SMAD6 variants in
craniosynostosis: genotype and phenotype evaluation

Abstract Purpose Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. Methods We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. Results We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10−7). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. Conclusion Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

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Loss-of-function variants in the cardiac Kv11.1 channel as a genetic biomarker for SUDEP

10.1101/2021.03.19.436102 ◽

2021 ◽

Author(s):

Ming S. Soh ◽

Richard D. Bagnall ◽

Mark F. Bennett ◽

Lauren E. Bleakley ◽

Erlina S. Mohamed Syazwan ◽

...

Keyword(s):

Functional Analysis ◽

Allele Frequency ◽

Predictive Biomarkers ◽

Loss Of Function ◽

Similar Frequency ◽

Channel Function ◽

Fold Enrichment ◽

A Minor ◽

Patients With Epilepsy ◽

The Impact

AbstractObjectiveTo compare the frequency and impact on channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-of-function KCNH2 variants as predictive biomarkers of SUDEP risk.MethodsWe searched for KCNH2 variants with a minor allele frequency of < 5%. Functional analysis in Xenopus laevis oocytes was performed for all KCNH2 variants identified.ResultsKCNH2 variants were found in 11.1% (10/90) of SUDEP individuals compared to 6.0% (20/332) of epilepsy controls (p = 0.11). Loss-of-function KCNH2 variants, defined as causing > 20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about three-fold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (< 1% allele frequency) associated with greater loss of function and an ∼11-fold enrichment in the SUDEP cohort (nominal p = 0.03). In silico tools were unable to predict the impact of a variant on function highlighting the need for electrophysiological analysis.ConclusionsThese data show that loss-of-function KCNH2 variants are enriched in SUDEP patients and suggest that cardiac mechanisms contribute to SUDEP risk. We propose that genetic screening in combination with functional analysis can identify loss-of-function KCNH2 variants that could act as biomarkers of an individual’s SUDEP risk.

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Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01294-4 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Anne Rovelet-Lecrux ◽

Sebastien Feuillette ◽

Laetitia Miguel ◽

Catherine Schramm ◽

Ségolène Pernet ◽

...

Keyword(s):

3D Structure ◽

Hek293 Cells ◽

Pathophysiological Mechanism ◽

Loss Of Function ◽

Missense Variants ◽

Major Player ◽

Functional Consequences ◽

Endosomal System ◽

The Impact ◽

Clear Increase

AbstractThe SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer’s disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

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Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease

10.1101/2021.06.18.448296 ◽

2021 ◽

Author(s):

Anne Rovelet-Lecrux ◽

Sebastien Feuillette ◽

Laetitia Miguel ◽

Catherine Schramm ◽

Segolene Pernet ◽

...

Keyword(s):

Protein Structure ◽

Alzheimer Disease ◽

Hek293 Cells ◽

Pathophysiological Mechanism ◽

Loss Of Function ◽

Missense Variants ◽

Major Player ◽

Functional Consequences ◽

Sorl1 Gene ◽

The Impact

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of SorLA protein. An initial screening, based on the overexpression of 71 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variations (R332W, S577P, and R654W) and two maturation-competent variations (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs were associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Finally, structural analysis of the impact of missense variations on SorLA protein structure indicated that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein structure resulting from changes in the interatomic interactions.

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A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor

Nucleic Acids Research ◽

10.1093/nar/gkz780 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10662-10677 ◽

Cited By ~ 12

Author(s):

Amélie Rodrigue ◽

Guillaume Margaillan ◽

Thiago Torres Gomes ◽

Yan Coulombe ◽

Gemma Montalban ◽

...

Keyword(s):

Functional Analysis ◽

Tumor Suppressor ◽

Homologous Recombination ◽

Parp Inhibitor ◽

Familial Pancreatic Cancer ◽

Missense Mutations ◽

Breast Cancer Patients ◽

Missense Variants ◽

Functional Assays ◽

The Impact

Abstract While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

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Functional analysis of patient-derived PALB2 missense variants of uncertain significance.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.1531 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 1531-1531

Author(s):

Shijie Wu ◽

Jiaojiao Zhou ◽

Yiding Chen

Keyword(s):

Breast Cancer ◽

Functional Analysis ◽

In Silico ◽

Cancer Genetic Counseling ◽

Wild Type ◽

Variants Of Uncertain Significance ◽

Missense Variants ◽

Pathogenic Variants ◽

Uncertain Significance ◽

The Impact

1531 Background: Inherited PALB2 pathogenic variants are associated with an increased lifetime risk for breast cancer development. However, the interpretation of numerous PALB2 missense variants of uncertain significance (VUS) identified in germline genetic testing remains a challenge. Here, we assessed the impact of breast cancer patient-derived VUS on PALB2 function and identified pathogenic PALB2 missense variants that may increase cancer risk. Methods: A total of seven potentially pathogenic PALB2 VUS identified in 2,279 breast cancer patients were selected for functional analysis. All these selected VUS were assessed by SIFT, Align-GVGD, and PolyPhen2 in silico and were predicted to be deleterious by at least two in silico algorithms. The p.L35P [c.104T > C] variant was also included, for which pathogenicity has been recently confirmed. The effects of the VUS on the homologous recombination (HR) activity of PALB2 were tested by U2OS/DR-GFP reporting system. Functional characterization was further validated by protein co-immunoprecipitation and RAD51 recruitment assay. Results: PALB2 variants p.L24F [c.72G > C] and p.L35P [c.104T > C] showed the most significant disruption to the HR activity of PALB2 relative to the wild-type condition, retaining only 52.2% ( p = 0.0013) and 8.5% ( p < 0.0001) of HR activity respectively. Moderate but statistically significant HR deficiency was observed for four other variants (p.P405A [c.1213C > G], p.T1012I [c.3035C > T], p.E1018D [c.3054G > C], and p.T1099M [c.3296C > T]). We found no statistical differences for the p.K628N [c.1884G > T] and p.R663C [c.1987C > T] in the HR activity compared to wild-type PALB2. The p.L24F and p.L35P variants compromised the BRCA1-PALB2 interaction and reduced RAD51 foci formation in response to DNA damage. Conclusions: We have identified a novel patient-derived pathogenic PALB2 missense variant, p.L24F [c.72G > C], that compromises PALB2-mediated HR activity. We suggest the integration of the identified pathogenic variants into breast cancer genetic counseling and individualized treatment regimens for better clinical outcomes.

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ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

International Journal of Molecular Sciences ◽

10.3390/ijms22052689 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2689

Author(s):

Jianmin Si ◽

Chris Van den Haute ◽

Evy Lobbestael ◽

Shaun Martin ◽

Sarah van Veen ◽

...

Keyword(s):

Membrane Integrity ◽

Ubiquitin Proteasome System ◽

Regulatory Function ◽

Membrane Association ◽

Loss Of Function ◽

Atypical Form ◽

Polyamine Transport ◽

Independent Functions ◽

Ubiquitin Proteasome ◽

The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

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A Melanocortin-4 Receptor Agonist Induces Skin and Hair Pigmentation in Patients with Monogenic Mutations in the Leptin-Melanocortin Pathway

Skin Pharmacology and Physiology ◽

10.1159/000516282 ◽

2021 ◽

pp. 1-10

Author(s):

Varvara Kanti ◽

Lia Puder ◽

Irina Jahnke ◽

Philipp Maximilian Krabusch ◽

Jan Kottner ◽

...

Keyword(s):

Leptin Receptor ◽

Skin Pigmentation ◽

Gene Mutations ◽

Continuous Treatment ◽

Whole Body ◽

Hair Color ◽

Phase 2 ◽

Loss Of Function ◽

The Impact ◽

Over Time

Background and Objectives: Gene mutations within the leptin-melanocortin signaling pathway lead to severe early-onset obesity. Recently, a phase 2 trial evaluated new pharmacological treatment options with the MC4R agonist setmelanotide in patients with mutations in the genes encoding proopiomelanocortin (POMC) and leptin receptor (LEPR). During treatment with setmelanotide, changes in skin pigmentation were observed, probably due to off-target effects on the closely related melanocortin 1 receptor (MC1R). Here, we describe in detail the findings of dermatological examinations and measurements of skin pigmentation during this treatment over time and discuss the impact of these changes on patient safety. Methods: In an investigator-initiated, phase 2, open-label pilot study, 2 patients with loss-of-function POMC gene mutations and 3 patients with loss-of-function variants in LEPR were treated with the MC4R agonist setmelanotide. Dermatological examination, dermoscopy, whole body photographic documentation, and spectrophotometric measurements were performed at screening visit and approximately every 3 months during the course of the study. Results: We report the results of a maximum treatment duration of 46 months. Skin pigmentation increased in all treated patients, as confirmed by spectrophotometry. During continuous treatment, the current results indicate that elevated tanning intensity levels may stabilize over time. Lips and nevi also darkened. In red-haired study participants, hair color changed to brown after initiation of setmelanotide treatment. Discussion: Setmelanotide treatment leads to skin tanning and occasionally hair color darkening in both POMC- and LEPR-deficient patients. No malignant skin changes were observed in the patients of this study. However, the results highlight the importance of regular skin examinations before and during MC4R agonist treatment.

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Preservation of epoxyeicosatrienoic acid bioavailability prevents renal allograft dysfunction and cardiovascular alterations in kidney transplant recipients

Scientific Reports ◽

10.1038/s41598-021-83274-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Thomas Duflot ◽

Charlotte Laurent ◽

Anne Soudey ◽

Xavier Fonrose ◽

Mouad Hamzaoui ◽

...

Keyword(s):

Endothelial Function ◽

Kidney Transplant ◽

Plasma Levels ◽

Renal Allograft ◽

Transplant Recipients ◽

Loss Of Function ◽

Kidney Transplant Recipients ◽

Allograft Dysfunction ◽

Allograft Function ◽

The Impact

AbstractThis study addressed the hypothesis that epoxyeicosatrienoic acids (EETs) synthesized by CYP450 and catabolized by soluble epoxide hydrolase (sEH) are involved in the maintenance of renal allograft function, either directly or through modulation of cardiovascular function. The impact of single nucleotide polymorphisms (SNPs) in the sEH gene EPHX2 and CYP450 on renal and vascular function, plasma levels of EETs and peripheral blood monuclear cell sEH activity was assessed in 79 kidney transplant recipients explored at least one year after transplantation. Additional experiments in a mouse model mimicking the ischemia–reperfusion (I/R) injury suffered by the transplanted kidney evaluated the cardiovascular and renal effects of the sEH inhibitor t-AUCB administered in drinking water (10 mg/l) during 28 days after surgery. There was a long-term protective effect of the sEH SNP rs6558004, which increased EET plasma levels, on renal allograft function and a deleterious effect of K55R, which increased sEH activity. Surprisingly, the loss-of-function CYP2C9*3 was associated with a better renal function without affecting EET levels. R287Q SNP, which decreased sEH activity, was protective against vascular dysfunction while CYP2C8*3 and 2C9*2 loss-of-function SNP, altered endothelial function by reducing flow-induced EET release. In I/R mice, sEH inhibition reduced kidney lesions, prevented cardiac fibrosis and dysfunction as well as preserved endothelial function. The preservation of EET bioavailability may prevent allograft dysfunction and improve cardiovascular disease in kidney transplant recipients. Inhibition of sEH appears thus as a novel therapeutic option but its impact on other epoxyfatty acids should be carefully evaluated.

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Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

Genetics in Medicine ◽

10.1038/s41436-021-01158-1 ◽

2021 ◽

Author(s):

Paolo Zanoni ◽

Katharina Steindl ◽

Deepanwita Sengupta ◽

Pascal Joset ◽

Angela Bahr ◽

...

Keyword(s):

Loss Of Function ◽

Mild Phenotype ◽

Psychological Issues ◽

Missense Variants ◽

Mild Developmental Delay ◽

Pathogenic Variants ◽

In Silico Modeling ◽

And Function ◽

Methylation Activity

Abstract Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

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