remitting spondylometaphyseal dysplasia secondary to lamin B receptor (LBR) gene mutations: further definition of the phenotypic heterogeneity of LBR-bone dysplasias.

We describe a boy who has an anadysplasia-like spondylometaphyseal dysplasia. By whole exome sequencing he was shown to have compound heterozygous mutations of LBR that codes for the lamin B receptor. He shares many similarities with a case previously described, but in whom the early natural history could not be established [Borovik et al., 2013]. Thus, in addition to Greenberg dysplasia (a perinatal lethal disorder), homozygosity or compound heterozygosity of mutations in LBR can result in a mild, spontaneously regressing bone dysplasia.

NBAS mutations cause a multisystem disorder involving bone, connective tissue, liver, immune system, and retina.

We report two unrelated patients with a multisystem disease involving liver, eye, immune system, connective tissue, and bone, caused by biallelic mutations in the neuroblastoma amplified sequence (NBAS) gene. Both presented as infants with recurrent episodes triggered by fever with vomiting, dehydration, and elevated transaminases. They had frequent infections, hypogammaglobulinemia, reduced natural killer cells, and the Pelger-Huët anomaly of their granulocytes. Their facial features were similar with a pointed chin and proptosis; loose skin and reduced subcutaneous fat gave them a progeroid appearance. Skeletal features included short stature, slender bones, epiphyseal dysplasia with multiple phalangeal pseudo-epiphyses, and small C1-C2 vertebrae causing cervical instability and myelopathy. Retinal dystrophy and optic atrophy were present in one patient. NBAS is a component of the synthaxin-18 complex and is involved in nonsense-mediated mRNA decay control. Putative loss-of-function mutations in NBAS are already known to cause disease in humans. A specific founder mutation has been associated with short stature, optic nerve atrophy and Pelger-Huët anomaly of granulocytes (SOPH) in the Siberian Yakut population. A more recent report associates NBAS mutations with recurrent acute liver failure in infancy in a group of patients of European descent. Our observations indicate that the phenotypic spectrum of NBAS deficiency is wider than previously known and includes skeletal, hepatic, metabolic, and immunologic aspects. Early recognition of the skeletal phenotype is important for preventive management of cervical instability.

Pseudo Pelger-Huët anomalies as potential biomarkers for acute exposure radiation dose in rhesus macaques (Macaca mulatta).

PURPOSE: The potential for malicious use of radiation, or radiation accidents could potentially lead to acute, high radiation doses to the public. Following acute accidental exposure to high doses of radiation, medical intervention is pivotal to the survivability of the patient, and the sooner the appropriate measures are taken the better the odds for survival. Early estimates of acute accidental radiation doses can be determined via biomarkers such as dicentric chromosome analysis or scenario reconstruction using computer software. However, both take valuable time and can be expensive. Increased frequencies of abnormal neutrophils in peripheral blood, referred to as pseudo Pelger-Huët anomalies (PPHAs), have been shown to be potential biomarkers of radiation exposure in several scenarios, including the 1958 Y-12 criticality accident and the radium dial painters. PPHAs are potentially a faster and cheaper quantitative biomarker for radiation exposure, and here they were evaluated in acutely exposed rhesus macaques. METHODS AND MATERIALS: Peripheral blood smears from acutely exposed rhesus macaques were evaluated for the percentage of neutrophils that displayed the PPHA morphology using light microscopy. Irradiated animals received 0 to 8.5 Gy total body radiation using one of two strategies: (1) linear accelerator-produced 6 MV photons delivered at 80 cGy/minute; or (2) Cobalt 60-produced gamma irradiation delivered at 60 cGy/min. Zero dose animals were used to determine a baseline percentage of PPHAs, and blood smears taken periodically throughout the lifetime of exposed animals post-irradiation were used to determine the persistence and biokinetics of PPHAs. RESULTS: The baseline prevalence of the PPHA in rhesus macaques was determined to be 0.58 ± 0.46%. The dose-response curve with doses ranging from 0 Gy to 8.5 Gy (LD90/30) displayed a strong positive correlation between PPHA percentage and acute radiation dose (R2 of 0.88 p = 3.62 × 10-22). Statistically significant differences were found when animals were separated into dose cohorts of 0, 4, 6.4-6.5, and 8-8.5 Gy. The biokinetics model utilized only 4 Gy exposures and blood smears taken periodically over 3.1 years post-irradiation. PPHA morphology increases quickly following irradiation and appears stable over 3.1 years post-irradiation. CONCLUSION: PPHA morphology was confirmed to be present in rhesus macaques, a dose-response relationship was constructed, and it is stable over 3 years post-irradiation. This study demonstrates that PPHA analysis can be a fast and cheap method of biodosimetry. Future studies will work to determine the accuracy of dose determination and lower limits of detection.

Blended Phenotype of Pelger-Huet Anomaly with Osteochondroma and Autosomal Recessive Deafness with Enlarged Vestibular Aqueduct.

Pelger-Huet anomaly (PHA) is a benign hematological anomaly that is characterized by impaired lobulation of neutrophils with a coarse nuclear chromatin. Skeletal abnormalities may accompany this anomaly. Autosomal recessive deafness-4 (DFNB4) with enlarged vestibular aqueduct (EVA) comprises a phenotypic spectrum of sensorineural hearing loss (SNHL). We report a case with SNHL, multiple skeletal anomalies including osteochondroma, developmental delay, and PHA. Molecular studies revealed a heterozygous pathogenic variant in the LBR gene and a homozygous likely pathogenic variant in the SLC26A4 gene. Due to these 2 variants, he was diagnosed with PHA and DFNB4 with EVA. If goiter develops, DFNB4 with EVA is named Pendred syndrome (PDS), so the patient will be followed up for this condition, and in the current literature, there is no case with PDS and PHA co-existence either. PHA may be accompanied by multiple skeletal abnormalities. In our case, there is also concomitance with osteochondroma. Although these are independent and distinct diagnoses, we present this case due to the concomitance of these situations.

Septic shock caused by Capnocytophaga canimorsus in a patient with heterozygous Pelger-Huët anomaly.

Capnocytophaga canimorsus is a Gram-negative bacillus of the commensal flora of dogs and cats that can cause infections in humans through bites, scratches or contact with oral secretions. It can be difficult to identify in clinical microbiology laboratories because of the need for specific culture media. We present the case of a patient with no relevant medical history who was admitted with septic shock, where blood smear examination was crucial for the etiologic diagnosis of Capnocytophaga canimorsus infection. The patient was also diagnosed Pelger-Huët anomaly, a condition causing a defect in neutrophil chemotaxis, which may have contributed to the severity of the infection.

Extracts Prepared from a Canadian Toxic Plant Induce Light-Dependent Perinuclear Vacuoles in Human Cells.

We are investigating plant species from the Canadian prairie ecological zone by phenotypic cell assays to discover toxins of biological interest. We provide the first report of the effects of extracts prepared from the shrub Symphoricarpos occidentalis in several human cell lines. S. occidentalis (Caprifoliaceae) extracts are cytotoxic, and, strikingly, treated cells undergo light-dependent vacuolation near the nucleus. The range of irradiation is present in standard ambient light and lies in the visible range (400-700 nm). Vacuolization in treated cells can be induced with specific wavelengths of 408 or 660 nm at 1 J/cm2 energies. Vacuolated cells show a striking phenotype of a large perinuclear vacuole (nuclear associated vacuole, NAV) that is distinct from vesicles observed by treatment with an autophagy-inducing agent. Treatment with S. occidentalis extracts and light induces an intense lamin A/C signal at the junction of a nuclear vacuole and the nucleus. Further study of S. occidentalis extracts and vacuolation provide chemical tools that may contribute to the understanding of nuclear envelope organization and human cell biology.

Peripheral Blood Smear Findings of COVID-19 Patients Provide Information about the Severity of the Disease and the Duration of Hospital Stay.

BACKGROUND: Data about the morphological changes in peripheral blood smears during COVID-19 infection and their clinical severity association are limited. We aimed to examine the characteristics of the cells detected in the pathological rate and/or appearance and whether these findings are related to the clinical course by evaluating the peripheral blood smear at the time of diagnosis in COVID-19 patients. METHODS: Clinical features, laboratory data, peripheral blood smear of fifty patients diagnosed with COVID-19 by PCR was evaluated at diagnosis. Peripheral smear samples of the patients were compared with the age and sex-matched 30 healthy controls. Pictures were taken from the patient's peripheral blood smear. Patients were divided into two groups. Mild and severe stage patient groups were compared in terms of laboratory data and peripheral smear findings. The relationship between the laboratory values of all patients and the duration of hospitalization was analyzed. RESULTS: The number of segmented neutrophils and eosinophils were low, pseudo-Pelger-Huet, pseudo-Pelger-Huet/mature lymphocyte ratio, atypical lymphocytes, monocytes with vacuoles, bands, and pyknotic neutrophils rates were higher in the peripheral blood smear of the patient group (p <0.05). Increased pseudo-Pelger-Huet anomaly, pseudo-Pelger Huet/mature lymphocyte ratio, a decreased number of mature lymphocytes, and eosinophils in peripheral blood smear were observed in the severe stage patients (p <0.05). A negative correlation was observed between hospitalization duration and mature lymphocyte and monocytes with vacuoles rates (p <0.05). CONCLUSION: A peripheral blood smear is an inexpensive, easily performed, and rapid test. Increased Pseudo-Pelger-Huet anomaly/mature lymphocyte rate suggests a severe stage disease, while high initial mature lymphocyte and monocytes with vacuoles rates at the time of diagnosis may be an indicator of shortened duration of hospitalization.

A 34-year-old Japanese patient exhibiting NBAS deficiency with a novel mutation and extended phenotypic variation.

Biallelic neuroblastoma amplified sequence (NBAS) gene mutations have recently been identified to cause a reduction in its protein expression and a broad phenotypic spectrum, from isolated short stature, optic nerve atrophy, and Pelger-Huët anomaly (SOPH) syndrome or infantile liver failure syndrome 2 to a combined, multi-systemic disease including skeletal dysplasia and immunological and neurological abnormalities. Herein, we report a 34-year-old patient with a range of phenotypes for NBAS deficiency due to compound heterozygous variants; one is a SOPH-specific variant, p.Arg1914His, and the other is a novel splice site variant, c.6433-2A>G. The patient experienced recurrent acute liver failure until early childhood. Hypogammaglobulinemia, a decrease in natural killer cells, and optic nerve atrophy were evident from infancy to childhood. In adulthood, the patient exhibited novel phenotypic features such as hepatic cirrhosis complicated by portal hypertension and autoimmune hemolytic anemia. The patient also suffered from childhood-onset insulin-requiring diabetes with progressive beta cell dysfunction. The patient had severe short stature and exhibited dysmorphic features compatible with SOPH, intellectual disability, and epilepsy. NBAS protein expression in the patient's fibroblasts was severely low. RNA expression analysis for the c.6433-2A>G variant showed that this variant activated two cryptic splice sites in intron 49 and exon 50, for which the predicted consequences at the protein level were an in-frame deletion/insertion, p.(Ile2199_Asn2202delins16), and a premature termination codon, p.(Ile2199Tyrfs*17), respectively. These findings indicate that NBAS deficiency is a multi-systemic progressive disease. The results of this study extend the spectrum of clinical and genetic findings related to NBAS deficiency.

Lamin B receptor-related disorder is associated with a spectrum of skeletal dysplasia phenotypes.

LBR (Lamin B Receptor) encodes a bifunctional protein important for cholesterol biosynthesis and heterochromatin organization on the inner nuclear membrane. Pathogenic variants in LBR are associated with marked phenotypic variability, ranging from the benign Pelger-Huët anomaly to lethal Greenberg Dysplasia. We performed trio exome sequencing (ES) on two patients with atypical variants of skeletal dysplasia and their unaffected parents. Patient 1 exhibited frontal bossing, mid-face hypoplasia, short stature with rhizomelic limb shortening, and relative macrocephaly at birth. Although remained short, Patient 1 later showed spontaneous improvement in her skeletal findings. Exome sequencing revealed two novel variants in LBR, c.1504C > G (p.Arg502Gly) in exon 12 and c.1748G > T (p.Arg583Leu) in exon 14, which were inherited from her unaffected father and mother, respectively. Sterol analysis revealed an increased level of cholesta­8,14­dien­3ß­ol to 2.9% of total sterols, consistent with a functional deficiency of 3ß­hydroxysterol Δ14­reductase. Patient 2 presented at birth with short stature and marked rhizomelic limb shortening but later exhibited decreasing severity of shortening of the long bones and improvement in the radiographic skeletal abnormalities although he continued to be significantly short at age 10 years. Exome sequencing revealed that Patient 2 is homozygous for a pathogenic variant c.1534C > T (p.Arg512Trp) in exon 12 of LBR, which was inherited from his unaffected consanguineous parents. This report provides further evidence for a phenotypic spectrum of LBR-associated disorders and expands the genotypic spectrum by describing 3 novel disease-causing variants that have not been previously associated with a disease. Moreover, our data on Patient 1 demonstrate that variants throughout the gene appear to influence both the sterol reductase and nuclear functions of LBR.

Inter-observer variance and the need for standardization in the morphological classification of myelodysplastic syndrome.

In this era of genome medicine, the sub-classification of myeloid neoplasms, including myelodysplastic syndrome (MDS), is now supported by genetic testing in selected cases. However, as the initial suspicion and primary diagnosis of the disease still largely relies on morphological features and numbers of hematopoietic cells, the establishment of a uniform diagnostic basis, especially for cell morphology, is essential. In this study, we collected nearly 100,000 hematopoietic cell images from 499 peripheral blood smear specimens from patients with MDS and used these to evaluate the standardization of morphological classification by medical technologists. The observers in this study ranged between two to eleven for each image, and the images were classified according to MDS criteria through a web-based system. We found considerable inter-observer variance in the assessment of dysplastic features. Observers did not recognize cytoplasmic hypo-granularity unless almost all granules in neutrophils were absent. Pseudo Pelger-Huët anomalies were also often overlooked, except for cells with a very typical "pince-nez" appearance. Taken together, this study suggests a requirement for further standardization in terms of morphological cell classification, and a need for the development of automatic cell classification-supporting devices for the accurate diagnosis of MDS.