Chronic Pancreatitis: The True Pathogenic Culprit within the SPINK1 N34S-Containing Haplotype Is No Longer at Large

A diverse range of loss-of-function variants in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) has been identified in patients with chronic pancreatitis (CP). The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser or N34S) variant (rs17107315:T>C) is one of the most important heritable risk factors for CP as a consequence of its relatively high prevalence worldwide (population allele frequency ≈ 1%) and its considerable effect size (odds ratio ≈ 11). The causal variant responsible for this haplotype has been intensively investigated over the past two decades. The different hypotheses tested addressed whether the N34S missense variant has a direct impact on enzyme structure and function, whether c.101A>G could affect pre-mRNA splicing or mRNA stability, and whether another variant in linkage disequilibrium with c.101A>G might be responsible for the observed association with CP. Having reviewed the currently available genetic and experimental data, we conclude that c.-4141G>T (rs142703147:C>A), which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L cis-regulatory module located ∼4 kb upstream of the SPINK1 promoter, can be designated as the causal variant beyond reasonable doubt. This case illustrates the difficulties inherent in determining the identity of the causal variant underlying an initially identified disease association.

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A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia

Human Molecular Genetics ◽

10.1093/hmg/ddz200 ◽

2019 ◽

Vol 28 (21) ◽

pp. 3543-3551

Author(s):

Carsten Rautengarten ◽

Oliver W Quarrell ◽

Karen Stals ◽

Richard C Caswell ◽

Elisa De Franco ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Missense Variant ◽

Sugar Transporter ◽

Loss Of Function ◽

Transport Activity ◽

Nucleotide Sugar ◽

Gene Encoding ◽

Hypomorphic Allele ◽

Nucleotide Sugar Transporter

Abstract We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a ‘Schneckenbecken-like dysplasia.’ The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.

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Lysosomal perturbations in dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

10.1101/734244 ◽

2019 ◽

Author(s):

Justyna Okarmus ◽

Helle Bogetofte ◽

Sissel Ida Schmidt ◽

Matias Ryding ◽

Silvia Garcia Lopez ◽

...

Keyword(s):

Dopaminergic Neurons ◽

Induced Pluripotent Stem Cell ◽

Autophagic Flux ◽

Compensatory Mechanism ◽

Loss Of Function ◽

Gene Encoding ◽

Tem Analysis ◽

Induced Pluripotent ◽

And Function ◽

The Impact

AbstractMutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson’s disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function mutations remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. In the present study, we used such cells to investigate the impact of PARK2 KO on the lysosomal compartment combining different approaches, such as mass spectrometry-based proteomics, electron microscopy (TEM) analysis and functional assays. We discovered a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology, displaying significantly enlarged and electron-lucent lysosomes as well as an increased total lysosomal content, which was exacerbated by mitochondrial stress. In addition, we found perturbed autophagic flux and decreased lysosomal enzyme activity suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was significantly increased suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons, thus revealing a new important connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

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The reversion variant (p.Arg90Leu) at the evolutionarily adaptive p.Arg90 site in CELA3B predisposes to chronic pancreatitis

10.1101/2020.11.28.20240135 ◽

2020 ◽

Author(s):

Emmanuelle Masson ◽

Vinciane Rebours ◽

Louis Buscail ◽

Frédérique Frete ◽

Mael Pagenault ◽

...

Keyword(s):

Chronic Pancreatitis ◽

Amino Acid ◽

Human Genome ◽

Low Frequency ◽

Missense Variant ◽

Loss Of Function ◽

Ancestral Allele ◽

Functional Effect ◽

Gain Of Function ◽

Missense Variants

ABSTRACTA gain-of-function missense variant in the CELA3B gene, p.Arg90Cys (c.268C>T), has recently been reported to cause pancreatitis in an extended pedigree. Herein, we sequenced the CELA3B gene in 644 genetically unexplained French chronic pancreatitis (CP) patients (all unrelated) and 566 controls. No predicted loss-of-function variants were identified. None of the six low frequency or common missense variants detected showed significant association with CP. Nor did the aggregate rare/very rare missense variants (n=14) show any significant association with CP. However, p.Arg90Leu (c.269G>T), which was found in 4 patients but no controls and affects the same amino acid as p.Arg90Cys, serves to revert p.Arg90 to the human elastase ancestral allele. Since p.Arg90Leu has previously been shown to exert a similar functional effect to p.Arg90Cys, our findings not only confirm the involvement of CELA3B in the etiology of CP but also pinpoint a new evolutionarily adaptive site in the human genome.

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Mitochondrial PCK2 Missense Variant in Shetland Sheepdogs with Paroxysmal Exercise-Induced Dyskinesia (PED)

Genes ◽

10.3390/genes11070774 ◽

2020 ◽

Vol 11 (7) ◽

pp. 774 ◽

Cited By ~ 1

Author(s):

Jasmin Nessler ◽

Petra Hug ◽

Paul J. J. Mandigers ◽

Peter A. J. Leegwater ◽

Vidhya Jagannathan ◽

...

Keyword(s):

Stress Reduction ◽

Mutant Allele ◽

Phosphoenolpyruvate Carboxykinase ◽

Missense Variant ◽

Nerve Biopsy ◽

Causal Variant ◽

Gluten Free ◽

Gene Encoding ◽

Muscle Nerve ◽

Exercise Induced

Four female Shetland Sheepdogs with hypertonic paroxysmal dyskinesia, mainly triggered by exercise and stress, were investigated in a retrospective multi-center investigation aiming to characterize the clinical phenotype and its underlying molecular etiology. Three dogs were closely related and their pedigree suggested autosomal dominant inheritance. Laboratory diagnostic findings included mild lactic acidosis and lactaturia, mild intermittent serum creatine kinase (CK) elevation and hypoglycemia. Electrophysiological tests and magnetic resonance imaging of the brain were unremarkable. A muscle/nerve biopsy revealed a mild type II fiber predominant muscle atrophy. While treatment with phenobarbital, diazepam or levetiracetam did not alter the clinical course, treatment with a gluten-free, home-made fresh meat diet in three dogs or a tryptophan-rich, gluten-free, seafood-based diet, stress-reduction, and acetazolamide or zonisamide in the fourth dog correlated with a partial reduction in, or even a complete absence of, dystonic episodes. The genomes of two cases were sequenced and compared to 654 control genomes. The analysis revealed a case-specific missense variant, c.1658G>A or p.Arg553Gln, in the PCK2 gene encoding the mitochondrial phosphoenolpyruvate carboxykinase 2. Sanger sequencing confirmed that all four cases carried the mutant allele in a heterozygous state. The mutant allele was not found in 117 Shetland Sheepdog controls and more than 500 additionally genotyped dogs from various other breeds. The p.Arg553Gln substitution affects a highly conserved residue in close proximity to the GTP-binding site of PCK2. Taken together, we describe a new form of paroxysmal exercise-induced dyskinesia (PED) in dogs. The genetic findings suggest that PCK2:p.Arg553Gln should be further investigated as putative candidate causal variant.

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Abstract 17: APOC3 A43T Variant Promotes ApoC-III Catabolism and Accelerates TG-rich Lipoprotein Clearance in Mice and Humans

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.17 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Sumeet A Khetarpal ◽

John S Millar ◽

Amritha Varshini ◽

Cecilia Vitali ◽

Xuemei Zeng ◽

...

Keyword(s):

Viral Vectors ◽

Missense Variant ◽

Protective Mechanism ◽

Loss Of Function ◽

Humanized Mouse ◽

Gene Encoding ◽

Hepatic Expression ◽

Apolipoprotein C ◽

Human Participants

Humans with loss-of-function (LoF) variants in APOC3 , the gene encoding apolipoprotein C-III (apoC-III), have significantly reduced plasma triglycerides (TG) and protection from coronary disease. These findings suggest that apoC-III may be a viable therapeutic target for decreasing vascular risk through TG reduction, and that elucidation of the protective mechanism of APOC3 LoF variants would inform such strategies. We report here the protective mechanism of the APOC3 A43T missense variant, one of four recently identified CAD-protective variants. By genotyping >8,000 human participants with low TG, we identified 17 APOC3 A43T carriers and phenotyped 6 carriers and 54 matched controls. A43T heterozygotes demonstrate a significant reduction in apoC-III levels relative to non-carriers (50% reduction, P<0.05), resulting in decreased plasma TG (50% reduction, P<0.05). We generated viral vectors expressing WT or A43T apoC-III and expressed these in humanized mouse models to further explore the mechanism of reduced apoC-III levels due to the A43T variant. Mice expressing human CETP and the apoC-III A43T variant exhibit reduced plasma apoC-III (50% reduction, P<0.0001) despite equal hepatic expression and secretion relative to controls expressing WT human apoC-III. These mice also exhibit reduced plasma TG and VLDL-C, and increased HDL-C relative to WT-expressing mice, fully recapitulating the protective lipoprotein profile of the human A43T carriers. Radioisotope-labeled apoC-III turnover studies showed that the A43T mutation causes a >3-fold higher apoC-III clearance rate in vivo (P<0.0001) due to defective integration into lipoprotein particles and accelerated renal catabolism (40% increase, P<0.01). This results in increased lipoprotein lipase (LPL) activity (27% increase, P<0.01) and faster chylomicron-TG clearance (97% increase, P<0.01) in vivo . We are currently performing analogous studies of WT vs. A43T apoC-III turnover and VLDL clearance in human APOC3 A43T carriers. Collectively, our results support the rationale for therapeutic efforts to target circulating apoC-III through disruption of its binding to lipoproteins, mirroring the genetics-driven approaches for targeting PCSK9 that have recently yielded novel therapies.

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Aldo-Keto Reductase 1C1 (AKR1C1) as the First Mutated Gene in a Family with Nonsyndromic Primary Lipedema

International Journal of Molecular Sciences ◽

10.3390/ijms21176264 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6264

Author(s):

Sandro Michelini ◽

Pietro Chiurazzi ◽

Valerio Marino ◽

Daniele Dell’Orco ◽

Elena Manara ◽

...

Keyword(s):

Subcutaneous Fat ◽

Missense Variant ◽

Bioinformatic Analysis ◽

Fat Deposition ◽

Upper Body ◽

Loss Of Function ◽

Gene Encoding ◽

Efficient Reduction ◽

Inactive Form ◽

Dynamics Simulations

Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.

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Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

Cell Death Discovery ◽

10.1038/s41420-019-0224-0 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Adrian Israel Lehvy ◽

Guy Horev ◽

Yarden Golan ◽

Fabian Glaser ◽

Yael Shammai ◽

...

Keyword(s):

Malignant Tumors ◽

Zinc Homeostasis ◽

Healthy Population ◽

Missense Mutations ◽

Protein Alignment ◽

Loss Of Function ◽

Intracellular Zinc ◽

Dismal Prognosis ◽

Zinc Levels ◽

And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

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The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400692 ◽

2019 ◽

Vol 10 (1) ◽

pp. 199-210 ◽

Cited By ~ 1

Author(s):

Chuanman Zhou ◽

Jintao Luo ◽

Xiaohui He ◽

Qian Zhou ◽

Yunxia He ◽

...

Keyword(s):

Plant Hormone ◽

Neuronal Excitability ◽

Resting Membrane Potential ◽

Loss Of Function ◽

Wild Type ◽

C Elegans ◽

Link Type ◽

Complex Functions ◽

And Function ◽

Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

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Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

The Journal of Membrane Biology ◽

10.1007/s00232-021-00184-z ◽

2021 ◽

Author(s):

Vitalii Kryvenko ◽

Olga Vagin ◽

Laura A. Dada ◽

Jacob I. Sznajder ◽

István Vadász

Keyword(s):

Endoplasmic Reticulum ◽

Intracellular Signaling ◽

Loss Of Function ◽

Α Subunit ◽

Rate Limiting Step ◽

Atpase Subunits ◽

A Cell ◽

Health And Disease ◽

And Function ◽

Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

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GWAS of serum ALT and AST reveals an association of SLC30A10 Thr95Ile with hypermanganesemia symptoms

Nature Communications ◽

10.1038/s41467-021-24563-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lucas D. Ward ◽

Ho-Chou Tu ◽

Chelsea B. Quenneville ◽

Shira Tsour ◽

Alexander O. Flynn-Carroll ◽

...

Keyword(s):

Bile Duct ◽

Extrahepatic Bile Duct ◽

Association Studies ◽

Missense Variant ◽

Deficiency Anemia ◽

Genome Wide Association Studies ◽

Loss Of Function ◽

Extrahepatic Bile Duct Cancer ◽

Hepatocellular Damage ◽

Increased Risk

AbstractUnderstanding mechanisms of hepatocellular damage may lead to new treatments for liver disease, and genome-wide association studies (GWAS) of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum activities have proven useful for investigating liver biology. Here we report 100 loci associating with both enzymes, using GWAS across 411,048 subjects in the UK Biobank. The rare missense variant SLC30A10 Thr95Ile (rs188273166) associates with the largest elevation of both enzymes, and this association replicates in the DiscovEHR study. SLC30A10 excretes manganese from the liver to the bile duct, and rare homozygous loss of function causes the syndrome hypermanganesemia with dystonia-1 (HMNDYT1) which involves cirrhosis. Consistent with hematological symptoms of hypermanganesemia, SLC30A10 Thr95Ile carriers have increased hematocrit and risk of iron deficiency anemia. Carriers also have increased risk of extrahepatic bile duct cancer. These results suggest that genetic variation in SLC30A10 adversely affects more individuals than patients with diagnosed HMNDYT1.

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