Genetic causes of Parkinson’s disease in the Maltese : a study of selected mutations in LRRK2, MTHFR, QDPR and SPR

The samples and data used in this study were collected as part of the 5th framework (FP5) EU funded Geoparkinson study, project number QLK4‐CT‐ 1999‐01133. The Maltese arm of this group included Prof Christian Scerri, Dr Joseph Borg, Dr Karen Cassar, Ms Wilma Cassar, Ms Ruth Galdies, Dr Norbert Vella, Dr Vicky Mifsud, Dr Josanne Aquilina and Dr Galea Debono.Background: Mutations in Leucine-rich repeat kinase 2 NM_198578 (LRRK2 c.6055G>A (p.G2019S), LRRK2 c.4321C>G (p.R1441G)) and alpha-synuclein NM_000345 (SNCA c.209G>A (p.A53T)) genes causing Parkinson's disease (PD) are common in Mediterranean populations. Variants in the Quinoid Dihydropteridine Reductase NM_000320 (QDPR c.68G>A (p.G23D)), Sepiapterin Reductase NM_003124 (SPR c.596-2A>G) and Methylenetetrahydrofolate Reductase NM_005957 (MTHFR c.677C>T and c.1298A>C) genes are frequent in Malta and potential candidates for PD. Methods: 178 cases and 402 control samples from Malta collected as part of the Geoparkinson project were genotyped for MTHFR polymorphisms, QDPR and SPR mutations. Only PD and parkinsonism cases were tested for SNCA and LRRK2 mutations. Results: LRRK2 c.4321C>G and SNCA c.209G>A were not detected. The LRRK2 c.6055G>A mutation was found in 3.1 % of Maltese PD cases. The QDPR mutation was found in both cases and controls and did not increase risk for PD. The SPR mutation was found in controls only. The odds ratios for MTHFR polymorphisms were not elevated. Conclusions: The LRRK2 c.6055G>A is a cause of PD in the Maltese, whilst QDPR c.68G>A, SPR c.596-2A>G and MTHFR c.677C>T and c.1298A>C are not important determinants of PD.The samples and data used in this study were collected as part of the 5th framework (FP5) EU funded Geoparkinson study, project number QLK4‐CT‐ 1999‐01133. This work was supported by research grants of SBW and RF from the University of Malta. The funders played no other part in the research or its interpretation.peer-reviewe

CYP2C19 genetic polymorphisms in Maltese patients on clopidogrel therapy

Introduction and Aims: The prevalence of CYP2C19 genetic polymorphisms in the Maltese population is not reported. The aims were to determine CYP2C19 *2 and *17 allele frequencies and CYP2C19 genotype distribution in a cohort of Maltese patients on clopidogrel and to compare observed frequencies of the CYP2C19 *2 allele and *2/*2 genotype to other populations bordering the Mediterranean Sea. Methods: Genotyping for the CYP2C19 *2 and *17 alleles in Maltese patients on clopidogrel was performed using TaqMan® drug metabolism assays. The frequency of both alleles and six genotypes (*1/*1, *1/*2, *2/*2, *1/*17, *17/*17, *2/*17) were determined. Observed frequencies of the CYP2C19 *2 allele and *2/*2 genotype were compared to fourteen populations bordering the Mediterranean Sea (p>0.05 indicated similar prevalence) Results: Frequencies of the CYP2C19 *2 and *17 alleles in the 244 patients genotyped were 12.3% and 15.4% respectively. CYP2C19 genotype distribution was: *1/*1 (52.1%), *1/*17 (22.5%), *1/*2 (18.0%), 2/*17 (6.6%), *17/*17 (0.8%) and *2/*2 (0). Prevalence of the *2 allele in the Maltese cohort was similar to all fourteen populations bordering the Mediterranean Sea, while prevalence of *2/*2 was similar to Egyptian, Moroccan, Southern French, Slovenian, Turkish and Tunisian populations (p>0.05). Conclusions: This study provides an indication of the prevalence of CYP2C19 polymorphisms in Maltese patients. The high percentage of patients with CYP2C19 IM or UM phenotype demonstrates that CYP2C19 genotyping could aid clinicians to individualise treatment with clopidogrel and other drugs metabolised by the CYP2C19 enzyme.peer-reviewe

CYP2C19*2 allele carrier status and coronary in-stent restenosis : is there an association?

Background and objective: The CYP2C19*2 allele is associated with reduced clopidogrel bioactivation, increasing the risk of complications after percutaneous coronary intervention (PCI), particularly stent thrombosis. Recently published data suggests that CYP2C19*2 allele carriers have a higher risk for in-stent restenosis (ISR) after endovascular treatment. Very few studies have investigated the relationship between CYP2C19*2 and coronary ISR, with no significant association reported. The objective of this study was to assess the relationship between CYP2C19*2 allele carrier status and coronary ISR. Methods: Patients with previous PCI with stenting and who were scheduled for elective PCI after coronary angiogram were recruited from the cardiac catheterization suite over a 12-month period. The angiography report of each patient was perused to identify patients requiring PCI due to ISR. For patients with angiography-confirmed ISR, date of previous PCI to the restenosed stent was noted. CYP2C19*2 genotyping was undertaken using a TaqMan® Drug Metabolism assay. The association between CYP2C19*2 allele carrier status and incidence of coronary ISR within 1 year was assessed using Fisher’s exact test (p < 0.05 significance) and by calculating the odds ratio (OR) with a 95% confidence interval (CI). Results: Of the 82 patients with previous PCI, 29 (35.4%) had angiography-confirmed ISR (12 carriers, 17 noncarriers of CYP2C19*2). In 13 (44.8%) of these patients, the restenosed stent was deployed within 1 year and the patients were on clopidogrel therapy at the time of repeat PCI (8 carriers, 5 non-carriers of CYP2C19*2). The association between CYP2C19*2 allele carrier status and ISR within 1 year was not statistically significant (Fisher’s exact p = 0.067; OR: 4.80, 95% CI: 0.98–23.54, p = 0.053). Conclusions: Despite a higher proportion of CYP2C19*2 allele carriers exhibiting ISR within 1 year compared to non-carriers, the association was not statistically significant. This result may be attributed to the small sample size, and larger prospective studies are recommended to further assess this association.peer-reviewe

Genotype-phenotype of autosomal dominant polycystic kidney disease in Malta

Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features.customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results.Results: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1.Conclusions: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.peer-reviewe

The distribution and prevalence of HPV genotype in Maltese women diagnosed with CIN 3 and cervical cancer

Introduction: The Human papilloma virus (HPV) is the causative agent of cervical carcinoma in women. There is a global variation of HPV genotypes that are highly carcinogenic. The aim of this study was to estimate the prevalence and type - specific distribution of HPV genotype in Maltese patients who were previously diagnosed with cervical intraepithelial neoplasia 3 (CIN 3) or cervical carcinoma.peer-reviewe

Identification of antibiotic resistance patterns in Helicobacter pylori strains isolated from gastric biopsies using real-time PCR and genotypic analysis

Background Helicobacter pylori (H. pylori) is associated with dyspepsia, mucus-associated lymphoid tissue lymphoma, gastritis, and peptic ulcer disease. Treatment in Malta consists of triple therapy, which consists of a proton pump inhibitor and 2 of the antibiotics amoxicillin, clarithromycin, metronidazole and fluoroquinolones. We aimed to determine the resistance rates for clarithromycin and fluoroquinolones in patients with H. pylori, and its incidence, in patients undergoing an esophagogastroduodenoscopy (EGD) using real-time polymerase chain reaction (RT-PCR). Methods Patients undergoing an EGD were recruited. A rapid urease test (RUT) was performed, and 4 gastric biopsies were also taken (2 from antrum, 2 from corpus) and analyzed using RT-PCR. Positive samples were tested for antibiotic resistance using amplification and reverse hybridization techniques. Results Two hundred patients (mean age 53.6 [range 20-92] years; 53.1% female) were recruited; the majority were (78%) non-smokers. H. pylori was identified in 21.0% of the patients. Fluoroquinolone resistance was detected in 21.4% of the patients. Clarithromycin resistance was observed in 26.2%, with dual resistance identified in 4.8% of the patients. A high concordance was present with patients testing negative for H. pylori with both RUT and RT-PCR (94.3%). Only 57.6% of patients tested positive with both tests. However, 92.9% of RT-PCR positive patients had a positive genotype HelicoDR test. Conclusions This data demonstrates a high rate of H. pylori resistance to both clarithromycin and fluoroquinolones. These should be avoided when treating H. pylori by utilizing different treatment regimes. Furthermore, we derived important data on the role of RT-PCR, which may be implemented in routine clinical practice.peer-reviewe

Dysbiosis in the gut microbiota in patients with inflammatory bowel disease during remission

Inflammatory bowel disease (IBD) is a chronic, relapsing, inflammatory disorder which comprises two main conditions: Crohn’s disease (CD) and ulcerative colitis (UC). Although the etiology of IBD has not been fully elucidated, the gut microbiota is hypothesized to play a vital role in its development. The aim of this cross-sectional study was to characterize the fecal microbiota in CD or UC patients in a state of remission to reveal potential factors sustaining residual levels of inflammation and triggering disease relapses. Ninety-eight IBD patients in a state of clinical remission (66 UC, 32 CD) and 97 controls were recruited, and stool samples, as well as detailed patient data, were collected. After DNA extraction, the variable regions V1 and V2 of the 16S rRNA gene were amplified and sequenced. Patients with IBD had a decrease in alpha diversity compared to that of healthy controls, and the beta diversity indices showed dissimilarity between the cohorts. Healthy controls were associated with the beneficial organisms unclassified Akkermansia species (Akkermansia uncl.), Oscillibacter uncl., and Coprococcus uncl., while flavonoid-degrading bacteria were associated with IBD. Network analysis identified highly central and influential disease markers and a strongly correlated network module of Enterobacteriaceae which was associated with IBD and could act as drivers for residual inflammatory processes sustaining and triggering IBD, even in a state of low disease activity. The microbiota in IBD patients is significantly different from that of healthy controls, even in a state of remission, which implicates the microbiota as an important driver of chronicity in IBD. IMPORTANCE Dysbiosis in inflammatory bowel disease (IBD) has been implicated as a causal or contributory factor to the pathogenesis of the disease. This study, done on patients in remission while accounting for various confounding factors, shows significant community differences and altered community dynamics, even after acute inflammation has subsided. A cluster of Enterobacteriaceae was linked with Crohn’s disease, suggesting that this cluster, which contains members known to disrupt colonization resistance and form biofilms, persists during quiescence and can lead to chronic inflammation. Flavonoid-degrading bacteria were also associated with IBD, raising the possibility that modification of dietary flavonoids might induce and maintain remission in IBD.peer-reviewe

Microbial dynamics in newly diagnosed and treatment naïve IBD patients in the Mediterranean

Background: Microbial communities have long been suspected to influence inflammatory processes in the gastrointestinal tract of patients with inflammatory bowel disease. However, these effects are often influenced by treatments and can rarely be analyzed in treatment-naïve onset cases. Specifically, microbial differences between IBD pathologies in new onset cases have rarely been investigated and can provide novel insight into the dynamics of the microbiota in Crohn’s disease (CD) and ulcerative colitis (UC). Methods: Fifty-six treatment-naïve IBD onset patients (67.3% CD, 32.7% UC) and 97 healthy controls were recruited from the Maltese population. Stool samples were collected after diagnosis but before administration of anti-inflammatory treatments. Fecal microbial communities were assessed via 16S rRNA gene sequencing and subjected to ecological analyses to determine disease-specific differences between pathologies and disease subtypes or to predict future treatment options. Results: We identified significant differences in community composition, variability, and diversity between healthy and diseased individuals—but only small to no differences between the newly diagnosed, treatment-naïve UC and CD cohorts. Network analyses revealed massive turn-over of bacterial interactions between healthy and diseased communities, as well as between CD and UC communities, as signs of disease-specific changes of community dynamics. Furthermore, we identified taxa and community characteristics serving as predictors for prospective treatments. Conclusion: Untreated and newly diagnosed IBD shows clear differences from healthy microbial communities and an elevated level of disturbance, but only the network perspective revealed differences between pathologies. Furthermore, future IBD treatment is to some extent predictable by microbial community characteristics.peer-reviewe

Repositioning of the global epicentre of non-optimal cholesterol

High blood cholesterol is typically considered a feature of wealthy western countries(1,2). However, dietary and behavioural determinants of blood cholesterol are changing rapidly throughout the world(3) and countries are using lipid-lowering medications at varying rates. These changes can have distinct effects on the levels of high-density lipoprotein (HDL) cholesterol and non-HDL cholesterol, which have different effects on human health(4,5). However, the trends of HDL and non-HDL cholesterol levels over time have not been previously reported in a global analysis. Here we pooled 1,127 population-based studies that measured blood lipids in 102.6 million individuals aged 18 years and older to estimate trends from 1980 to 2018 in mean total, non-HDL and HDL cholesterol levels for 200 countries. Globally, there was little change in total or non-HDL cholesterol from 1980 to 2018. This was a net effect of increases in low- and middle-income countries, especially in east and southeast Asia, and decreases in high-income western countries, especially those in northwestern Europe, and in central and eastern Europe. As a result, countries with the highest level of non-HDL cholesterol-which is a marker of cardiovascular riskchanged from those in western Europe such as Belgium, Finland, Greenland, Iceland, Norway, Sweden, Switzerland and Malta in 1980 to those in Asia and the Pacific, such as Tokelau, Malaysia, The Philippines and Thailand. In 2017, high non-HDL cholesterol was responsible for an estimated 3.9 million (95% credible interval 3.7 million-4.2 million) worldwide deaths, half of which occurred in east, southeast and south Asia. The global repositioning of lipid-related risk, with non-optimal cholesterol shifting from a distinct feature of high-income countries in northwestern Europe, north America and Australasia to one that affects countries in east and southeast Asia and Oceania should motivate the use of population-based policies and personal interventions to improve nutrition and enhance access to treatment throughout the world.Peer reviewe

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707