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Mutations in ADNP result in Helsmoortel-Van der Aa syndrome. Here, we describe the first de novo intronic deletion, affecting the splice-acceptor site of the first coding ADNP exon in a five-year-old girl with developmental delay and autism. Whereas exome sequencing failed to detect the non-coding deletion, genome-wide CpG methylation analysis revealed an episignature suggestive of a Helsmoortel-Van der Aa syndrome diagnosis. This diagnosis was further supported by PhenoScore, a novel facial recognition software package. Subsequent whole-genome sequencing resolved the three-base pair ADNP deletion c.[-5-1_-4del] with transcriptome sequencing showing this deletion leads to skipping of exon 4. An N-terminal truncated protein could not be detected in transfection experiments with a mutant expression vector in HEK293T cells, strongly suggesting this is a first confirmed diagnosis exclusively due to haploinsufficiency of the ADNP gene. Pathway analysis of the methylome indicated differentially methylated genes involved in brain development, the cytoskeleton, locomotion, behavior, and muscle development. Along the same line, transcriptome analysis identified most of the differentially expressed genes as upregulated, in line with the hypomethylated CpG episignature and confirmed the involvement of the cytoskeleton and muscle development pathways that are also affected in patient cell lines and animal models. In conclusion, this novel mutation for the first time demonstrates that Helsmoortel-Van der Aa syndrome can be caused by a loss-of-function mutation. Moreover, our study elegantly illustrates the use of EpiSignatures, WGS and Phenoscore as novel complementary diagnostic tools in case a of negative WES result., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Background: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation., Results: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain., Conclusion: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target., (© 2024. The Author(s).)

Here, we identified the causal mutation in the MRX20 family, one of the larger X-linked pedigrees that have been described in which no gene had been identified up till now. In 1995, the putative disease gene had been mapped to the pericentromeric region on the X chromosome, but no follow-up studies were performed. Here, whole exome sequencing (WES) on two affected and one unaffected family member revealed the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM&#95;021120.4) that segregated with the affected individuals in the family. DLG3 mutations have been consequently associated with intellectual disability and are a plausible explanation for the clinical abnormalities observed in this family. In addition, we identified two other variants co-segregating with the phenotype: a stop gain mutation in SSX1 (c.358G>T/p.(Glu120Ter)) (NM&#95;001278691.2) and a nonsynonymous SNV in USP27X (c.56 A>G/p.(Gln19Arg)) (NM&#95;001145073.3). RNA sequencing revealed 14 differentially expressed genes (p value < 0.1) in 7 affected males compared to 4 unaffected males of the family, including four genes known to be associated with neurological disorders. Thus, in this paper we identified the c.195del/p.(Thr66ProfsTer55) mutation in the DLG3 gene (NM&#95;021120.4) as likely responsible for the phenotype observed in the MRX20 family., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Expanded CGG-repeats have been linked to neurodevelopmental and neurodegenerative disorders, including the fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS). We hypothesized that as of yet uncharacterised CGG-repeat expansions within the genome contribute to human disease. To catalogue the CGG-repeats, 544 human whole genomes were analyzed. In total, 6101 unique CGG-repeats were detected of which more than 93% were highly variable in repeat length. Repeats with a median size of 12 repeat units or more were always polymorphic but shorter repeats were often polymorphic, suggesting a potential intergenerational instability of the CGG region even for repeats units with a median length of four or less. 410 of the CGG repeats were associated with known neurodevelopmental disease genes or with strong candidate genes. Based on their frequency and genomic location, CGG repeats may thus be a currently overlooked cause of human disease.

Background: Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and the most commonly identified monogenic cause of autism. Recent studies have shown that long-term pathological consequences of FXS are not solely confined to the central nervous system (CNS) but rather extend to other physiological dysfunctions in peripheral organs. To gain insights into possible immune dysfunctions in FXS, we profiled a large panel of immune-related biomarkers in the serum of FXS patients and healthy controls., Methods: We have used a sensitive and robust Electro Chemi Luminescence (ECL)-based immunoassay to measure the levels of 52 cytokines in the serum of n = 25 FXS patients and n = 29 healthy controls. We then used univariate statistics and multivariate analysis, as well as an advanced unsupervised clustering method, to identify combinations of immune-related biomarkers that could discriminate FXS patients from healthy individuals., Results: While the majority of the tested cytokines were present at similar levels in FXS patients and healthy individuals, nine chemokines, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26 and CXCL10, were present at much lower levels in FXS patients. Using robust regression, we show that six of these biomarkers (CCL2, CCL3, CCL11, CCL22, CCL26 and CXCL10) were negatively associated with FXS diagnosis. Finally, applying the K-sparse unsupervised clustering method to the biomarker dataset allowed for the identification of two subsets of individuals, which essentially matched the FXS and healthy control categories., Conclusions: Our data show that FXS patients exhibit reduced serum levels of several chemokines and may therefore exhibit impaired immune responses. The present study also highlights the power of unsupervised clustering methods to identify combinations of biomarkers for diagnosis and prognosis in medicine.

Pathogenic variant in COCH are a known cause of DFNA9 autosomal dominant progressive hearing loss and vestibular dysfunction with adult onset. Hitherto, only dominant nonsynonymous variants and in-frame deletions with a presumed dominant negative or gain-of-function effect have been described. Here, we describe two brothers with congenital prelingual deafness and a homozygous nonsense c.292C>T(p.Arg98*) COCH variant, suggesting a loss-of-function effect. Vestibular dysfunction starting in the first decade was observed in the older patient. The heterozygous parents and sibling have normal hearing and vestibular function, except for the mother, who shows vestibular hyporeflexia and abnormal smooth pursuit tests, most likely due to concomitant disease. This is the first report of autosomal recessive inheritance of cochlea-vestibular dysfunction caused by a pathogenic variant in the COCH gene. An earlier onset of hearing impairment and vestibular dysfunction compared to the dominant hearing loss causing COCH variants is observed.

Objectives: The objective of this study is to clarify the role of (G4C2)n expansions in the etiology of Parkinson disease (PD) in the worldwide multicenter Genetic Epidemiology of Parkinson's Disease (GEO-PD) cohort., Methods: C9orf72 (G4C2)n repeats were assessed in a GEO-PD cohort of 7,494 patients diagnosed with PD and 5,886 neurologically healthy control individuals ascertained in Europe, Asia, North America, and Australia., Results: A pathogenic (G4C2)n>60 expansion was detected in only 4 patients with PD (4/7,232; 0.055%), all with a positive family history of neurodegenerative dementia, amyotrophic lateral sclerosis, or atypical parkinsonism, while no carriers were detected with typical sporadic or familial PD. Meta-analysis revealed a small increase in risk of PD with an increasing number of (G4C2)n repeats; however, we could not detect a robust association between the C9orf72 (G4C2)n repeat and PD, and the population attributable risk was low., Conclusions: Together, these findings indicate that expansions in C9orf72 do not have a major role in the pathogenesis of PD. Testing for C9orf72 repeat expansions should only be considered in patients with PD who have overt symptoms of frontotemporal lobar degeneration/amyotrophic lateral sclerosis or apparent family history of neurodegenerative dementia or motor neuron disease., (© 2014 American Academy of Neurology.)

Background: Autosomal dominant dopa-responsive dystonia (AD-DRD) is caused by a biochemical defect primarily resulting from guanosine triphosphate cyclohydrolase 1 gene (GCH1) mutations. Few families have been reported without mutations in GCH1., Methods: Genome-wide linkage analysis and positional cloning to identify the genetic defect in a Belgian AD-DRD family was carried out., Results and Conclusion: In this study, we report on the identification and characterization of a novel 24-kb deletion spanning exon 1 and the 5' regulatory region of GCH1 causing a wide spectrum of motor and nonmotor symptoms in a large Belgian AD-DRD family. This large-scale deletion of regulatory sequences leads to decreased GCH1 activity in all carriers, most probably resulting from allelic loss of transcription. We mapped the breakpoints of this deletion to the nucleotide level, allowing the development of a straightforward polymerase chain reaction assay for fast, efficient detection of this large deletion, which will prove valuable for preimplantation genetic diagnosis., (Copyright © 2012 Movement Disorder Society.)

VPS35 was recently identified as a novel autosomal dominant gene for Parkinson disease. In this study, we aimed to determine the contribution of simple and complex VPS35 variations to the genetic etiology of the spectrum of Lewy body disorders (LBD) in a Flanders-Belgian patient cohort (n = 677). We identified 3 novel missense variations in addition to 1 silent and 1 intronic variation predicted to activate a cryptic splice site, but no copy number variations. Despite the absence of these rare variations in the control group (n = 800), we could not attain convincing evidence for pathogenicity by segregation analysis or in silico predictions. Hence, our data do not support a major role for VPS35 variations in the genetic etiology of Lewy body disorders in the Flanders-Belgian population., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Based on the substantial overlap in clinical and pathological characteristics of dementia with Lewy bodies (DLB) and Parkinson disease with dementia (PDD) with Alzheimer disease (AD) and Parkinson disease (PD) we hypothesized that these disorders might share underlying genetic factors. The contribution of both sequence and copy number variants (CNVs) in known AD and PD genes to the genetic etiology of DLB and PDD however is currently unclear. Therefore, we performed a gene-based mutation analysis of all major AD and PD genes in 99 DLB and 75 PDD patients, including familial and sporadic forms, from Flanders, Belgium. Also, copy number variants in APP, SNCA, and PARK2 were determined. In the AD genes we detected proven pathogenic missense mutations in PSEN1 and PSEN2, and 2 novel missense variants in PSEN2 and MAPT. In the PD genes we identified 1 SNCA duplication, the LRRK2 R1441C founder mutation and 4 novel heterozygous missense variants with unknown pathogenicity. Our results suggest a contribution of established AD and PD genes to the genetic etiology of DLB and PDD though to a limited extent. They do support the hypothesis of a genetic overlap between members of the Lewy body disease spectrum, but additional genes still have to exist., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region., Methods: We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers., Findings: In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5-4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes., Interpretation: We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum., Funding: Full funding sources listed at end of paper (see Acknowledgments)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The second most frequent form of neurodegenerative dementia after Alzheimer's disease is dementia with Lewy bodies (DLB). Since informative DLB families are scarce, little is presently known about the molecular genetic etiology of DLB. We recently mapped the first locus for DLB on chromosome 2q35-q36 in a multiplex Belgian family, DR246, with autopsy-proven DLB pathology in a region of 9.2 Mb. Here, we describe the ascertainment of additional DR246 family members and significant finemapping of the DLB locus to 3.3 Mb based on informative meiotic recombinants. Extensive sequencing of the 42 positional candidate genes within the DLB region did not identify a simple pathogenic mutation that co-segregated with disease in family DR246. Also high resolution analysis of copy number variations in the DLB locus did not provide evidence for a complex mutation. In conclusion, we confirmed the DLB locus at 2q35-q36 as a genetic entity but candidate gene-based sequencing and copy number variation analysis did not identify the pathogenic mutation in family DR246. Other detection strategies will be needed to reveal the underlying mutation explaining the linkage of DLB to 2q35-q26. Possibly the disease mutation in this family acts through a more complex mechanism than generally envisaged for monogenic disorders. Nevertheless, identifying the first familial DLB gene is likely to contribute an entry point into the pathogenic cascades underlying DLB pathology.

Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and a complex inflammatory cascade within the intestinal muscularis. Treatment with carbon monoxide (CO)--inhaled or intraperitonea--has been shown to ameliorate bowel dysmotility caused by surgical manipulation of the gut in experimental animals. Recent evidence indicates that CO exerts its anti-inflammatory effects through the induction of peroxisome proliferator-activated receptor (PPAR)-gamma, a nuclear receptor whose activation has been linked to several physiological pathways, including those related to the regulation of intestinal inflammation. The purpose of this study was to evaluate pharmacological activation of PPARgamma in a murine model of postoperative ileus by use of the PPARgamma agonist rosiglitazone. Postoperative bowel dysmotility was induced by surgical manipulation of the colon. The functional severity of postoperative ileus was significantly ameliorated in mice pretreated with rosiglitazone (0.3 to 10 mg/kg i.p.); this was associated with a down-regulation of pro-inflammatory cytokines/chemokines, inducible nitric oxide synthase activity, cyclooxygenase-2 activity, as well as a decrease in leukocyte recruitment into the muscularis of both colon and jejunum. These anti-inflammatory effects were preceded by a PPARgamma-dependent down-regulation of early growth response (Egr)-1, a key regulator of inflammatory gene expression. In conclusion, these results indicate that rosiglitazone significantly attenuates postoperative ileus in mice by suppression of the muscularis inflammatory cascade through a PPARgamma-dependent down-regulation of Egr-1 and encourage the further clinical evaluation of synthetic PPARgamma agonists as pharmacological tool to prevent this postoperative event.

Background and Aims: Treatment with carbon monoxide (CO) inhalation has been shown to ameliorate postoperative ileus (POI) in rodents and swine. The aim of this study was to investigate whether CO liberated from water-soluble CO-releasing molecules (CO-RMs) can protect against POI in mice and to elucidate the mechanisms involved., Methods: Ileus was induced by surgical manipulation of the small intestine (IM). Intestinal contractility-transit was evaluated by video-fluorescence imaging. Leucocyte infiltration (myeloperoxidase), inflammatory parameters (ELISA), oxidative stress (lipid peroxidation), and haem oxygenase (HO)/inducible nitric oxide synthase (iNOS) enzyme activity were measured in the intestinal mucosa and muscularis propria., Results: Intestinal contractility and transit were markedly restored when manipulated mice were pre-treated with CO-RMs. Intestinal leucocyte infiltration, expression levels of interleukin 6 (IL6), monocyte chemoattractant protein-1 and intercellular adhesion molecule-1, as well as iNOS activity were reduced by treatment with CORM-3 (a transition metal carbonyl that releases CO very rapidly); whereas expression of IL10/HO-1 was further increased when compared to nontreated manipulated mice. Moreover, treatment with CORM-3 markedly reduced oxidative stress and extracellular signal-related kinase (ERK)1/2 activation in both mucosa (early response) and muscularis (biphasic response). The p38 mitogen-activated protein kinase inhibitor SB203580 abolished CORM-3-mediated HO-1 induction. The HO inhibitor chromium mesoporphyrin only partially reversed the protective effects of CORM-3 on inflammation/oxidative stress in the muscularis, but completely abrogated CORM-3-mediated inhibition of the early "oxidative burst" in the mucosa., Conclusions: Pre-treatment with CO-RMs markedly reduced IM-induced intestinal muscularis inflammation. These protective effects are, at least in part, mediated through induction of HO-1, in a p38-dependent manner, as well as reduction of ERK1/2 activation. In addition, CORM-induced HO-1 induction reduces the early "oxidative burst" in the mucosa following IM.

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCalpha1beta1 heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCalpha1/alpha2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCalpha1 knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCalpha1 KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5'-hydroxymethyl-2'-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCalpha1 KO mice. Both in WT and sGCalpha1 KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCalpha1 KO mice. These data indicate that besides the predominant sGCalpha1beta1 isoform, also the less abundantly expressed sGCalpha2beta1 isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCalpha1 KO mice. Prolonged administration of CO-either by the addition of CORM-2 or by continuous infusion of CO-mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.

Background: In animal models of postoperative ileus (POI), inflammation of the intestine plays an important role in the pathogenesis of POI. Changes in alpha(2)-adrenoceptors and nitrergic regulation have been proposed to be implicated. The aim of our study was to investigate the presynaptic alpha(2)-receptor-mediated control of cholinergic nerve activity, the nitrergic nerve activity, and the possible role of soluble guanylate cyclase (sGC) during the inflammatory phase of POI., Methods: Ileus was induced by anesthesia and manipulation of the rat jejunum. Rats were treated with the sGC inhibitors methylene blue or ODQ; nonoperated animals served as controls. After 24 h, plasma and jejunal tissue were collected for biochemical assays, nitric oxide synthase-1 (NOS-1)-immunohistochemistry, acetylcholine (Ach)-release experiments, and muscle tension experiments., Results: In all operated animal groups, myeloperoxidase activity was significantly increased, which indicates initiation of an inflammatory response. The alpha(2)-adrenoceptor agonist UK14,304 reduced electrically induced Ach-release similarly in operated and nonoperated animals. In strips of operated animals, electrically induced nitrergic relaxations were decreased, whereas relaxations induced by exogenous nitric oxide (NO) remained unchanged compared with control. The number of myenteric neurons and the percentage of NOS-1-positive neurons were not influenced. Plasmatic cyclic guanosine monophosphate (cGMP) levels were decreased in all operated groups, whereas jejunal cGMP levels were unchanged compared with nonoperated controls; treatment with sGC inhibitors did not reduce plasmatic cGMP levels., Conclusions: This study demonstrates that presynaptic alpha(2)-receptor mediated control of intestinal cholinergic nerve activity is unchanged during manipulation-induced inflammation. However, this inflammation induces impaired nitrergic neurotransmission related to decreased NOS-1 activity in the nitrergic nerves.

Pathogen genotyping of polyclonal infections is limited by 2 major drawbacks: (1). how to establish whether multiple mutations detected in 1 gene belong to the same clone and (2). how to evaluate the proportion of different genotypes in a given sample. For drug-resistance genotyping in Plasmodium falciparum malaria, we address these problems by using a fluorogenic assay that combines fluorescence-resonance energy transfer, between fluorophores present on a probe and a polymerase-chain-reaction primer, and a melt-curve analysis. We demonstrate that this tool allows a more accurate insight into the P. falciparum populations present in complex biological samples.