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Purpose: Defects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants in known and candidate cohesinopathy genes from a clinical exome perspective., Methods: We retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization., Results: Pathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS., Conclusion: CES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR) at the cell surface and reroutes the internalized LDLR to intracellular degradation. In this study, we have shown that PCSK9-mediated degradation of the full-length 160 kDa LDLR generates a 17 kDa C-terminal LDLR fragment. This fragment was not generated from mutant LDLRs resistant to PCSK9-mediated degradation or when degradation was prevented by chemicals such as ammonium chloride or the cysteine cathepsin inhibitor E64d. The observation that the 17 kDa fragment was only detected when the cells were cultured in the presence of the γ-secretase inhibitor DAPT indicates that this 17 kDa fragment undergoes γ-secretase cleavage within the transmembrane domain. The failure to detect the complementary 143 kDa ectodomain fragment is likely to be due to its rapid degradation in the endosomal lumen. The 17 kDa C-terminal LDLR fragment was also generated from a Class 5 mutant LDLR undergoing intracellular degradation. Thus, one may speculate that an LDLR with bound PCSK9 and a Class 5 LDLR with bound LDL are degraded by a similar mechanism that could involve ectodomain cleavage in the endosome.

Objective: To study whether mutations in the SORT1 gene could be a cause of autosomal dominant hypercholesterolemia and to study the effect of sortilin on the binding and internalization of low density lipoprotein (LDL)., Methods: 842 unrelated hypercholesterolemic subjects without mutations in genes known to cause autosomal dominant hypercholesterolemia, were screened for mutations in the SORT1 gene by DNA sequencing. Transfections of wild-type or mutant SORT1 plasmids in HeLa T-REx cells and the use of siRNA were used to study the effect of sortilin on the number of cell-surface LDL receptors and on the binding and internalization of LDL., Results: A total of 45 mutations in the SORT1 gene were identified of which 15 were missense mutations. Eight of these were selected for in vitro studies, of which none had a major impact on the amount of LDL bound to the cell surface. There was a positive correlation between the amount of sortilin on the cell surface and the amount of LDL bound. The observation that a mutant sortilin which is predominantly found on the cell surface rather than in post-Golgi compartments, bound very high amounts of LDL, indicates that sortilin does not increase the binding of LDL through an intracellular mechanism. Rather, our data indicate that sortilin binds LDL on the cell surface., Conclusion: Even though sortilin binds and internalizes LDL by receptor-mediated endocytosis, mutations in the SORT1 gene are unlikely to cause autosomal dominant hypercholesterolemia and may only have a marginal effect on plasma LDL cholesterol levels., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. When human PCSK9 is injected into LDLR-deficient mice, PCSK9 is still rapidly cleared by the liver. This finding may suggest that PCSK9 is physiologically also cleared by receptors other than the LDLR. An alternative explanation could be that PCSK9 has undergone modifications during purification and is cleared by scavenger receptors on liver endothelial sinusoidal cells when injected into mice. If the only mechanism for clearing PCSK9 in humans is through the LDLR, one would expect that differences in the number of LDLRs would affect the plasma levels of low-density lipoprotein cholesterol (LDLC) and PCSK9 in a similar fashion. In this study, levels of LDLC and PCSK9 were measured in familial hypercholesterolemia (FH) homozygotes, FH heterozygotes, and normocholesterolemic subjects. The ratio between the levels of LDLC and PCSK9 was 1.7-fold higher in FH heterozygotes and 3-fold higher in FH homozygotes than in the normocholesterolemic subjects. Thus, defective LDLRs have a greater impact on the levels of LDLC than on the levels of PCSK9. By assuming that the rate of PCSK9 synthesis is similar in the 3 groups, this finding suggests that in humans, plasma PCSK9 is also cleared by LDLR-independent mechanisms., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE(814) and/or ExD(837) had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.

The low density lipoprotein receptor (LDLR) binds and internalizes low density lipoprotein (LDL). At the mildly acidic pH of the sorting endosomes, LDL is released from the receptor and the receptor recycles back to the cell membrane. Mutations in the LDLR gene may disrupt the normal function of the LDLR in different ways. Class 5 mutations result in receptors that are able to bind and internalize LDL, but they fail to release LDL in the sorting endosomes and fail to recycle. Instead they are rerouted to the lysosomes for degradation. However, the underlying mechanism remains to be determined. To study the role of the cytoplasmic domain of the LDLR for rerouting Class 5 mutants to the lysosomes, we have performed studies to determine whether Class 5 mutants caused by mutations E387K or V408M are degraded when the cytoplasmic domain has been altered or deleted. As determined by confocal laser-scanning microscopy, these mutant LDLR were inserted into the cell membrane and were able to internalize LDL. As determined by Western blot analysis, Class 5 mutants without a cytoplasmic domain still were degraded after binding LDL. Thus, the cytoplasmic domain does not play a role in rerouting Class 5 mutant LDLR to the lysosomes. Rather, one may speculate that sterical hindrance may prevent Class 5 mutants with bound LDL from entering the narrow recycling tubules of the sorting endosome., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31-53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32-40 and 48-50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs. Deletion of the acidic residues of the longest negatively charged segment increased PCSK9's ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9's activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9's activity by 36%. These findings indicate that the inhibitory effect of residues 31-53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9's activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The low density lipoprotein receptor (LDLR) family is a family of structurally related cell surface receptors with conserved exon/intron organization. Several members of this family have been shown to undergo alternative splicing. However, no alternative splicing of the LDLR pre-mRNA has so far been described., Methods: In the present study alternative splicing of human LDLR pre-mRNA has been studied in eight different tissues and four different cell lines using reverse transcription (RT) PCR. A quantitative real-time PCR with exon-exon boundary spanning primers was established to measure the relative amount of two novel isoforms., Results: Several novel isoforms were identified by RT-PCR of which the isoforms lacking exon 4 or 12 were two of the most prominent. Although highly detectable by RT-PCR, the quantification by real-time PCR revealed low levels of these isoforms., Conclusions: Novel isoforms of LDLR mRNA are described. Quantification by real-time PCR of two of the alternatively spliced isoforms revealed low amount of these isoforms in the examined tissues and cell lines. Further investigations are needed to evaluate if these isoforms represent functional transcripts of LDLR mRNA.