Cysteine modification reveals an allosteric inhibitory site on the CAL PDZ domain.

Biosci Rep. 2018 Feb 22;:

Authors: Zhao Y, Cushing PR, Smithson DC, Pellegrini M, Pletnev AA, Al-Ayyoubi S, Grassetti AV, Gerber SA, Guy RK, Madden DR

Abstract
Protein-protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that play important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of a PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and mediates an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action exploits the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein-protein binding modules.

PMID: 29472314 [PubMed - as supplied by publisher]

The relationship between energy intake and body-growth in children with cystic fibrosis.

Clin Nutr. 2018 Feb 15;:

Authors: Woestenenk JW, Dalmeijer GW, van der Ent CK, Houwen RH

Abstract
BACKGROUND & AIMS: Body-growth, expressed as weight- and height gain, is a strong predictor of morbidity and mortality in patients with cystic fibrosis (CF). Whether current historically based recommendations on a high-energy diet are sufficient for optimal growth is questionable. We therefore assessed the longitudinal relation between body-growth and routine energy intake in paediatric CF patients.
METHODS: Included were patients with CF, aged 2-10 years of whom we obtained 969 measurements of weight and height along with dietary records, and 786 coefficient of fat absorption measurements (CFA). We described body-growth, energy intake, macronutrient intake and the long-term effect of energy intake and coefficient of fat absorption on body-growth during the 8-year follow-up period.
RESULTS: Enrolled were 191 children with CF who had a compromised growth when compared to healthy children. The dietary intake was ≥110% estimated average requirement (EAR) in 47% of the measurements (457/969) and did not (fully) achieve the recommended high-energy level (110-200% EAR). Further, the intake expressed as EAR decreased with increasing age. Cross-sectionally, boys and girls with higher caloric intakes had higher weight-for-age (WFA). The caloric intake explained 18 and 6% of the variation. Further, boys with higher caloric intakes had also higher height-for-age-adjusted-for-target-height (HFA/TH) or BMI. The caloric intake explained 6 or 7% of the variation. Longitudinally, caloric intake was associated with both WFA in boys and girls, and with BMI in boys. Each 100 calories increased intake would result in a 0.01 (girls)-0.02 increase in z-score WFA and 0.03 increase in z-score BMI. We found no significant association between CFA and WFA, HFA/TH or BMI. The contribution of protein, fat and carbohydrates was not associated with WFA, nor with HFA/TH or BMI.
CONCLUSION: Even at this relatively early age, a compromised growth in children with CF was found when compared to healthy children. The energy intake was below 110% EAR in 47% of the measurements, and appeared to be insufficient to prevent suboptimal body-growth over the 8-years of follow-up.

PMID: 29472121 [PubMed - as supplied by publisher]

A novel mutation of dipeptidyl aminopeptidase-like protein-6 in a family with suspicious idiopathic ventricular fibrillation.

QJM. 2018 Feb 20;:

Authors: Ding DB, Fan LL, Xiao Z, Huang H, Chen YQ, Guo S, Liu ZH, Xiang R

Abstract
Background: Sudden cardiac death (SCD) occurs in a broad spectrum of cardiac pathologies and is an important cause of mortality in the general population. Idiopathic ventricular fibrillation (IVF) is a rare but important factor resulting in SCD. It is diagnosed in a resuscitated cardiac arrest victim underlying unknown cause, with documented ventricular fibrillation. Previous studies have demonstrated that mutations in dipeptidyl aminopeptidase-like protein-6 (DPP6) and cardiac sodium channel Nav1.5 (SCN5A) are the most important genetic factors involve in IVF.
Methods: In this study, we employed whole-exome sequencing in combination with arrhythmia-related gene filtering to identify the genetic lesion for a family suffering from suspicious IVF, syncope and SCD. We then generated the plasmids of DPP6-pcDNA3.1 + (WT and c.1578G>C/p.Q526H). Kv4.3-pcDNA3.1+ was co-transfected together with/without DPP6-pcDNA3.1 + (WT and/or c.1578G>C/p.Q526H) into HEK293 cells to perform the patch clamp experiments.
Results: A novel missense mutation (c.1578G>C/p.Q526H) of DPP6 was identified and co-segregated with affected patients in this family. Patch clamp experiments suggested that this novel mutation might result in a gain of function and disturb the efflux of potassium ion.
Conclusion: Our study not only reported the second missense mutation of DPP6 in heart disease and expanded the spectrum of DPP6 mutations, but also contribute to the genetic diagnosis and counseling of families with suspicious IVF, syncope and SCD.

PMID: 29474731 [PubMed - as supplied by publisher]

AP4 deficiency: A novel form of neurodegeneration with brain iron accumulation?

Neurol Genet. 2018 Feb;4(1):e217

Authors: Roubertie A, Hieu N, Roux CJ, Leboucq N, Manes G, Charif M, Echenne B, Goizet C, Guissart C, Meyer P, Marelli C, Rivier F, Burglen L, Horvath R, Hamel CP, Lenaers G

Abstract
Objective: To describe the clinico-radiological phenotype of 3 patients harboring a homozygous novel AP4M1 pathogenic mutation.
Methods: The 3 patients from an inbred family who exhibited early-onset developmental delay, tetraparesis, juvenile motor function deterioration, and intellectual deficiency were investigated by magnetic brain imaging using T1-weighted, T2-weighted, T2*-weighted, fluid-attenuated inversion recovery, susceptibility weighted imaging (SWI) sequences. Whole-exome sequencing was performed on the 3 patients.
Results: In the 3 patients, brain imaging identified the same pattern of bilateral SWI hyposignal of the globus pallidus, concordant with iron accumulation. A novel homozygous nonsense mutation was identified in AP4M1, segregating with the disease and leading to truncation of half of the adap domain of the protein.
Conclusions: Our results suggest that AP4M1 represents a new candidate gene that should be considered in the neurodegeneration with brain iron accumulation (NBIA) spectrum of disorders and highlight the intersections between hereditary spastic paraplegia and NBIA clinical presentations.

PMID: 29473051 [PubMed]

Familial monophasic acute transverse myelitis due to the pathogenic variant in VPS37A.

Neurol Genet. 2018 Feb;4(1):e213

Authors: Mealy MA, Nam TS, Pardo SJ, Pardo CA, Sobreira NL, Avramopoulos D, Valle D, Burns KH, Levy M

Abstract
Objective: To identify genetic differences among siblings with a family history of idiopathic transverse myelitis (ITM).
Methods: We compared whole-exome sequencing (WES) on germline samples from the 2 affected sisters with ITM with 3 of their healthy siblings.
Results: The 2 sisters with ITM both had acute onset of sensory loss in the legs, weakness, and bowel/bladder dysfunction. The first developed ITM at age 15 years with a clinical nadir of complete paralysis, which slowly recovered over a few years. MRI demonstrated a persistent T2 lesion in the lower thoracic cord. The second developed ITM at age 50 years with a nadir of sensory loss from T6 down and paraparesis in the legs, associated with an MRI lesion at T6. She also made a partial recovery with treatment. Both sisters are homozygous for a missense variant in VPS37A (c.700C>A, p.Leu234Ile) identified by WES. We performed targeted sequencing of VPS37A in an additional 86 samples from patients with ITM and 175 with other diseases to investigate the p.Leu234Ile variant. We identified another patient with ITM homozygous for the same rare variant. No patients with multiple sclerosis, neuromyelitis optica, other neurologic conditions, or any healthy controls in public databases were homozygous for this variant.
Conclusions: A rare missense variant in VPS37A may predispose to development of ITM. Further studies are necessary to determine the frequency of this variant in the patient population and the mechanism through which it contributes to the risk of disease.

PMID: 29473047 [PubMed]

Related Articles

Modeling Genomic Instability and Selection Pressure in a Mouse Model of Melanoma.

Cell Rep. 2017 May 16;19(7):1304-1312

Authors: Kwong LN, Zou L, Chagani S, Pedamallu CS, Liu M, Jiang S, Protopopov A, Zhang J, Getz G, Chin L

Abstract
Tumor evolution is an iterative process of selection for pro-oncogenic aberrations. This process can be accelerated by genomic instability, but how it interacts with different selection bottlenecks to shape the evolving genomic landscape remains understudied. Here, we assessed tumor initiation and therapy resistance bottlenecks in mouse models of melanoma, with or without genomic instability. At the initiation bottleneck, whole-exome sequencing revealed that drug-naive tumors were genomically silent, and this was surprisingly unaffected when genomic instability was introduced via telomerase inactivation. We hypothesize that the strong engineered alleles created low selection pressure. At the therapy resistance bottleneck, strong selective pressure was applied using a BRAF inhibitor. In the absence of genomic instability, tumors acquired a non-genomic drug resistance mechanism. By contrast, telomerase-deficient, drug-resistant melanomas acquired highly recurrent copy number gains. These proof-of-principle experiments demonstrate how different selection pressures can interact with genomic instability to impact tumor evolution.

PMID: 28514651 [PubMed - indexed for MEDLINE]

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GUCA1A mutation causes maculopathy in a five-generation family with a wide spectrum of severity.

Genet Med. 2017 Aug;19(8):945-954

Authors: Chen X, Sheng X, Zhuang W, Sun X, Liu G, Shi X, Huang G, Mei Y, Li Y, Pan X, Liu Y, Li Z, Zhao Q, Yan B, Zhao C

Abstract
PURPOSE: The aim of this study was to investigate the genetic basis and pathogenic mechanism of variable maculopathies, ranging from mild photoreceptor degeneration to central areolar choroidal dystrophy, in a five-generation family.
METHODS: Clinical characterizations, whole-exome sequencing, and genome-wide linkage analysis were carried out on the family. Zebrafish models were used to investigate the pathogenesis of GUCA1A mutations.
RESULTS: A novel mutation, GUCA1A p.R120L, was identified in the family and predicted to alter the tertiary structure of guanylyl cyclase-activating protein 1, a photoreceptor-expressed protein encoded by the GUCA1A gene. The mutation was shown in zebrafish to cause significant disruptions in photoreceptors and retinal pigment epithelium, together with atrophies of retinal vessels and choriocapillaris. Those phenotypes could not be fully rescued by exogenous wild-type GUCA1A, suggesting a likely gain-of-function mechanism for p.R120L. GUCA1A p.D100E, another mutation previously implicated in cone dystrophy, also impaired the retinal pigment epithelium and photoreceptors in zebrafish, but probably via a dominant negative effect.
CONCLUSION: We conclude that GUCA1A mutations could cause significant variability in maculopathies, including central areolar choroidal dystrophy, which represents a severe pattern of maculopathy. The diverse pathogenic modes of GUCA1A mutations may explain the phenotypic diversities.Genet Med advance online publication 26 January 2017.

PMID: 28125083 [PubMed - indexed for MEDLINE]

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Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles.

Eur J Prev Cardiol. 2017 Mar;24(5):492-504

Authors: Gregson JM, Freitag DF, Surendran P, Stitziel NO, Chowdhury R, Burgess S, Kaptoge S, Gao P, Staley JR, Willeit P, Nielsen SF, Caslake M, Trompet S, Polfus LM, Kuulasmaa K, Kontto J, Perola M, Blankenberg S, Veronesi G, Gianfagna F, Männistö S, Kimura A, Lin H, Reilly DF, Gorski M, Mijatovic V, CKDGen consortium, Munroe PB, Ehret GB, International Consortium for Blood Pressure, Thompson A, Uria-Nickelsen M, Malarstig A, Dehghan A, CHARGE inflammation working group, Vogt TF, Sasaoka T, Takeuchi F, Kato N, Yamada Y, Kee F, Müller-Nurasyid M, Ferrières J, Arveiler D, Amouyel P, Salomaa V, Boerwinkle E, Thompson SG, Ford I, Wouter Jukema J, Sattar N, Packard CJ, Shafi Majumder AA, Alam DS, Deloukas P, Schunkert H, Samani NJ, Kathiresan S, MICAD Exome consortium, Nordestgaard BG, Saleheen D, Howson JM, Di Angelantonio E, Butterworth AS, Danesh J, EPIC-CVD consortium and the CHD Exome+ consortium

Abstract
Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10-25) with carriage of any of the four loss-of-function variants, by 45% ( p < 10-300) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10-12) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10-300). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.

PMID: 27940953 [PubMed - indexed for MEDLINE]

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Congenital Sucrase-isomaltase Deficiency: A Novel Compound Heterozygous Mutation Causing Aberrant Protein Localization.

J Pediatr Gastroenterol Nutr. 2017 May;64(5):770-776

Authors: Haberman Y, Di Segni A, Loberman-Nachum N, Barel O, Kunik V, Eyal E, Kol N, Hout-Siloni G, Kochavi B, Avivi C, Schvimer M, Rechavi G, Anikster Y, Barshack I, Weiss B

Abstract
OBJECTIVES: Congenital diarrheal disorders is a group of inherited enteropathies presenting in early life and requiring parenteral nutrition. In most cases, genetics may be the key for precise diagnosis. We present an infant girl with chronic congenital diarrhea that resolved after introduction of fructose-based formula but had no identified mutation in the SLC5A1 gene. Using whole exome sequencing (WES) we identified other mutations that better dictated dietary adjustments.
METHODS: WES of the patient and her parents was performed. The analysis focused on recessive model including compound heterozygous mutations. Sanger sequencing was used to validate identified mutations and to screen the patient's newborn sister and grandparents. Expression and localization analysis were performed in the patient's duodenal biopsies using immunohistochemistry.
RESULTS: Using WES we identified a new compound heterozygote mutation in sucrase-isomaltase (SI) gene; a maternal inherited known V577G mutation, and a novel paternal inherited C1531W mutation. Importantly, the newborn offspring carried similar compound heterozygous mutations. Computational predictions suggest that both mutations highly destabilize the protein. SI expression and localization studies determined that the mutated SI protein was not expressed on the brush border membrane in the patient's duodenal biopsies, verifying the diagnosis of congenital sucrase-isomaltase deficiency (CSID).
CONCLUSIONS: The novel compound heterozygote V577G/C1531W SI mutations lead to lack of SI expression in the duodenal brush border, confirming the diagnosis of CSID. These cases of CSID extend the molecular spectrum of this condition, further directing a more adequate dietary intervention for the patient and newborn sibling.

PMID: 27749612 [PubMed - indexed for MEDLINE]

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Nonlinear tumor evolution from dysplastic nodules to hepatocellular carcinoma.

Oncotarget. 2017 Jan 10;8(2):2076-2082

Authors: Joung JG, Ha SY, Bae JS, Nam JY, Gwak GY, Lee HO, Son DS, Park CK, Park WY

Abstract
Dysplastic nodules are premalignant neoplastic nodules found in explanted livers with cirrhosis. Genetic signatures of premalignant dysplastic nodules (DNs) with concurrent hepatocellular carcinoma (HCC) may provide an insight in the molecular evolution of hepatocellular carcinogenesis. We analyzed four patients with multifocal nodular lesions and cirrhotic background by whole-exome sequencing (WES). The genomic profiles of somatic single nucleotide variations (SNV) and copy number variations (CNV) in DNs were compared to those of HCCs. The number and variant allele frequency of somatic SNVs of DNs and HCCs in each patient was identical along the progression of pathological grade. The somatic SNVs in DNs showed little conservation in HCC. Additionally, CNVs showed no conservation. Phylogenetic analysis based on SNVs and copy number profiles indicated a nonlinear segregation pattern, implying independent development of DNs and HCC in each patient. Thus, somatic mutations in DNs may be developed separately from other malignant nodules in the same liver, suggesting a nonlinear model for hepatocarcinogenesis from DNs to HCC.

PMID: 27409339 [PubMed - indexed for MEDLINE]

Funding Opportunity PA-18-680 from the NIH Guide for Grants and Contracts. This FOA is to be used to request administrative supplements for awards made to PAR-12-085 and PAR-15-167 "NIBIB Research Education Programs for Residents and Clinical Fellows program". This Funding Opportunity Announcement (FOA) does not allow appointed trainees to lead an independent clinical trial, but does allow them to obtain research experience in a clinical trial led by a mentor or co-mentor.

Funding Opportunity PA-18-678 from the NIH Guide for Grants and Contracts. The purpose of this concept initiative is to promote etiologic research investigating novel and innovative hypotheses on emerging risk factors (biological, environmental, and social) and their interplay with established risk factors (e.g., viral hepatitis) associated with the development of liver cancer (hepatocellular carcinoma and other histological subtypes) in the United States.

Funding Opportunity PA-18-677 from the NIH Guide for Grants and Contracts. The purpose of this concept initiative is to promote etiologic research investigating novel and innovative hypotheses on emerging risk factors (biological, environmental, and social) and their interplay with established risk factors (e.g., viral hepatitis) associated with the development of liver cancer (hepatocellular carcinoma and other histological subtypes) in the United States.

Funding Opportunity PAR-18-679 from the NIH Guide for Grants and Contracts. The purpose of the NIAID Physician-Scientist Pathway to Independence Award (K99/R00) program is to increase and maintain a strong cohort of new and talented independent physician-scientists. This program is designed to facilitate a timely transition of outstanding postdoctoral researchers with a clinical doctorate degree from mentored, postdoctoral research positions to independent, tenure-track or equivalent faculty positions. The program will provide independent NIAID research support during this transition to help awardees launch competitive, independent research careers in biomedical fields and thereby help to address the national physician-scientist workforce shortage.

Notice NOT-AT-18-006 from the NIH Guide for Grants and Contracts

Notice NOT-GM-18-020 from the NIH Guide for Grants and Contracts

Notice NOT-GM-18-019 from the NIH Guide for Grants and Contracts

Notice NOT-NS-18-046 from the NIH Guide for Grants and Contracts

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