Germline homozygous missense DEPDC5 variants cause severe refractory early-onset epilepsy, macrocephaly and bilateral polymicrogyria.

DEPDC5 (DEP Domain-Containing Protein 5) encodes an inhibitory component of the mammalian target of rapamycin (mTOR) pathway and is commonly implicated in sporadic and familial focal epilepsies, both non-lesional and in association with focal cortical dysplasia. Germline pathogenic variants are typically heterozygous and inactivating. We describe a novel phenotype caused by germline biallelic missense variants in DEPDC5. Cases were identified clinically. Available records, including magnetic resonance imaging and electroencephalography, were reviewed. Genetic testing was performed by whole exome and whole-genome sequencing and cascade screening. In addition, immunohistochemistry was performed on skin biopsy. The phenotype was identified in nine children, eight of which are described in detail herein. Six of the children were of Irish Traveller, two of Tunisian and one of Lebanese origin. The Irish Traveller children shared the same DEPDC5 germline homozygous missense variant (p.Thr337Arg), whereas the Lebanese and Tunisian children shared a different germline homozygous variant (p.Arg806Cys). Consistent phenotypic features included extensive bilateral polymicrogyria, congenital macrocephaly and early-onset refractory epilepsy, in keeping with other mTOR-opathies. Eye and cardiac involvement and severe neutropenia were also observed in one or more patients. Five of the children died in infancy or childhood; the other four are currently aged between 5 months and 6 years. Skin biopsy immunohistochemistry was supportive of hyperactivation of the mTOR pathway. The clinical, histopathological and genetic evidence supports a causal role for the homozygous DEPDC5 variants, expanding our understanding of the biology of this gene.

A Nationwide Study of GATA2 Deficiency in Norway-the Majority of Patients Have Undergone Allo-HSCT.

PURPOSE: GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT. METHODS: All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records. RESULTS: Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively. CONCLUSION: Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described.

Benefits of clinical criteria and high-throughput sequencing for diagnosing children with syndromic craniosynostosis.

An accurate diagnosis of syndromic craniosynostosis (CS) is important for personalized treatment, surveillance, and genetic counselling. We describe detailed clinical criteria for syndromic CS and the distribution of genetic diagnoses within the cohort. The prospective registry of the Norwegian National Unit for Craniofacial Surgery was used to retrieve individuals with syndromic CS born between 1 January 2002 and 30 June 2019. All individuals were assessed by a clinical geneticist and classified using defined clinical criteria. A stepwise approach consisting of single-gene analysis, comparative genomic hybridization (aCGH), and exome-based high-throughput sequencing, first filtering for 72 genes associated with syndromic CS, followed by an extended trio-based panel of 1570 genes were offered to all syndromic CS cases. A total of 381 individuals were registered with CS, of whom 104 (27%) were clinically classified as syndromic CS. Using the single-gene analysis, aCGH, and custom-designed panel, a genetic diagnosis was confirmed in 73% of the individuals (n = 94). The diagnostic yield increased to 84% after adding the results from the extended trio-based panel. Common causes of syndromic CS were found in 53 individuals (56%), whereas 26 (28%) had other genetic syndromes, including 17 individuals with syndromes not commonly associated with CS. Only 15 individuals (16%) had negative genetic analyses. Using the defined combination of clinical criteria, we detected among the highest numbers of syndromic CS cases reported, confirmed by a high genetic diagnostic yield of 84%. The observed genetic heterogeneity encourages a broad genetic approach in diagnosing syndromic CS.

A Nationwide Study of Norwegian Patients with Hereditary Angioedema with C1 Inhibitor Deficiency Identified Six Novel Mutations in SERPING1.

Hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) is characterized by relapsing, non-pruritic swelling in skin and submucosal tissue. Symptoms can appear in early infancy when diagnosis is more difficult. In the absence of a correct diagnosis, treatment of abdominal attacks often lead to unnecessary surgery, and laryngeal edema can cause asphyxiation. A cohort study of 52 patients from 25 unrelated families in Norway was studied. Diagnosis of C1-INH-HAE was based on international consensus criteria including low functional and/or antigenic C1-INH values and antigenic C4. As SERPING1 mutations in Norwegian patients with C1-INH-HAE are largely undescribed and could help in diagnosis, we aimed to find and describe these mutations. Mutation analysis of the SERPING1 gene was performed by Sanger sequencing of all protein coding exons and exon-intron boundaries. Samples without detected mutation were further analyzed by multiplex ligation-dependent probe amplification to detect deletions and duplications. Novel mutations suspected to lead to splice defects were analyzed on the mRNA level. Fifty-two patients from 25 families were included. Forty-four (84,6%) suffered from C1-INH-HAE type I and eight (15,4%) suffered from C1-INH-HAE type II. Pathogenic or likely pathogenic mutations were found in 22/25 families (88%). Thirteen unique mutations were detected, including six previously undescribed. There were three missense mutations including one mutation affecting the reactive center loop at codon 466, three nonsense mutations, three small deletions/duplications, three gross deletions, and one splice mutation.

A 19-year-old man with relapsing bilateral pneumothorax, hemoptysis, and intrapulmonary cavitary lesions diagnosed with vascular Ehlers-Danlos syndrome and a novel missense mutation in COL3A1.

A 19-year-old sportsman experienced a right-sided pneumothorax and hemoptysis after having had an intermittent cough and blood-tinged sputum for 2 months. A chest CT scan revealed small cavitary lesions in both lungs. The relapsing pneumothorax was treated with a chest tube twice, as well as surgically after the second relapse. Two months after surgery, the patient developed a cough, fever, and high C-reactive protein levels. At that time, large consolidations had developed in the right lung, while the left lung subsequently collapsed due to pneumothorax. The patient's physical appearance and anamnestic information led us to suspect a genetic connective tissue disease. A sequencing analysis of the COL3A1 gene identified a novel, de novo missense mutation that confirmed the diagnosis of vascular Ehlers-Danlos syndrome (EDS). This atypical presentation of vascular EDS with intrathoracic complications shows that enhanced awareness is required and demonstrates the usefulness of the genetic analyses that are clinically available for several hereditary connective tissue disorders.

Affinity selection using filamentous phage display.

Display of peptides on filamentous phage, phage display, is an in vitro selection technique well suited for identification of therapeutic peptide binders for a huge variety of protein targets. The peptides are identified in a process where phage libraries are subjected to affinity selection towards a particular protein target. A successful outcome of an affinity selection is dependent on proper surveillance of the phage life cycle, to make sure that the selection is based on affinity for the target, not on bias in phage propagation rate. In this chapter we present two approaches for protein target presentation and a protocol for phage rescue and propagation, which includes several controls to ensure that all phages initially eluted from the protein target are given equal conditions during the following amplification and selection steps.

Design and characterization of targeted ultrasound microbubbles for diagnostic use.

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 µm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties.

Analysis of LDLR mRNA in patients with familial hypercholesterolemia revealed a novel mutation in intron 14, which activates a cryptic splice site.

Familial hypercholesterolemia (FH) is caused by a defective low-density lipoprotein receptor (LDLR), and >1000 mutations in LDLR have been identified. However, in some patients with clinically defined FH, no mutation can be detected within the exons and adjacent intronic segments of the LDLR. We have analyzed RNA extracted from blood samples of patients with clinically defined FH and identified an aberrantly spliced mRNA containing an 81-bp insert from intron 14. The aberrant splicing was caused by a novel intronic mutation, c.2140+86C>G, which activated a cryptic splice site. Although the cryptic splice site does not completely surpass the normal splice site, the mutation was found to cosegregate with high cholesterol levels in a family, which supports the notion that c.2140+86C>G causes FH. The insertion of 81 bp in LDLR mRNA encodes an in-frame insertion of 27 amino acids in the LDLR. However, the insertion was found to hamper LDLR activity by preventing the receptor from leaving the endoplasmic reticulum, probably because of misfolding of the protein. In patients with clinically defined hypercholesterolemia, despite normal results from sequencing of exonic regions of the LDLR gene, characterization of the LDLR mRNA might identify the underlying genetic defect.

Splice-site mutation c.313+1, G>A in intron 3 of the LDL receptor gene results in transcripts with skipping of exon 3 and inclusion of intron 3.

BACKGROUND: Familial hypercholesterolemia (FH) patients with the splice site mutation c.313+1, G>A in intron 3 of the low density lipoprotein receptor (LDLR) gene, present with a phenotype similar to that of FH patients in general. However, a mild phenotype would have been expected from the published data showing that the mutation only causes skipping of exon 3. METHODS: Epstein Barr virus-transformed lymphocytes from eight c.313+1, G>A heterozygotes and two c.313+1, G>A homozygotes were subjected to studies of the LDLR at the mRNA and protein levels. RESULTS: Mutation c.313+1, G>A not only causes skipping of exon 3, but also causes inclusion of intron 3. No functional LDLR was produced from the transcript with inclusion of intron 3. The transcript with skipping of exon 3 produced a receptor which had markedly reduced ability to internalize low density lipoprotein. CONCLUSION: The findings that the mutation c.313+1, G>A in the LDLR gene also generates a mutant transcript with inclusion of intron 3, explains why the mutation c.313+1, G>A may result in a severe phenotype.

Functional analysis of the synonymous R385R mutation in the low-density lipoprotein receptor gene.

Familial hypercholesterolemia is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. The synonymous mutation R385R has been shown to introduce a cryptic splice site in exon 9. The aims of this study were to establish to what extent the cryptic splice site is selected ahead of the normal splice site and to determine if the aberrant transcript is degraded by nonsense-mediated mRNA decay. The relative amount of the aberrant transcript was determined by real-time PCR and found to vary from 25% to 45% in heterozygous familial hypercholesterolemia individuals. Epstein-Barr virus-transformed lymphocytes were established from one heterozygous patient, and treatment of these cells with cycloheximide increased the amount of aberrant transcript, indicating that the aberrant transcripts are degraded by nonsense-mediated mRNA decay. Cloning of reverse transcriptase-PCR products from one of the heterozygous patients and introduction of the R385R mutation into a minigene reporter construct revealed an almost exclusive use of the cryptic splice site in the mutated allele. Thus, the synonymous mutation R385R converts the mutated allele to a null allele unable to produce functional mRNA.

Nonsense-mediated decay of human LDL receptor mRNA.

OBJECTIVE: The objective of this project was to determine whether nonsense mutation in the low density lipoprotein receptor (LDLR) induces nonsense-mediated mRNA decay (NMD). MATERIAL AND METHODS: Four known nonsense mutations (W23X, S78X, E207X and W541X) in the LDLR gene, which are found in Norwegian familial hypercholesterolaemia (FH) patients, were investigated. Epstein-Barr virus (EBV) transformed lymphocytes from patients heterozygous for these mutations in the LDLR gene were analysed. Flow cytometric analysis was used to determine the amount and function of the cell surface LDLRs. The expression of LDLR mRNA in lymphocytes was quantified by real-time polymerase chain reaction (PCR). The presence of NMD was tested using the inhibitors gentamicin, emetine or cycloheximide. RESULTS: Cells from heterozygous FH patients with nonsense mutations in the LDLR gene contained significantly less LDLR protein (p<0.05). In addition, flow cytometric analysis revealed that these patients had a reduced LDL-uptake compared to controls (p<0.005). Cells from heterozygous FH patients with nonsense mutations W23X, S78X or W541X in the LDLR gene showed significantly decreased levels of LDLR mRNA (p<0.005). LDLR mRNA was reduced in the mutant lymphocyte S78X prior to treatment with pharmacological inhibitors, and after treatment the level of LDLR mRNA increased to the same level as that of normal cells. CONCLUSION: In the present study, NMD was confirmed in the LDLR gene. Translation inhibitors showed reduced NMD caused by nonsense mutated LDLR transcripts. Knowledge of NMD might have an important impact in clinical medicine as genetic intervention develops.

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density.

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P(BAD) promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 x 10(9) unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.

Genome-wide expression analysis of cells expressing gain of function mutant D374Y-PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum cholesterol. The possibility that PCSK9 also functions in other pathways needs to be addressed. We have transfected HepG2 cells with mutant D374Y-PCSK9 or control vector. Gene expression signatures were determined using the Affymetrix GeneChip technology, and the expression pattern of selected genes was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Data was normalized and analyzed using a model-based background adjustment for oligonucleotide expression arrays, then filtered based upon expression within treatments group, and subjected to moderated t-statistics. Five hundred twenty transcripts had altered expression levels between D374Y-PCSK9 and control vector. Among the 520 probes on our top list, 312 were found to have an assigned Gene Ontology (GO) term, and 96 were found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Genome-wide expression profiling revealed that "steroid biosynthesis," "sterol metabolism," and "cholesterol biosynthsis" were affected by D374Y-PCSK9. Also, the GO biological process terms "response to stresss," "response to virus," "response to unfolded protein," and "immune response" were influenced by D374Y-PCSK9. Our results suggest that D374Y-PCSK9 results in up-regulation of genes involved in sterol biosynthesis and down-regulation of stress-response genes and specific inflammation pathways.

Model system for phenotypic characterization of sequence variations in the LDL receptor gene.

BACKGROUND: Sequence variations in the LDL receptor (LDLR) gene cause defects of LDLR protein production and function through different molecular mechanisms. Here we describe a cell model system for the phenotypic characterization of sequence variations in the LDLR gene. Well-known sequence variations belonging to LDLR classes 2 to 5 (p.G565V, p.I161D, p.Y828C, and p.V429M) were studied in CHO and HepG2 cells. METHODS: Expression of LDLR protein on the cell surface was detected by use of fluorescence-conjugated antibodies against the LDLR and the LDLR activity was measured by incubating the cells with fluorescently labeled and radiolabeled LDL. The intracellular locations of the LDLR mutants and wild-type were also investigated. RESULTS: The class 2A p.G565V sequence variant exhibited an intracellular distribution of LDLR with no active receptors on the cell surface. Both the class 3 p.I161D and class 4 p.Y828C sequence variants gave surface staining but had a reduced ability to bind or internalize LDL, respectively. By determining the intracellular locations of the receptors we were able to visualize the accumulation of the class 5 p.V429M sequence variant in endosomes by means of a specific marker, as well as confirming that the class 4 p.Y828C variant was not localized in clathrin-coated pits. Flow cytometry allowed us quantitatively to determine the amount and activity of receptors. To confirm the results of binding and cell association of fluorescently labeled LDL analyzed by flow cytometry, assays using 125I-labeled LDL were performed. In addition to a useful and valid alternative to radiolabeled LDL, the unique properties of fluorescently labeled LDL allowed a variety of detection technologies to be used. CONCLUSIONS: This new approach enables phenotypic characterization of sequence variations in the LDLR gene. The assays developed may be valuable for confirming the pathogenicity of novel missense sequence variations found throughout the LDLR gene.

Effect of mutations in the PCSK9 gene on the cell surface LDL receptors.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through 'loss-of-function' and 'gain-of-function' mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.

Reduced secretion of triacylglycerol in CaCo-2 cells transfected with intestinal fatty acid-binding protein.

The fatty acid-binding proteins are hypothesized to be involved in cellular fatty acid transport and trafficking. We established CaCo-2 cells stably transfected with intestinal fatty acid-binding protein (I-FABP) and examined how the expression of this protein may influence fatty acid metabolism. I-FABP expression was detectable in I-FABP-transfected cells, whereas parent CaCo-2 cells as well as mock-transfected cells failed to express detectable levels of I-FABP mRNA or protein at any stage of differentiation. For studies of lipid metabolism, cells were incubated with [14C]oleic acid in taurocholate micelles containing monoolein, and distribution of labeled fatty acid in cellular and secreted lipids was examined. In one transfected cell clone, expressing the highest level of I-FABP, labeled cellular triacylglycerol increased approximately twofold as compared to control cells. The level of intracellular triacylglycerol in two other I-FABP-transfected clones resembled that of control cells. However, secretion of triacylglycerol was markedly reduced in all the I-FABP-expressing cell lines. Our data suggest that increased expression of I-FABP leads to reduced triacylglycerol secretion in intestinal cells.