Identification of a functional missense variant in the matrix metallopeptidase 10 (MMP10) gene in two families with premature myocardial infarction.

A positive family history is a major independent risk factor for atherosclerosis, and genetic variation is an important aspect of cardiovascular disease research. We identified a heterozygous missense variant p.L245P in the MMP10 gene in two families with premature myocardial infarction using whole-exome sequencing. The aim of this study was to investigate the consequences of this variant using in-silico and functional in-vitro assays. Molecular dynamics simulations were used to analyze protein interactions, calculate free binding energy, and measure the volume of the substrate-binding cleft of MMP10-TIMP1 models. The p.L245P variant showed an altered protein surface, different intra- and intermolecular interactions of MMP10-TIMP1, a lower total free binding energy between MMP10-TIMP1, and a volume-minimized substrate-binding cleft of MMP10 compared to the wild-type. For the functional assays, human THP-1 cells were transfected with plasmids containing MMP10 cDNA carrying the p.L245P and wild-type variant and differentiated into macrophages. Macrophage adhesion and migration assays were then conducted, and pro-inflammatory chemokine levels were evaluated. The p.L245P variant led to macrophages that were more adherent, less migratory, and secreted higher levels of the pro-inflammatory chemokines CXCL1 and CXCL8 than wild-type macrophages. Thus, the p.L245P variant in MMP10 may influence the pathogenesis of atherosclerosis in families with premature myocardial infarction by altering protein - protein interactions, macrophage adhesion and migration, and expression of pro-inflammatory chemokines, which may increase plaque rupture. These results could contribute to the development of selective MMP10 inhibitors and reduce the risk of atherosclerosis in families with a history of premature myocardial infarction.

Complete genome sequence of Oligella urethralis MSHR-50412PR, isolated from an ear discharge swab of a child with chronic suppurative otitis media.

Oligella urethralis are opportunistic pathogens typically associated with genitourinary infections. Here, we report the complete genome for an Oligella urethralis isolate recovered from ear discharge of a child with chronic suppurative otitis media (strain MSHR-50412PR). The genome comprises 2.58 Mb, with 2,448 coding sequences and 46.26% average GC content.

Lineage-informative microhaplotypes for spatio-temporal surveillance of Plasmodium vivax malaria parasites.

Challenges in understanding the origin of recurrent Plasmodium vivax infections constrains the surveillance of antimalarial efficacy and transmission of this neglected parasite. Recurrent infections within an individual may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or new inoculations (reinfection). Molecular inference of familial relatedness (identity-by-descent or IBD) based on whole genome sequence data, together with analysis of the intervals between parasitaemic episodes ("time-to-event" analysis), can help resolve the probable origin of recurrences. Whole genome sequencing of predominantly low-density P. vivax infections is challenging, so an accurate and scalable genotyping method to determine the origins of recurrent parasitaemia would be of significant benefit. We have developed a P. vivax genome-wide informatics pipeline to select specific microhaplotype panels that can capture IBD within small, amplifiable segments of the genome. Using a global set of 615 P. vivax genomes, we derived a panel of 100 microhaplotypes, each comprising 3-10 high frequency SNPs within <200 bp sequence windows. This panel exhibits high diversity in regions of the Asia-Pacific, Latin America and the horn of Africa (median HE = 0.70-0.81) and it captured 89% (273/307) of the polyclonal infections detected with genome-wide datasets. Using data simulations, we demonstrate lower error in estimating pairwise IBD using microhaplotypes, relative to traditional biallelic SNP barcodes. Our panel exhibited high accuracy in predicting the country of origin (median Matthew's correlation coefficient >0.9 in 90% countries tested) and it also captured local infection outbreak and bottlenecking events. The informatics pipeline is available open-source and yields microhaplotypes that can be readily transferred to high-throughput amplicon sequencing assays for surveillance in malaria-endemic regions.

Can non-typeable Haemophilus influenzae carriage surveillance data infer antimicrobial resistance associated with otitis media?

Importance: In remote communities of the Northern Territory, Australia, children experience high rates of otitis media (OM), commonly caused by non-typeable Haemophilus influenzae (NTHi). Few data exist on antibiotic susceptibility of NTHi from OM. Objective: To determine whether population-level nasopharyngeal NTHi antibiotic susceptibility data could inform antibiotic treatment for OM. Methods: NTHi isolates (n = 92) collected from ear discharge between 2003 and 2013 were selected to time- and age-match NTHi isolates from the nasopharyngeal carriage (n = 95). Antimicrobial susceptibility were tested. Phylogenomic trees and a genome-wide association study (GWAS) were performed to determine the similarity of nasopharyngeal and ear isolates at a population level. Results: Among 174 NTHi isolates available for antimicrobial susceptibility testing, 10.3% (18/174) were resistant to ampicillin and 9.2% (16/174) were resistant to trimethoprim-sulfamethoxazole. Small numbers of isolates (≤3) were resistant to tetracycline, chloramphenicol, or amoxicillin-clavulanic acid. There was no statistical difference in the proportion of ampicillin-resistant (P = 0.11) or trimethoprim-sulfamethoxazole-resistant isolates (P = 0.70) between ear discharge and nasopharynx-derived NTHi isolates. Three multi-drug resistant NTHi isolates were identified. Phylogenomic trees showed no clustering of 187 Haemophilus influenzae isolates based on anatomical niche (nasopharynx or ear discharge), and no genetic variations that distinguished NTHi derived from ear discharge and nasopharyngeal carriage were evident in the GWAS. Interpretation: In this population-level study, nasopharyngeal and ear discharge isolates did not represent distinct microbial populations. These results support tracking of population-level nasopharyngeal NTHi antibiotic resistance patterns to inform clinical management of OM in this population.

Melioidosis in the remote Katherine region of northern Australia.

Melioidosis is endemic in the remote Katherine region of northern Australia. In a population with high rates of chronic disease, social inequities, and extreme remoteness, the impact of melioidosis is exacerbated by severe weather events and disproportionately affects First Nations Australians. All culture-confirmed melioidosis cases in the Katherine region of the Australian Top End between 1989-2021 were included in the study, and the clinical features and epidemiology were described. The diversity of Burkholderia pseudomallei strains in the region was investigated using genomic sequencing. From 1989-2021 there were 128 patients with melioidosis in the Katherine region. 96/128 (75%) patients were First Nations Australians, 72/128 (56%) were from a very remote region, 68/128 (53%) had diabetes, 57/128 (44%) had a history of hazardous alcohol consumption, and 11/128 (9%) died from melioidosis. There were 9 melioidosis cases attributable to the flooding of the Katherine River in January 1998; 7/9 flood-associated cases had cutaneous melioidosis, five of whom recalled an inoculating event injury sustained wading through flood waters or cleaning up after the flood. The 126 first-episode clinical B. pseudomallei isolates that underwent genomic sequencing belonged to 107 different sequence types and were highly diverse, reflecting the vast geographic area of the study region. In conclusion, melioidosis in the Katherine region disproportionately affects First Nations Australians with risk factors and is exacerbated by severe weather events. Diabetes management, public health intervention for hazardous alcohol consumption, provision of housing to address homelessness, and patient education on melioidosis prevention in First Nations languages should be prioritised.

Emergence of Burkholderia pseudomallei Sequence Type 562, Northern Australia.

Since 2005, the range of Burkholderia pseudomallei sequence type 562 (ST562) has expanded in northern Australia. During 2005-2019, ST562 caused melioidosis in 61 humans and 3 animals. Cases initially occurred in suburbs surrounding a creek before spreading across urban Darwin, Australia and a nearby island community. In urban Darwin, ST562 caused 12% (53/440) of melioidosis cases, a proportion that increased during the study period. We analyzed 2 clusters of cases with epidemiologic links and used genomic analysis to identify previously unassociated cases. We found that ST562 isolates from Hainan Province, China, and Pingtung County, Taiwan, were distantly related to ST562 strains from Australia. Temporal genomic analysis suggested a single ST562 introduction into the Darwin region in ≈1988. The origin and transmission mode of ST562 into Australia remain uncertain.

Whole-genome sequencing of Burkholderia pseudomallei from an urban melioidosis hot spot reveals a fine-scale population structure and localised spatial clustering in the environment.

Melioidosis is a severe disease caused by the environmental bacterium Burkholderia pseudomallei that affects both humans and animals throughout northern Australia, Southeast Asia and increasingly globally. While there is a considerable degree of genetic diversity amongst isolates, B. pseudomallei has a robust global biogeographic structure and genetic populations are spatially clustered in the environment. We examined the distribution and local spread of B. pseudomallei in Darwin, Northern Territory, Australia, which has the highest recorded urban incidence of melioidosis globally. We sampled soil and land runoff throughout the city centre and performed whole-genome sequencing (WGS) on B. pseudomallei isolates. By combining phylogenetic analyses, Bayesian clustering and spatial hot spot analysis our results demonstrate that some sequence types (STs) are widespread in the urban Darwin environment, while others are highly spatially clustered over a small geographic scale. This clustering matches the spatial distribution of clinical cases for one ST. Results also demonstrate a greater overall isolate diversity recovered from drains compared to park soils, further supporting the role drains may play in dispersal of B. pseudomallei STs in the environment. Collectively, knowledge gained from this study will allow for better understanding of B. pseudomallei phylogeography and melioidosis source attribution, particularly on a local level.

Melioidosis fatalities in captive slender-tailed meerkats (Suricata suricatta): combining epidemiology, pathology and whole-genome sequencing supports variable mechanisms of transmission with one health implications.

BACKGROUND: Melioidosis is a tropical infectious disease which is being increasingly recognised throughout the globe. Infection occurs in humans and animals, typically through direct exposure to soil or water containing the environmental bacterium Burkholderia pseudomallei. Case clusters of melioidosis have been described in humans following severe weather events and in exotic animals imported into melioidosis endemic zones. Direct transmission of B. pseudomallei between animals and/or humans has been documented but is considered extremely rare. Between March 2015 and October 2016 eight fatal cases of melioidosis were reported in slender-tailed meerkats (Suricata suricatta) on display at a Wildlife Park in Northern Australia. To further investigate the melioidosis case cluster we sampled the meerkat enclosure and adjacent park areas and performed whole-genome sequencing (WGS) on all culture-positive B. pseudomallei environmental and clinical isolates. RESULTS: WGS confirmed that the fatalities were caused by two different B. pseudomallei sequence types (STs) but that seven of the meerkat isolates were highly similar on the whole-genome level. Used concurrently with detailed pathology data, our results demonstrate that the seven cases originated from a single original source, but routes of infection varied amongst meerkats belonging to the clonal outbreak cluster. Moreover, in some instances direct transmission may have transpired through wounds inflicted while fighting. CONCLUSIONS: Collectively, this study supports the use of high-resolution WGS to enhance epidemiological investigations into transmission modalities and pathogenesis of melioidosis, especially in the instance of a possible clonal outbreak scenario in exotic zoological collections. Such findings from an animal outbreak have important One Health implications.

A cluster of melioidosis infections in hatchling saltwater crocodiles (Crocodylus porosus) resolved using genome-wide comparison of a common north Australian strain of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative saprophytic bacillus and the aetiological agent of melioidosis, a disease of public-health importance throughout Southeast Asia and northern Australia. Infection can occur in humans and a wide array of animal species, though zoonotic transmission and case clusters are rare. Despite its highly plastic genome and extensive strain diversity, fine-scale investigations into the population structure of B. pseudomallei indicate there is limited geographical dispersal amongst sequence types (STs). In the 'Top End' of northern Australia, five STs comprise 90 % of the overall abundance, the most prevalent and widespread of which is ST-109. In May 2016, ST-109 was implicated in two fatal cases of melioidosis in juvenile saltwater crocodiles at a wildlife park near Darwin, Australia. To determine the probable source of infection, we sampled the crocodile enclosures and analysed the phylogenetic relatedness of crocodile and culture-positive ST-109 environmental park isolates against an additional 135 ST-109 B. pseudomallei isolates from the Top End. Collectively, our whole-genome sequencing (WGS) and pathology findings confirmed B. pseudomallei detected in the hatchling incubator as the likely source of infection, with zero SNPs identified between clinical and environmental isolates. Our results also demonstrate little variation across the ST-109 genome, with SNPs in recombinogenic regions and one suspected case of ST homoplasy accounting for nearly all observed diversity. Collectively, this study supports the use of WGS for outbreak source attribution in highly recombinogenic pathogens, and confirms the epidemiological and phylogenetic insights that can be gained from high-resolution sequencing platforms.

CtGEM typing: Discrimination of Chlamydia trachomatis ocular and urogenital strains and major evolutionary lineages by high resolution melting analysis of two amplified DNA fragments.

Chlamydia trachomatis infects the urogenital tract (UGT) and eyes. Anatomical tropism is correlated with variation in the major outer membrane protein encoded by ompA. Strains possessing the ocular ompA variants A, B, Ba and C are typically found within the phylogenetically coherent "classical ocular lineage". However, variants B, Ba and C have also been found within three distinct strains in Australia, all associated with ocular disease in children and outside the classical ocular lineage. CtGEM genotyping is a method for detecting and discriminating ocular strains and also the major phylogenetic lineages. The rationale was facilitation of surveillance to inform responses to C. trachomatis detection in UGT specimens from young children. CtGEM typing is based on high resolution melting analysis (HRMA) of two PCR amplified fragments with high combinatorial resolving power, as defined by computerised comparison of 65 whole genomes. One fragment is from the hypothetical gene defined by Jali-1891 in the C. trachomatis B_Jali20 genome, while the other is from ompA. Twenty combinatorial CtGEM types have been shown to exist, and these encompass unique genotypes for all known ocular strains, and also delineate the TI and T2 major phylogenetic lineages, identify LGV strains and provide additional resolution beyond this. CtGEM typing and Sanger sequencing were compared with 42 C. trachomatis positive clinical specimens, and there were no disjunctions. CtGEM typing is a highly efficient method designed and tested using large scale comparative genomics. It divides C. trachomatis into clinically and biologically meaningful groups, and may have broad application in surveillance.

A genomic exploration identifies mechanisms that may explain adverse cardiovascular effects of COX-2 inhibitors.

Cyclooxygenase-2 inhibitors (coxibs) are characterized by multiple molecular off-target effects and increased coronary artery disease (CAD) risk. Here, we systematically explored common variants of genes representing molecular targets of coxibs for association with CAD. Given a broad spectrum of pleiotropic effects of coxibs, our intention was to narrow potential mechanisms affecting CAD risk as we hypothesized that the affected genes may also display genomic signals of coronary disease risk. A Drug Gene Interaction Database search identified 47 gene products to be affected by coxibs. We traced association signals in 200-kb regions surrounding these genes in 84,813 CAD cases and 202,543 controls. Based on a threshold of 1 × 10-5 (Bonferroni correction for 3131 haplotype blocks), four gene loci yielded significant associations. The lead SNPs were rs7270354 (MMP9), rs4888383 (BCAR1), rs6905288 (VEGFA1), and rs556321 (CACNA1E). By additional genotyping, rs7270354 at MMP9 and rs4888383 at BCAR1 also reached the established GWAS threshold for genome-wide significance. The findings demonstrate overlap of genes affected by coxibs and those mediating CAD risk and points to further mechanisms, which are potentially responsible for coxib-associated CAD risk. The novel approach furthermore suggests that genetic studies may be useful to explore the clinical relevance of off-target drug effects.

Systematic analysis of variants related to familial hypercholesterolemia in families with premature myocardial infarction.

Familial hypercholesterolemia (FH) is an oligogenic disorder characterized by markedly elevated low-density lipoprotein cholesterol (LDLC) levels. Variants in four genes have been reported to cause the classical autosomal-dominant form of the disease. FH is largely under-diagnosed in European countries. As FH increases the risk for coronary artery disease (CAD) and myocardial infarction (MI), it might be specifically overlooked in the large number of such patients. Here, we systematically examined the frequency of potential FH-causing variants by exome sequencing in 255 German patients with premature MI and a positive family history for CAD. We further performed co-segregation analyses in an average of 5.5 family members per MI patient. In total, we identified 11 potential disease-causing variants that co-segregate within the families, that is, 5% of patients with premature MI and positive CAD family history had FH. Eight variants were previously reported as disease-causing and three are novel (LDLR.c.811G>A p.(V271I)), PCSK9.c.610G>A (p.(D204N)) and STAP1.c.139A>G (p.(T47A))). Co-segregation analyses identified multiple additional family members carrying one of these FH variants and the clinical phenotype of either FH (n=2) or FH and premature CAD (n=15). However, exome sequencing also revealed that some variants in FH genes, which have been reported to cause FH, do not co-segregate with FH. The data reveal that a large proportion of FH patients escape the diagnosis, even when they have premature MI. Hence, systematic molecular-genetic screening for FH in such patients may reveal a substantial number of cases and thereby allow a timely LDLC-lowering in both FH/MI patients as well as their variant-carrying family members.

Whole-exome sequencing in an extended family with myocardial infarction unmasks familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal-dominant disease leading to markedly elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature myocardial infarction (MI). Mutation carriers display variable LDL cholesterol levels, which may obscure the diagnosis. We examined by whole-exome sequencing a family in which multiple myocardial infarctions occurred at a young age with unclear etiology. METHODS: Whole-exome sequencing of three affected family members, validation of the identified variant with Sanger-sequencing, and subsequent co-segregation analysis in the family. RESULTS: The index patient (LDL cholesterol 188 mg/dL) was referred for molecular-genetic investigations. He had coronary artery bypass graft (CABG) at the age of 59 years; 12 out of 15 1st, 2nd and 3rd degree relatives were affected with coronary artery disease (CAD) and/or premature myocardial infarction (MI). We sequenced the whole-exome of the patient and two cousins with premature MI. After filtering, we were left with a potentially disease causing variant in the LDL receptor (LDLR) gene, which we validated by Sanger-sequencing (nucleotide substitution in the acceptor splice-site of exon 10, c.1359-1G > A). Sequencing of all family members available for genetic analysis revealed co-segregation of the variant with CAD (LOD 3.0) and increased LDLC (>190 mg/dL), following correction for statin treatment (LOD 4.3). Interestingly, mutation carriers presented with highly variable corrected (183-354 mg/dL) and on-treatment LDL levels (116-274 mg/dL) such that the diagnosis of FH in this family was made only after the molecular-genetic analysis. CONCLUSION: Even in families with unusual clustering of CAD FH remains to be underdiagnosed, which underscores the need for implementation of systematic screening programs. Whole-exome sequencing may facilitate identification of disease-causing variants in families with unclear etiology of MI and enable preventive treatment of mutation carriers in a more timely fashion.