We 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 (, Suppl. Table 1
). Detailed clinical examination was obtained from fetus A; fetus B has been described previously.25
Sterol 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.
Figure 1 Phenotype and identified mutations. (A) Post mortem appearance of fetus A at 16 + 3 weeks pregnancy. Note the edema, extreme micromelia of all four limbs and roentgenographic moth-eaten appearance of tubular bones. The thorax is deformed and narrow. Note (more ...)
Sequence analysis revealed frameshift and missense mutations in the LBR gene.
We sequenced LBR
and identified mutations in all three families (, 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 carriers of the missense mutation p.N547D. Even though no material was available from fetus C to show homozygosity for mutation p.N547D, consanguinity of the parents and the presence of the same mutation in another fetus with Greenberg dysplasia28
indicate that this mutation was causative. We proved that the nucleotide changes were not present in 150 controls, thereby making a polymorphism unlikely. For the missense mutations, we tested another 150 controls, to further reduce the possibility that they were rare variants.
The missense mutations reside in the sterol reductase domain and affect evolutionary conserved residues (). We tested the potential relevance of the identified missense mutations by an interspecies comparison. The p.N547D and p.R583Q mutations both change residues that are evolutionary extremely conserved among LBR and other sterol reductases, indicating their functional relevance (Suppl. Fig. 1B
). Sequencing of DHCR7
did not reveal alterations in any of the families, excluding a second hit in another gene of the C14 sterol reductase family.
LBR missense mutation did not alter nuclear shape in neutrophils.
Since both missense mutations reside in the sterol reductase domain of the lamin B receptor, disruption of the sterol reductase function seemed likely. However, the position of these mutations made effects on the second function of the lamin B receptor, namely nuclear structure, less likely. Based on the assumption that the amino acid substitution does not affect essential regions for modification, transport, or lamin B receptor anchoring, we expected nuclear morphology to remain unchanged. To test this hypothesis, we obtained blood from the parents of fetus B. The blood smear of the father indeed showed apparently normal neutrophils with multisegmented nuclei, whereas the mother had an obvious heterozygous Pelger anomaly with nuclear hyposegmentation (). This state of affairs was confirmed by sequence analyses. The mother with the Pelger anomaly was the carrier of the nonsense mutation p.V11EfsX24. The father, with the normal neutrophils on blood smears, carried the heterozygous missense mutation p.R583Q, indicating that this mutation affects sterol metabolism but not nuclear shape. No blood smears from the parents of fetus A and C were available.
Figure 2 Neutrophils show normal nuclei with multiple segments in the father with heterozygous missense mutation p.R583Q and in controls, indicating that this mutation does not affect nuclear shape. In contrast, the mother is a heterozygous carrier of a nonsense (more ...)
Both missense mutations failed to compensate for C14 sterol reductase deficiency in yeast.
The human wildtype lamin B receptor can complement for the sterol reductase function in yeast.5,30
LBR belongs to the C14 sterol reductase family as does yeast ERG24. The ERG24 deficient yeast has an altered sterol metabolism where the normal end product, ergosterol, is missing and the abnormal metabolite ignosterol is produced instead. Since ergosterol is essential, ERG24 mutants show impaired growth (, Suppl. Fig. 2
Figure 3 Design and results of the yeast rescue experiment. (A) Systematic view of the rescue experiment. (B) Analysis of human LBR constructs by GC/MS. hLBRwt is able to complement ERG24 deficiency in yeast. hLBR_N547D is partially able to complement ERG24 deficiency, (more ...)
We first demonstrated that wildtype human LBR and wildtype yeast ERG24 rescued the ERG24 deficient yeast phenotype in our system (data not shown). We then introduced the missense mutations p.N547D and p.R583Q, respectively, and transformed yeast with these mutant variants. We found that the mutants failed to rescue the yeast ergosterol phenotype (). As expected, wildtype yeast showed metabolites of the normal pathway, lanosterol, fecosterol and ergosterol. The same applied for ERG24 deficient yeast rescued by transformation with the yeast ERG24 gene or by the human LBR wildtype gene. The human LBR mutant p.N547D also produced fecosterol and ergosterol in an amount similar to the wildtype rescue, indicating a partial compensation. However, p.N547D caused a significant accumulation of the abnormal metabolites 4-methylzymosterol and ignosterol. Human mutation p.R583Q failed to produce the normal end product ergosterol in significant amounts. Instead, we observed a huge accumulation of the abnormal end product ignosterol. Both mutations increased the total amount of pathway metabolites, probably to provide at least trace amounts of ergosterol that is necessary for a number of essential cellular functions in yeast. Analyses of growth pattern confirmed these results with p.N547D partially restoring growth and p.R583Q failing to do so ().
In fibroblasts, LBR is not only located at the nuclear rim but also shows significant non-nuclear localization.
The lamin B receptor is a protein of the inner nuclear membrane. Accordingly predominant localization was so far only reported in the nucleus. To test the hypothesis that malformations observed in Greenberg dysplasia result from effects other than nuclear sterol synthesis, we studied the cellular distribution of the protein. The abnormal sterol metabolite in Greenberg dysplasia was initially identified by growing Greenberg fetal fibroblasts in lipid-depleted serum.18
We therefore reasoned that fibroblasts are a reasonable cell type to test this hypothesis and indeed found extensive localization of the lamin B receptor outside the nucleus ( and B
). De-novo endogeneous sterol synthesis takes place in the endoplasmatic reticulum (ER). Accordingly, we found a co-localization of the non-nuclear LBR with calnexin as an ER membrane component (representative localization in ; more cells are shown in Suppl. Fig. 3
). Further, we tested this in HeLa cells that have a higher growth rate and a higher expression level of LBR compared to fibroblasts. We found extensive cytoplasmic localization of LBR co-localizing with calnexin in immunostaining also in this cell type. Further, the western blot of fractionated HeLa cells revealed LBR in both the nuclear and cytoplasmic fraction whereas the nuclear marker lamin B was only present in the nuclear fraction (Suppl. Fig. 4
Figure 4 Localization of the lamin B receptor in human control fibroblasts. (A) Lamin B receptor staining (gp-anti-LBR_N-term) of the nuclear rim and in addition in the cytoplasm in control fibroblasts. The non-nuclear LBR showed a partial co-localization with (more ...)
LBR is expressed in human osteoclasts and osteoblast-like cells.
The Greenberg phenotype manifests as hydrops and severe skeletal dysplasia with shortening of long bones and altered growth plates in early fetal development. Therefore, we analyzed RNA levels in potentially disease-relevant human cell lines. We found expression of LBR in fibroblasts, lymphoblastoid cells, and to a very high degree in human in vitro differentiated osteoclasts (OC) and human osteosarcoma cells (HOS) which are osteoblast-like ().
Figure 5 Expression of the lamin B receptor in different cell types and in development. (A) Relative LBR mRNA expression in different human cell lines, calibrated to fibroblasts. The highest expression is found in cell types associated with skeletal development, (more ...)
LBR is strongly expressed in liver, skin, brain as well as in specific regions of the developing cartilage and bone in mouse embryos.
To analyze Lbr expression in vivo we studied wildtype mouse embryos at developmental stages consistent with the earliest manifestations in human Greenberg fetuses which have been reported as early as in gw 13 by ultrasound. In situ hybridization in wildtype mouse embryos at embryonic day E12.5 (corresponding to human gw 8 + 2) and qPCR of mouse tissues at postnatal day P4 showed strong expression of Lbr-RNA in the liver, lung, midgut, skin, brain, as well as in developing cartilage ( and C). To further analyze Lbr expression in growth plate cartilage we performed immunohistochemistry on E15.5 mouse forelimb sections in comparison to the chondrogenic marker Sox9 (). Lbr is expressed throughout growth plate cartilage, with weaker expression in hypertrophic chondrocytes. At the sites of trabecular bone formation Lbr expression was also seen in osteoblasts. In addition to the cartilage/bone expression, a signal for Lbr protein was also observed in muscle and in connective tissue fibroblasts. Consistent with the findings in human fibroblast cultures, we found, in addition to localization at the nuclear rim, a non-nuclear staining in connective tissue fibroblasts and also in Lbr-expressing cells of the developing cartilage and bone ( and magnification in B).
Figure 6 Expression analysis of Lbr protein in wildtype mouse fore limb on embryonic day E15.5. (A) Lbr (red) is strongly expressed in skin and in growth plate cartilage with exception of the hypertrophic zone. Immunostaining for the cartilage marker Sox9 (green) (more ...)