Impact of genetic and non-genetic factors on phenotypic diversity in NBAS-associated disease.

Biallelic pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause a pleiotropic multisystem disorder. Three clinical subgroups have been defined correlating with the localisation of pathogenic variants in the NBAS gene: variants affecting the C-terminal region of NBAS result in SOPH syndrome (short stature, optic atrophy, Pelger-Huët anomaly), variants affecting the Sec 39 domain are associated with infantile liver failure syndrome type 2 (ILFS2) and variants affecting the ß-propeller domain give rise to a combined phenotype. However, there is still unexplained phenotypic diversity across the three subgroups, challenging the current concept of genotype-phenotype correlations in NBAS-associated disease. Therefore, besides examining the genetic influence, we aim to elucidate the potential impact of pre-symptomatic diagnosis, emergency management and other modifying variables on the clinical phenotype. We investigated genotype-phenotype correlations in individuals sharing the same genotypes (n = 30 individuals), and in those sharing the same missense variants with a loss-of-function variant in trans (n = 38 individuals). Effects of a pre-symptomatic diagnosis and emergency management on the severity of acute liver failure (ALF) episodes also were analysed, comparing liver function tests (ALAT, ASAT, INR) and mortality. A strong genotype-phenotype correlation was demonstrated in individuals sharing the same genotype; this was especially true for the ILFS2 subgroup. Genotype-phenotype correlation in patients sharing only one missense variant was still high, though at a lower level. Pre-symptomatic diagnosis in combination with an emergency management protocol leads to a trend of reduced severity of ALF. High genetic impact on clinical phenotype in NBAS-associated disease facilitates monitoring and management of affected patients sharing the same genotype. Pre-symptomatic diagnosis and an emergency management protocol do not prevent ALF but may reduce its clinical severity.

Characterization of a novel non-canonical splice site variant (c.886-5T>A) in NBAS and description of the associated phenotype.

BACKGROUND: Biallelic pathogenic variants in the neuroblastoma-amplified sequence (NBAS) gene manifest in a broad spectrum of disorders, including, but not limited to recurrent acute liver failure, skeletal dysmorphism, susceptibility to infections, and SOPH syndrome with its cardinal symptoms of short stature, optic atrophy, and Pelger-Huët anomaly. We aimed to present clinical and genetic characteristics of two sisters (20 and 15 years old) who were diagnosed with optic atrophy and cone dystrophy in childhood. Genome sequencing revealed two novel variants in NBAS in compound heterozygous state in both sisters, namely a 1-bp deletion predicted to result in a premature termination codon (c.5104del; p.(Met1702*)), and a non-canonical splice site variant of unclear significance (c.886-5T>A; p.?). RESULTS: Clinical examination and history revealed cone dystrophy, optic atrophy, and Pelger-Huët anomaly, but no short stature, recurrent acute liver failure, or susceptibility to infections. RNA analysis revealed that the c.886-5T>A variant results in two aberrant transcripts that are predicted to lead to in frame amino acid changes in the ß-propeller region of the protein. CONCLUSION: We hypothesize that the phenotype of our subjects, which appears to be at the end of the spectrum of NBAS-related disorders, could be explained by residual protein function mediated by the non-canonical splice site variant c.886-5T>A. Our study contributes to the existing knowledge on the genotypic and phenotypic spectrum of NBAS-related disorders.

MDS/AML with del5q: An acquired "laminopathy"?

In this issue of Cell Stem Cell, Reilly et al. propose loss of LMNB1, the gene encoding lamin B1, often deleted in MDS/AML, as a novel genetic basis for the abnormal nuclear shape of neutrophils (known as acquired Pelger-Huët anomaly) and a cause of HSPC fate alterations promoting malignancy.

Lamin B1 deletion in myeloid neoplasms causes nuclear anomaly and altered hematopoietic stem cell function.

Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.

Pyoderma gangrenosum associated with pseudo-Pelger-Huet anomaly in a patient with idiopathic myelofibrosis.

Pseudo-Pelger-Huët anomaly is a condition in which almost all the granulocytes are hyposegmented and/or hypogranulated. It is typically recognized in peripheral blood smears and represents a marker of several disorders, such as myeloproliferative diseases and myelodysplasia. The occurrence of the pseudo-Pelger-Huët anomaly in the cutaneous infiltrate of pyoderma gangrenosum is very rare. We describe the case of a 70-year-old man with idiopathic myelofibrosis who developed pyoderma gangrenosum. Histological examination showed an infiltrate consisting of granulocytic elements with features of dysmaturity and segmentation anomalies (hypo- and hypersegmented forms), suggestive of pseudo-Pelger-Huët anomaly. Methylprednisolone treatment resulted in progressive improvement of pyoderma gangrenosum.

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.

Severe SOPH syndrome due to a novel NBAS mutation in a 27-year-old woman-Review of this pleiotropic, autosomal recessive disorder: Mystery solved after two decades.

Autosomal recessive SOPH syndrome was first described in the Yakuts population of Asia by Maksimova et al. in 2010. It arises from biallelic pathogenic variants in the NBAS gene and is characterized by severe postnatal growth retardation, senile facial appearance, small hands and feet, optic atrophy with loss of visual acuity and color vision, and normal intelligence (OMIM #614800). The presence of Pelger-Hüet anomaly in this disorder led to its name as an acronym for Short stature, Optic nerve atrophy, and Pelger-Hüet anomaly. Recent publications have further contributed to the characterization of this syndrome through additional phenotype-genotype correlations. We review the clinical features described in these publications and report on a 27-year-old woman with dwarfism with osteolysis and multiple skeletal problems, minor anomalies, immunodeficiency, diabetes mellitus, and multiple secondary medical problems. Her condition was considered an unknown autosomal recessive disorder for many years until exome sequencing provided the diagnosis by revealing a founder disease-causing variant that was compound heterozygous with a novel pathogenic variant in NBAS. Based on the major clinical features of this individual and others reported earlier, a revision of the acronym is warranted to facilitate clinical recognition.

SOPH syndrome in three affected individuals showing similarities with progeroid cutis laxa conditions in early infancy.

Individuals affected with autosomal recessive cutis laxa type 2B and 3 usually show translucent skin with visible veins and abnormal elastic fibers, intrauterine and/or postnatal growth restriction and a typical triangular facial gestalt. Here we describe three unrelated individuals in whom such a cutis laxa syndrome was suspected, especially after electron microscopy revealed immature and less dense dermal elastic fibers in one of them. However, one of these children also displayed optic atrophy and two hypogammaglobulinemia. All had elevated liver enzymes and acute liver failure during febrile episodes leading to early demise in two of them. The only surviving patient had been treated with immunoglobulins. Through exome sequencing we identified mutations in NBAS, coding for a protein involved in Golgi-to-ER transport. NBAS deficiency causes several rare conditions ranging from isolated recurrent acute liver failure to a multisystem disorder mainly characterized by short stature, optic nerve atrophy and Pelger-Huët anomaly (SOPH). Since we subsequently verified Pelger-Huët anomaly in two of the patients the diagnosis SOPH syndrome was unequivocally proven. Our data show that SOPH syndrome can be regarded as a differential diagnosis for the progeroid forms of cutis laxa in early infancy and that possibly treatment of the hypogammaglobulinemia can be of high relevance for the prognosis.

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.

[Short stature, optic nerve atrophy and Pelger-Huët anomaly syndrome with antibody immunodeficiency and aplastic anemia: a case report and literature review].

Objective: To investigate the clinical features and genetic characteristics of cases with NBAS gene defects. Method: Characteristics of clinical materials, immunological data and gene mutation of the first case in China with NBAS gene mutation were retrospectively analyzed. The related literature was searched by using search terms'NBAS'. Result: A 2-year-four-month old girl, was admitted due to 'fever and pallor for one day'. There was an intrauterine growth retardation at her fetal stage. Since her birth, she had suffered from recurrent infections and development delay was accompanied by persistent liver dysfunction. Her head circumference and height were 43.5 cm and 60 cm, respectively. She seemed pale. She had progeroid appearance with loose skin, sparse hair, proptosis and low-set ears. The cranial suture did no close and the anterior fontanel was about 6 cm×5 cm. Abdominal palpation showed that the liver was 2 cm below the right costal margin, and the spleen was 1.5 cm below the left rib. Both alanine aminotransferase(100-1 991 IU/L) and aspartate aminotransferase (191-1 367 IU/L) were persistently abnormal. Visual evoked potentials and fundus examination revealed optic nerve atrophy. Bone mineral density assessment showed osteoporosis. The IgG level was 2.0 g/L (3.41-19.6) and absolute count of CD19(+)B cells was 231.27/µl (608.8-2 167.7) . Her hemoglobin level was 53 g/L. Bone marrow smear showed serious hypoplasia in erythroid cell. The gene sequencing results showed NBAS gene c.5741C> T, pR1914H and c.6496-6497insA, p.S2166Ffs* 2 compound heterozygous mutations. A total of 8 literatures were collected including 57 cases with NBAS gene homozygous or compound heterozygous mutation. These 57 cases were characterized by short stature(88%, 50/57) , Pelger-Huët anomaly (75%, 43/57) , skeletal dysplasia (74%, 42/57), optic nerve atrophy (72%, 41/57), abnormality of liver enzymes or acute liver failure (42%,24/57), abnormalities of immune system(19%, 11/57), development delay of mental, language or sports(11%, 6/57). Other clinical manifestations such as progeroid appearance, proptosis and hypotonia were also common. NBAS gene c.5741G>A homozygous mutation accounted for 61% (35/57) cases. Conclusion: Cases with NBAS gene defects often manifests as short stature, optic nerve atrophy, Pelger-Huët anomaly, skeletal dysplasia, recurrent infections, abnormality of liver enzymes, progeroid appearance, proptosis, hypotonia and immunodeficiency. Gene sequencing analysis showed NBAS gene homozygous or compound heterozygous mutations, and homozygous mutation of c.5741G>A was most common.

The Pseudo-Pelger HuËt Cell-A New Permanent Radiation Biomarker.

Using archival peripheral blood slides obtained from patients in the 1958 Y-12 criticality accident, the authors have recently described the pseudo-Pelger Huët anomaly (PHA) in neutrophils as a new radiation-induced biomarker. The current work provides additional evidence that PHA is also a permanent biomarker, potentially useful in retrospective dosimetry. In the Y-12 cohort, the high dose group (n = 5, 2.98-4.61 Gy-Eq) exhibited 13.0 ± 0.85 % Pelger Huët cells (mean ± SEM) in the neutrophil population compared to 6.8 ± 1.6 % in the low dose group (n = 3, 0.29-0.86 Gy-Eq; p = 0.008). An age and gender-matched control group (n = 8) exhibited 3.6 ± 0.9 % PH cells. Results of a one-way ANOVA show that the high dose group is statistically different from both the low dose group and the control group (p = 0.002). In the Y-12 cohort, PHA appears <12 h post-accident and is permanent for more than 16 y. Similar long-term persistence of the PHA mutation has been obtained from examination of peripheral blood slides from the 1971 Co accident at the Variable Dose Rate Irradiation Facility (VDRIF) in Oak Ridge, TN. In order to investigate the pseudo-PH cell as a biomarker in animal studies under well controlled dosimetry, peripheral blood slides were obtained from animals in a nonhuman primate (NHP) (Macaca mulatta) total-body irradiation (TBI) model (Co γ rays at 0.6 Gy min; dose range 1-8.5 Gy, LD50/60 6.44 Gy). In the NHP studies, the first measurement of PHA is taken at 5 h post-irradiation, then daily for days 1-5 and every 5-10 d thereafter. In the TBI model, the PH cell appears quickly (<5 h) post-irradiation, and the dose-dependent PH percentage is constant from 1 d over the 60-d monitoring period of the experiments. Using the average of data from 1-60 d, a linear dose response (PHA % slope = 0.49 ± 0.07 % Gy, r = 0.92) is obtained over the dose range 0-8.5 Gy. The authors conclude that ionizing radiation induces dose-dependent internuclear bridges in circulating neutrophils, and this morphological change can be used both as an acute phase biomarker and as a tool for retrospective dosimetry.

Detection of Perlger-Huet anomaly based on augmented fast marching method and speeded up robust features.

Pelger-Huet anomaly (PHA) and Pseudo Pelger-Huet anomaly (PPHA) are neutrophil with abnormal morphology. They have the bilobed or unilobed nucleus and excessive clumping chromatin. Currently, detection of this kind of cell mainly depends on the manual microscopic examination by a clinician, thus, the quality of detection is limited by the efficiency and a certain subjective consciousness of the clinician. In this paper, a detection method for PHA and PPHA is proposed based on karyomorphism and chromatin distribution features. Firstly, the skeleton of the nucleus is extracted using an augmented Fast Marching Method (AFMM) and width distribution is obtained through distance transform. Then, caryoplastin in the nucleus is extracted based on Speeded Up Robust Features (SURF) and a K-nearest-neighbor (KNN) classifier is constructed to analyze the features. Experiment shows that the sensitivity and specificity of this method achieved 87.5% and 83.33%, which means that the detection accuracy of PHA is acceptable. Meanwhile, the detection method should be helpful to the automatic morphological classification of blood cells.

Appearance of pseudo-Pelger Huet anomaly after accidental exposure to ionizing radiation in vivo.

To evaluate the morphology of formed elements of human blood after exposure to ionizing radiation in vivo, archival smears of peripheral blood from eight individuals involved in the 1958 Y-12 criticality accident at Oak Ridge, Tennessee, were examined manually by light microscopy. For each case, increased interlobar bridging was observed in nuclei of the myeloid cells, many of which were bilobed and morphologically similar to Pelger Huet (PH) cells. The high-dose group (n = 5, 2.98-4.61 Gy-Eq) exhibited 13.0 ± 0.85% PH cells (mean ± SEM) in the neutrophil population compared to 6.8 ± 1.6% in the low-dose group (n = 3, 0.29-0.86 Gy-Eq; p = 0.008). An age- and gender-matched control group (n = 8) exhibited 3.6 ± 0.9% PH cells. Results of a one-way ANOVA show that the high-dose group is statistically different from both the low-dose group and the control group (p = 0.002). However, the low-dose group is not statistically different from the control group (p = 0.122). The mean number of nuclear lobes in blood neutrophils was also enumerated as a function of time after exposure and was found to be diminished, consistent with incomplete nuclear segmentation that is characteristic of the Pelger Huet anomaly (PHA). In contrast to these changes in myeloid cells, the morphology of erythrocytes and platelets appeared to be normal. The authors conclude that ionizing radiation induces abnormal morphology of circulating neutrophils, which is similar to the pseudo-PHA that is acquired in disorders such as myelodysplastic syndrome, acute myeloid leukemia, and leukemoid reactions. Potential molecular mechanisms by which radiation induces this morphological change are discussed. From this cohort, the biomarker appears to be present early post-accident (<9 h) and stable at least up to 16 y post-accident. Assessment of circulating pseudo-Pelger Huet cells is being investigated as a potential biodosimetric tool.

Pelger-huet anomaly and a mild skeletal phenotype secondary to mutations in LBR.

The Lamin B receptor (LBR) gene has been described to encode a bifunctional protein. Mutations in the LBR gene can affect neutrophil segmentation and sterol reductase activity and have been associated with two different recognized clinical conditions, Pelger-Huet anomaly (PHA) and Greenberg skeletal dysplasia. PHA is a benign autosomal co-dominant laminopathy resulting in bilobed neutrophil nuclei in heterozygotes, and unsegmented (ovoid) neutrophil nuclei in homozygotes. Some putative PHA homozygotes have been reported with minor skeletal malformations. Greenberg skeletal dysplasia is a severe autosomal recessive, perinatal lethal dwarfing disorder in which heterozygous carriers are usually without clinical manifestations. We here report a girl who has bilobed neutrophil nuclei and a mild skeletal dysplasia. Mutation analysis showed two novel mutations in the LBR gene: c.651_653 delinsTGATGAGAAA (p.Ile218Aspfs*19) and c.1757G > A (p.Arg586His). These mutations were found to be in trans, and, thus, she is a compound heterozygote. Sterol analysis found trace amounts of cholesta-8,14-dien-3beta-ol, which is normally undetected in healthy individuals. This and previously reported cases suggest that mutations in LBR can result in a continuum of phenotypic manifestations.