The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology.To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis.We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development. ResultsWe studied three fetuses that all fulfilled the clinical criteria of Greenberg dysplasia, namely intrauterine growth retardation, massive generalized edema (hydrops), extreme shortening of long bones (tetrabrachymelia) with a moth-eaten appearance of tubular bones, ectopic calcification centers and a narrow thorax (Fig. 1A, Suppl. Table 1). Detailed clinical examination was obtained from fetus A; fetus B has been described previously. 25Sterol analyses were performed in muscle tissue of fetus B and revealed the abnormal sterol metabolite 5α-cholest-8,14-dien-3β-ol, 25 that was previously shown to be associated with Greenberg dysplasia.18 Sterol analysis was not available for the other two fetuses.Sequence analysis revealed frameshift and missense mutations in the LBR gene. We sequenced LBR and identified mutations in all three families (Fig. 1B, sequence traces and segregation in Suppl. Fig. 1A). Fetus A showed a homozygous frameshift mutation c.1492delT that is predicted to change residues 468 to 474 and to create a premature stop in codon 475 (p.Y468TfsX475). Fetus B revealed two different mutations, c.32delTGGT and c.1748G>A. The first is a deletion of 4 base pairs causing a frame shift with subsequent premature stop in codon 24 (p.V11EfsX24). The second is a missense mutation replacing arginine by glutamine at residue 583 (R583Q). Both parents of fetus C were carrie...

The Lamin B receptor (LBR) is a pivotal architectural protein in the nuclear envelope. Mutations in the Lamin B receptor lead to nuclear hyposegmentation (Pelger-Huët anomaly). We have exactly quantified the nuclear lobulation in neutrophils from individuals with 0, 1, 2 and 3 functional copies of the lamin B receptor gene and analyzed the effect of different mutation types. Our data demonstrate that there is a highly significant gene-dosage effect between the gene copy number and the nuclear segmentation index of neutrophils. This finding is paralleled by a dose-dependent increase in LBR protein and staining intensity of the nuclear membrane in corresponding lymphoblastoid cell lines, which demonstrates a significant correlation on the protein level as well. We further show that LBR expression continually increases during granulopoiesis in vitro from human precursor cells with ovoid nuclei to multi-segmented neutrophil nuclei 11 days later, indicating relevance for regular human granulopoiesis. Altogether, LBR is a unique model that will allow the systematic study of gene-dosage effects and of modifying endogeneous and exogeneous factors on granulopoiesis.

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The nuclear morphology of neutrophils depends on different endogenous and exogenous factors, which can lead to hypo- or hypersegmentation of the normally 2-4 segmented nucleus. Hyposegmentation can be due to mutations in the LBR-gene (Pelger-Huët-Anomaly) or can be induced, for example, by colchicine treatment. The range of this phenotypic variation is known as "norm of reaction," which can be of major relevance for clinical diagnosis and therapeutic intervention. In this project, we studied the norm of reaction in 26 subjects with 0-3 wild type LBR alleles. In addition, the phenotypic variation was analyzed in 3 patients with Familial Mediterranean Fever (FMF), before and after colchicine treatment. We measured the phenotype nuclear segmentation of neutrophils based on two conventional qualitative methods, the "rule of threads" and the "rule of thirds." In addition, we tested a morphometric quantitative approach, the "circularity index." The circularity index was superior in cases with hyposegmentation; the rule of thirds with respect to hypersegmentation. Approximately 65% of the observed phenotypic variance was explainable by the number of LBR wild type alleles. The gene-dosage effect followed a non-additive, hysteresis-like characteristic with lower and upper plateaus. Colchicine treatment had a clear, although minor phenotypic effect compared to the number of LBR wild type genes or the mutation type. Thus, the nuclear morphology of granulocytes and its norm of reaction can be regarded as an excellent model both for detailing the interplay between endogenous and exogenous factors and for clinical diagnostic purposes. © 2016 International Clinical Cytometry Society.