Zrinka Biloglav

Epidemiology and etiology of childhood pneumonia

Childhood pneumonia is the leading single cause of mortality in children aged less than 5 years. The incidence in this age group is estimated to be 0.29 episodes per child-year in developing and 0.05 episodes per child-year in developed countries. This translates into about 156 million new episodes each year worldwide, of which 151 million episodes are in the developing world. Most cases occur in India (43 million), China (21 million) and Pakistan (10 million), with additional high numbers in Bangladesh, Indonesia and Nigeria (6 million each). Of all community cases, 7-13% are severe enough to be life-threatening and require hospitalization. Substantial evidence revealed that the leading risk factors contributing to pneumonia incidence are lack of exclusive breastfeeding, undernutrition, indoor air pollution, low birth weight, crowding and lack of measles immunization. Pneumonia is responsible for about 19% of all deaths in children aged less than 5 years, of which more than 70% take place in sub-Saharan Africa and south-east Asia. Although based on limited available evidence, recent studies have identified Streptococcus pneumoniae, Haemophilus influenzae and respiratory syncytial virus as the main pathogens associated with childhood pneumonia.

SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout

Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200–500 μM) compared with other mammals (3–120 μM). About 70% of daily urate disposal occurs via the kidneys, and in 5–25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7–5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

Genetic determinants of circulating sphingolipid concentrations in European populations

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic β-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08×10−66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1–3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10−4 or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases.

Genome-Wide Association Study Identifies Novel Loci Associated with Circulating Phospho- and Sphingolipid Concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10−204) and 10 loci for sphingolipids (smallest P-value = 3.10×10−57). After a correction for multiple comparisons (P-value<2.2×10−9), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.

Genome-wide linkage analysis of serum creatinine in three isolated European populations

There is increasing evidence for a role of genetic predisposition in the etiology of kidney disease, but linkage scans have been poorly replicated. Here we performed a genome-wide linkage analysis of serum creatinine on 2859 individuals from isolated villages in South Tyrol (Italy), Rucphen (The Netherlands) and Vis Island (Croatia), populations that have been stable and permanently resident in their region. Linkage of serum creatinine levels to loci on chromosomes 7p14, 9p21, 11p15, 15q15-21, 16p13, and 18p11 was successfully replicated in at least one discovery population or in the pooled analysis. A novel locus was found on chromosome 10p11. Linkage to chromosome 22q13, independent of diabetes and hypertension, was detected over a region containing the non-muscle myosin heavy chain type II isoform A (MYH9) gene (LOD score=3.52). In non-diabetic individuals, serum creatinine was associated with this gene in two of the three populations and in meta-analysis (SNP rs11089788, P-value=0.0089). In populations sharing a homogeneous environment and genetic background, heritability of serum creatinine was higher than in outbred populations, with consequent detection of a larger number of loci than reported before. Our finding of a replicated association of serum creatinine with the MYH9 gene, recently linked to pathological renal conditions in African Americans, suggests that this gene may also influence kidney function in healthy Europeans.

3000 years of solitude: extreme differentiation in the island isolates of Dalmatia, Croatia

Communities with increased shared ancestry represent invaluable tools for genetic studies of complex traits. ‘1001 Dalmatians’ research program collects biomedical information for genetic epidemiological research from multiple small isolated populations (‘metapopulation’) in the islands of Dalmatia, Croatia. Random samples of 100 individuals from 10 small island settlements (n<2000 inhabitants) were collected in 2002 and 2003. These island communities were carefully chosen to represent a wide range of distinct and well-documented demographic histories. Here, we analysed their genetic make-up using 26 short tandem repeat (STR) markers, at least 5 cM apart. We found a very high level of differentiation between most of these island communities based on Wrights fixation indexes, even within the same island. The model-based clustering algorithm, implemented in STRUCTURE, defined six clusters with very distinct genetic signatures, four of which corresponded to single villages. The extent of background LD, assessed with eight linked markers on Xq13-21, paralleled the extent of differentiation and was also very high in most of the populations under study. For each population, demographic history was characterised and 12 ‘demographic history’ variables were tentatively defined. Following stepwise regression, the demographic history variable that most significantly predicted the extent of LD was the proportion of locally born grandparents. Strong isolation and endogamy are likely to be the main forces maintaining this highly structured overall population.

Setting research priorities to reduce global mortality from preterm birth and low birth weight by 2015

Aim This paper aims to identify health research priorities that could improve the rate of progress in reducing global neonatal mortality from preterm birth and low birth weight (PB/LBW), as set out in the UNs Millennium Development Goal 4. Methods We applied the Child Health and Nutrition Research Initiative (CHNRI) methodology for setting priorities in health research investments. In the process coordinated by the World Health Organization in 2007–2008, 21 researchers with interest in child, maternal and newborn health suggested 82 research ideas that spanned across the broad spectrum of epidemiological research, health policy and systems research, improvement of existing interventions and development of new interventions. The 82 research questions were then assessed for answerability, effectiveness, deliverability, maximum potential for mortality reduction and the effect on equity using the CHNRI method. Results The top 10 identified research priorities were dominated by health systems and policy research questions (eg, identification of LBW infants born at home within 24–48 hours of birth for additional care; approaches to improve quality of care of LBW infants in health facilities; identification of barriers to optimal home care practices including care seeking; and approaches to increase the use of antenatal corticosteriods in preterm labor and to improve access to hospital care for LBW infants). These were followed by priorities for improvement of the existing interventions (eg, early initiation of breastfeeding, including feeding mode and techniques for those unable to suckle directly from the breast; improved cord care, such as chlorhexidine application; and alternative methods to Kangaroo Mother Care (KMC) to keep LBW infants warm in community settings). The highest-ranked epidemiological question suggested improving criteria for identifying LBW infants who need to be cared for in a hospital. Among the new interventions, the greatest support was shown for the development of new simple and effective interventions for providing thermal care to LBW infants, if KMC is not acceptable to the mother. Conclusion The context for this exercise was set within the MDG4, requiring an urgent and rapid progress in mortality reduction from low birth weight, rather than identifying long-term strategic solutions of the greatest potential. In a short-term context, the health policy and systems research to improve access and coverage by the existing interventions, coupled with further research to improve effectiveness, deliverability and acceptance of existing interventions, and epidemiological research to address the key gaps in knowledge, were all highlighted as research priorities.

Quantifying the increase in average human heterozygosity due to urbanisation

The human population is undergoing a major transition from a historical metapopulation structure of relatively isolated small communities to an outbred structure. This process is predicted to increase average individual genome-wide heterozygosity (h) and could have effects on health. We attempted to quantify this increase in mean h. We initially sampled 1001 examinees from a metapopulation of nine isolated villages on five Dalmatian islands (Croatia). Village populations had high levels of genetic differentiation, endogamy and consanguinity. We then selected 166 individuals with highly specific personal genetic histories to form six subsamples, which could be ranked a priori by their predicted level of outbreeding. The measure h was then estimated in the 166 examinees by genotyping 1184 STR/indel markers and using two different computation methods. Compared to the value of mean h in the least outbred sample, values of h in the remaining samples increased successively with predicted outbreeding by 0.023, 0.038, 0.058, 0.067 and 0.079 (P<0.0001), where these values are measured on the same scale as the inbreeding coefficient (but opposite sign). We have shown that urbanisation was associated with an average increase in h of up to 0.08–0.10 in this Croatian metapopulation, regardless of the method used. Similar levels of differentiation have been described in many populations. Therefore, changes in the level of heterozygosity across the genome of this magnitude may be common during isolate break-up in humans and could have significant health effects through the established genetic mechanism of hybrid vigour/heterosis.

Haematological malignancies in childhood in Croatia: Investigating the theories of depleted uranium, chemical plant damage and 'population mixing'

Some of potential causes proposed to explain the reported increase of haematological malignancies in childhood during or after the war period in several countries include depleted uranium, chemical pollution and population mixing theory. The aim of this study was to define the population of Croatian children aged 0–14 years who were potentially exposed to each of those risks during the war and to investigate any possible association between the exposure and the incidence of haematological malignancies. The authors analyzed the data reported by the Cancer Registry of Croatia during the pre-war period (1986–1990), war period (1991–1995) and post-war period (1996–1999). In the group of 10 counties potentially exposed to depleted uranium and two counties where chemical war damage occurred, no significant difference in incidence of the studied haematological malignancies was noted in comparison to pre-war period. The incidence of lymphatic leukaemia significantly increased in four counties where population mixing had occurred during the war period, supporting the ‘mixing theory’. In those counties, the incidence of Hodgkins lymphoma decreased during and after the war. In Croatia as a whole, decreases in incidence of myeloid leukaemias during war and non-Hodgkin lymphoma after the war were noted.

Setting priorities for development of emerging interventions against childhood pneumonia, meningitis and influenza

Acute lower respiratory infections, which broadly include pneumonia and bronchiolitis, are still the leading cause of childhood mortality. ALRI contributed to 18% of all deaths in children younger than five years of age in 2008 [1], and the main pathogens responsible for high mortality were Streptococcus pneumoniae, Haemophilus influenzae and respiratory syncytial virus [2-4]. In addition, meningitis was estimated to contribute up to 200 000 deaths each year, and influenza anywhere between 25 000 and 110 000 [1,5]. It is widely acknowledged that a major portion of this mortality should be avoidable if universal coverage of all known effective interventions could be achieved. However, some evaluations of the implementation of World Health Organization’s (WHO) Integrated Management of Childhood Illness (IMCI) strategy, which promotes improved access to a trained health provider who can administer “standard case management”, have shown somewhat disappointing results [6-8]. Only a minority of all children with life-threatening episodes of pneumonia, meningitis and influenza in developing countries have access to trained health providers and receive appropriate treatment [6-8]. Thus, novel strategies for control of pneumonia that balance investments in scaling up of existing interventions and the development of novel approaches, technologies and ideas are clearly needed.