MD Friederike Mackensen
email: mackensen@uveitiszentrum.de

MD Felix David

Vedat Schwenger

MD Lynnelle K Smith

MD Raj Rajalingam

PhD Ralph D Levinson

MD Carrie R Austin

Donald Houghton

MD Tammy M Martin

PhD James T Rosenbaum

HLA-DRB1*0102 is associated with TINU Syndrome and bilateral, sudden onset anterior uveitis but not with interstitial nephritis alone

Keywords: uveitis, interstitial nephritis, TINU, HLA, genetics

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Tubulointerstitial nephritis and uveitis (TINU) was first described in 1975 by Dobrin [START_REF] Dobrin | Acute eosinophilic interstitial nephritis and renal failure with bone marrow-lymph node granulomas and anterior uveitis. A new syndrome[END_REF].

The syndrome is rare even in specialized uveitis clinics, but it was shown to be more frequent in patients with sudden onset, anterior, bilateral uveitis, especially when the patients are less than 20 years of age [START_REF] Mackensen | Enhanced recognition, treatment, and prognosis of tubulointerstitial nephritis and uveitis syndrome[END_REF]. Even this study might have underestimated the syndrome´s incidence [START_REF] Mackensen | Enhanced recognition, treatment, and prognosis of tubulointerstitial nephritis and uveitis syndrome[END_REF] because laboratory evaluations needed for the clinical diagnosis of TINU are often not obtained or not obtained in a timely manner. The uveitis of the TINU Syndrome can have a prolonged course of several months to years [START_REF] Mandeville | The tubulointerstitial nephritis and uveitis syndrome[END_REF] but generally has a favorable prognosis and usually can be treated with local therapy alone [START_REF] Mackensen | Enhanced recognition, treatment, and prognosis of tubulointerstitial nephritis and uveitis syndrome[END_REF]. Still, the possibility of missing renal disease and the consequence of not treating it is of some concern. Even though earlier publications reported the benign outcome of nephritis in TINU and suggested that possibly no treatment would be necessary [START_REF] Vohra | Tubulointerstitial nephritis and uveitis in children and adolescents. Four new cases and a review of the literature[END_REF], more recent publications of serial renal biopsies in patients with biopsy proven TINU showed histological changes of acute inflammation despite systemic prednisone treatment even 6-9 months after the first diagnosis [START_REF] Tanaka | Repeat renal biopsy in a girl with tubulointerstitial nephritis and uveitis syndrome[END_REF][START_REF] Yanagihara | Serial renal biopsies in three girls with tubulointerstitial nephritis and uveitis syndrome[END_REF]. Later biopsies in one case showed scarring [START_REF] Yanagihara | Serial renal biopsies in three girls with tubulointerstitial nephritis and uveitis syndrome[END_REF]. Moreover, there have been recent reports of the development of end-stage renal failure in patients with idiopathic tubulointerstitial nephritis not treated with systemic steroid therapy [START_REF] Suzuki | Repeat renal biopsy in children with severe idiopathic tubulointerstitial nephritis[END_REF]. Even if the tissue changes might be minimal, any residual tissue damage might lead to chronic kidney disease, hypertension and similar problems.

As uveitis patients often are referred to specialized centers several months after onset of disease, a time-independent marker would be helpful to suggest the diagnosis and give prognostic information to the patient.

Previously, we demonstrated a very strong association of HLA DRB1*0102 with definite TINU [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF]. Now, we tested the hypothesis that DRB1*0102 may be associated with isolated bilateral, sudden onset, anterior uveitis (U), isolated tubulointerstitial nephritis (TIN) or both.

Material and Methods

IRB approval was obtained for all institutions. Patients with sudden onset, anterior, bilateral uveitis (U) and inadequate criteria to meet a diagnosis of TINU (allowing maximum one unspecific criterion, e.g.minor urinary abnormalities) [START_REF] Mandeville | The tubulointerstitial nephritis and uveitis syndrome[END_REF] (see Table 1) Table 1: Diagnostic criteria for TINU (modified from Mandeville et al) [START_REF] Mandeville | The tubulointerstitial nephritis and uveitis syndrome[END_REF] Uveitis + Biopsy proven TIN or ≥1 of the following: 1) abnormal renal function 2) abnormal Urinalysis 3) systemic disease > 2 weeks, non-specific lab abnormalities were identified by database search in 2 centers (OHSU, Heidelberg) and asked for informed consent.. At the same time pathology reports from 1998 on were reviewed to identify subjects with biopsy proven TIN (see Fig. 1) in these centers. The principal pathologic abnormalities in the kidney biopsy were interstitial edema and infiltration by lymphocytes, plasma cells, macrophages and, variable numbers of eosinophils.

Small noncaseating granulomas were present in some cases. Focal proximal tubular cell injury and regeneration were sometimes apparent. To qualify, the glomeruli had to appear normal histologically. Excluded were patients with other systemic diseases that might have caused the nephritis as e.g. sarcoidosis, lupus, Sjogren's, and systemic infections. Equally excluded were patients with a likely or suspected hypersensitivity reaction/disease or renal toxicity, and anyone with known anatomic and/or functional lower urinary tract disease or patients with both TIN and uveitis. We identified 33 eligible TIN patients from a total of 43 biopsy proven TIN patients in that period. Patients were excluded for: Hantan virus infection (n=5), uveitis, sarcoidosis, multiple myeloma, missing chart and salofalk induced nephritis in a patient with crohn´s (each n=1). 20 patients could be contacted and finally 14 agreed to take part in the study. TIN patients were given a questionnaire to rule out uveitis at the time of nephritis diagnosis and if possible an ophthalmologic exam was performed at the time patients came in for the blood draw. Blood specimens were shipped to a single center (OHSU) where DNA was isolated from peripheral blood leukocytes using standard salt extraction techniques. HLA typing was performed on genomic DNA at another center (UCLA) by a Luminex-based PCR-SSO typing method (One Lambda Inc Canoga Park, CA Product information LABType® SSO Typing Tests). HLA frequencies were compared to normal published controls (dbMHC Europe cohort: http://www.ncbi.nlm.nih.gov/projects/gv/mhc/ihwg.cgi) and to the previously published TINU cohort [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF].

Chi-square (2-sided with Yates) and Mann-Whitney U (MWU) tests were applied and uncorrected p < 0.005 was used as cut-off for significance in multiple comparisons for HLA testing evaluation and p < 0.05 in single comparisons.

Results

We included 28 subjects with U and 14 with TIN. Demographic and clinical data are given in Table 2. The median age of onset in the U group was lower than in the TIN group (MWU p= 0.0055). Gender and race distributions were similar across the groups. However, a gender difference was seen in the group with age of onset ≤ 20 years where there were 58% males in the U group and only females in the TIN group.

Minor urinary findings such as a few cells or mild proteinuria as evidenced solely by beta-2 microglobulin elevations were found in 5 subjects with U. Other, non-specific laboratory abnormalities were found in 8. None out of the U group showed a creatinine rise or other signs of renal dysfunction.

Thirteen of the 14 TIN patients filled out the questionnaire. None had symptoms typical for uveitis. Nine reported seeing an ophthalmologist around the time of nephritis diagnosis without uveitis having been found.

The frequencies of HLA-DRB1 allelic variants are shown in Table 3 for the U and TIN cohorts recruited for this study as well as the TINU cohort previously published [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF] and a published control group (European dbMHC). As in the published TINU cohort, there was an increased allele frequency of HLA DRB1*0102 in the isolated U cohort compared to normal controls (10.7% vs. 0.6%; p <0.0001; RR 14.3 (6.9-29.8)). All DRB1*0102 positive individuals in the U group were heterozygous for this allele. All positive patients were Caucasian. Interestingly, when the subset of the U cohort with early disease onset (≤20 years old, n=12) was considered, the allele frequency of DRB1*0102 was higher than that of the entire U group (16.7%, see Table 4). None of the TIN patients showed this HLA subtype (see Table 3 and4). More than half (58.3%) of the U subjects ≤ 20 years old were male (Table 2), and there were some differences in DRB1*0102 allele frequencies between genders overall (8.3% in female versus 15% in male subjects) and even more pronounced in the age group ≤ 20 years old (4.2% in female versus 12.5% in male subjects). But the numbers in the subgroups were small. An association with the HLA-DRB1*08 allele was also seen in the U cohort with an allele frequency of 10.7% versus 2.7% in normal controls (p=0.0019; RR 4.0 (1.8-9.0)). In contrast, the HLA-DRB1*08 was not different from controls in the TINU cohort (allele frequency 2.8%, p=0.9903). No age differences were seen here, but we noticed a slightly higher frequency in males (see Table 4).Five of the patients positive for this allele were Caucasian and only one African American.

In the U vs. control comparisons, only HLA-DRB1*0102 and *08 showed statistically different frequencies. When comparing TIN vs. control, no differences were significant for any of the DRB1 alleles (see Table 5).

Discussion

Our results indicate that HLA-DRB1*0102 is an independent risk factor for the uveitis characteristic of the TINU phenotype, i.e., bilateral, anterior and of sudden onset.

Interestingly, this allele was found even more frequently in the group of patients 20 years old and younger.

Since DRB1*0102 was not found in the interstitial nephritis group, this allele is not a risk factor for the renal disease that occurs in the absence of uveitis. DRB1*0102 is an isolated risk factor for uveitis independent of clinically detectable renal disease.

A possible interpretation of our data is that many patients with bilateral, sudden onset, anterior uveitis might have a subclinical form of TINU. This latter conclusion is supported by observations from Japan demonstrating biopsy confirmed TINU in patients with normal renal function [START_REF] Goda | Clinical features in tubulointerstitial nephritis and uveitis (TINU) syndrome[END_REF]. Our patients were seen at referral centers. Due to referral patterns, urinalysis and other studies of renal function were not always obtained at the onset of uveitis and in a subset of our patients especially, urinalysis was never performed (n=15) or not available for our interpretation. A diagnosis of TINU might have been established in some of our patients if these studies had been done in a timely manner. Still, in the subgroup with the DRB1*0102, laboratory tests (urinalysis) had been performed more often than in the other subgroups and there was no statistically significant difference in the frequency of minor laboratory or urinary changes between the uveitis subgroup with the DRB1*0102 compared to those with other DRB1 types. An alternative explanation of our data is that DRB1*0102 is a risk factor for bilateral, anterior, sudden onset uveitis regardless of whether this is associated with interstitial nephritis. Levinson and coworkers published a case series of mestizo patients with Vogt-Koyanaghi-Harada syndrome, a uveitis subtype that often presents first with a bilateral anterior uveitis, where they also found a high frequency of DRB1*0102 [START_REF] Levinson | HLA-DRB1 and -DQB1 alleles in mestizo patients with Vogt-Koyanagi-Harada's disease in Southern California[END_REF] We used strict exclusion criteria for the TIN cohort, to obtain a clearcut phenotype.

So it may be that in other subgroups of TIN, eg. those where the treating physicians had thought an infection or a medication to be the cause of the nephritis, DRB1*0102 might have been found. Admittedly the proof of a systemic infection or a triggering medication is not always easy and thus the physician's interpretation may be debatable. In the planning of the study we had still decided to exclude these patients as we would exclude uveitis patients where an infection was thought to cause the uveitis as this in our opinion would be a different disease subtype. As only 10 out of 43 patients with TIN were excluded, and only five due to an assumed infectious origin, we do not think we introduced too high a bias.Prior to the 18 TINU cases published by Levinson, et al. [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF], several other small series (3-8 cases) or sibling reports examined HLA data for disease association. These studies provide evidence for possible TINU associations with HLA-A*02 [START_REF] Goda | Clinical features in tubulointerstitial nephritis and uveitis (TINU) syndrome[END_REF][START_REF] Howarth | Tubulointerstitial nephritis and uveitis in monozygotic twin boys[END_REF][START_REF] Takemura | Course and outcome of tubulointerstitial nephritis and uveitis syndrome[END_REF], -A*24 [START_REF] Takemura | Course and outcome of tubulointerstitial nephritis and uveitis syndrome[END_REF][START_REF] Gorrono-Echebarria | The tuberculointerstitial nephritis and uveitis (TINU) syndrome is associated with HLA-DR14 in Spanish patients[END_REF][START_REF] Iitsuka | HLA tissue types in patients with acute tubulointerstitial nephritis accompanying uveitis[END_REF], and the Cw3 serotype [START_REF] Takemura | Course and outcome of tubulointerstitial nephritis and uveitis syndrome[END_REF][START_REF] Iitsuka | HLA tissue types in patients with acute tubulointerstitial nephritis accompanying uveitis[END_REF]. Regarding HLA-DRB1, studies have implicated DRB1*14 (or the DR6 serotype) [START_REF] Gorrono-Echebarria | The tuberculointerstitial nephritis and uveitis (TINU) syndrome is associated with HLA-DR14 in Spanish patients[END_REF][START_REF] Gafter | Tubulointerstitial nephritis and uveitis: association with suppressed cellular immunity[END_REF][START_REF] Tanaka | Tubulointerstitial nephritis and uveitis syndrome in two siblings[END_REF], DRB1*01 [START_REF] Sanchez-Burson | Tubulointerstitial nephritis and uveitis syndrome in Southern Spain[END_REF], DRB1*0101 [START_REF] Howarth | Tubulointerstitial nephritis and uveitis in monozygotic twin boys[END_REF][START_REF] Li | Human Leucocyte Antigen DQ Alpha Heterodimers and Human Leucocyte Antigen DR Alleles in Tubulointerstitial Nephritis and Uveitis Syndrome[END_REF], and DRB1*0102 [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF] with TINU. The later study result was further defined by the DRB1*01-/DQA1*01-/DQB1*05 haplotype [START_REF] Levinson | Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome[END_REF].

Single case reports of monozygotic twins [START_REF] Howarth | Tubulointerstitial nephritis and uveitis in monozygotic twin boys[END_REF][START_REF] Gianviti | Acute tubulointerstitial nephritis occurring with 1-year lapse in identical twins[END_REF] or siblings [START_REF] Tanaka | Tubulointerstitial nephritis and uveitis syndrome in two siblings[END_REF][START_REF] Biester | Tubulointerstitial Nephritis and Uveitis in Siblings[END_REF] and even in a mother and her son [START_REF] Dusek | Tubulointerstitial nephritis and uveitis syndrome in a mother and her son[END_REF] diagnosed with TINU syndrome several years apart have been reported, underlining the genetic component. As the family members did not fall ill at the same time a common environmental influence seems unlikely. The report of monozygotic twins in which one sibling had acute interstitial nephritis without uveitis and the other sibling had TINU syndrome implies that the same genotype in two individuals may result in different disease presentations, perhaps depending on the nature of the environmental triggers [START_REF] Gianviti | Acute tubulointerstitial nephritis occurring with 1-year lapse in identical twins[END_REF] and other non-MHC genetic factors.

The HLA shared epitope is known to be a rheumatoid arthritis (RA) susceptibility factor and consists of several HLA-DRB1 alleles, including -DRB1*0101 and *0102.

While much of the genetic effect of the shared epitope is attributed to -DRB1*04 alleles, evidence exists for -DRB1*01 to play a specific role in the predisposition to late-onset RA [START_REF] Gonzalez-Gay | Influence of human leukocyte antigen-DRB1 on the susceptibility and severity of rheumatoid arthritis[END_REF]. In addition to RA, other immune-mediated diseases in which -DRB1*01 has been implicated include Crohn's disease [START_REF] Danze | Association of HLA class II genes with susceptibility to Crohn's disease[END_REF], atopic asthma [START_REF] Torio | HLA class II genotypic frequencies in atopic asthma: association of DRB1*01-DQB1*0501 genotype with Artemisia vulgaris allergic asthma[END_REF], autoimmune idiopathic hypoparathyroidism [START_REF] Goswami | Prevalence of calcium sensing receptor autoantibodies in patients with sporadic idiopathic hypoparathyroidism[END_REF], and allergic pollinosis in response to the mugwort allergen [START_REF] Jahn-Schmid | Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01[END_REF]. With regard to uveitis conditions other than TINU, VKH has been found to be associated with -DRB1*0101 [START_REF] Alaez | Strong association of HLA class II sequences in Mexicans with Vogt-Koyanagi-Harada's disease[END_REF] and -DRB1*0102 [START_REF] Levinson | HLA-DRB1 and -DQB1 alleles in mestizo patients with Vogt-Koyanagi-Harada's disease in Southern California[END_REF], both in Mestizo patients. Behcet's disease has been linked to -DRB1*01 in the Mestizo population [START_REF] Soto-Vega | Class I and class II MHC polymorphisms in Mexican patients with Behcet's disease[END_REF] as well. HLA-DRB1 associations in a Hungarian juvenile idiopathic arthritis cohort included -DRB1*01 which was more prevalent in rheumatoid factorpositive polyarthritic patients [START_REF] Pazar | Role of HLA-DRB1 and PTPN22 genes in susceptibility to juvenile idiopathic arthritis in Hungarian patients[END_REF]. In addition to inflammatory or allergic diseases, -DRB1*01 is an important contributor to infectious disease responses. For example, -DRB1*01 is associated with spontaneous clearance of hepatitis C virus [START_REF] Barrett | Association of the HLA-DRB1*01 allele with spontaneous viral clearance in an Irish cohort infected with hepatitis C virus via contaminated anti-D immunoglobulin[END_REF], acute human parvovirus B19 infection [START_REF] Kerr | Association of symptomatic acute human parvovirus B19 infection with human leukocyte antigen class I and II alleles[END_REF], rheumatic heart disease [START_REF] Gundogdu | Human leukocyte antigen (HLA) class I and II alleles in Turkish patients with rheumatic heart disease[END_REF], and idiopathic bronchiectasis [START_REF] Boyton | Human leucocyte antigen class II association in idiopathic bronchiectasis, a disease of chronic lung infection, implicates a role for adaptive immunity[END_REF]. Collectively, these data serve to highlight the fundamental role of HLA-DRB1*01 in important and varied aspects of immune-mediated responses.

The most known HLA (class-I) association with uveitis certainly is HLA-B27 for acute, unilateral, anterior uveitis (AAU) in approximately 50% of patients [START_REF] Brewerton | Acute anterior uveitis and HL-A 27[END_REF]. This correlation is even higher at 90% in patients of European descent with ankylosing spondylitis [START_REF] Brewerton | Ankylosing spondylitis and HL-A27[END_REF]. However, the relative genetic contribution from the major histocompatibility complex (MHC) has been estimated at 31-45% [START_REF] Brown | Genetics and the pathogenesis of ankylosing spondylitis[END_REF][START_REF] Brown | Breakthroughs in genetic studies of ankylosing spondylitis[END_REF][START_REF] Brown | A genome-wide screen for susceptibility loci in ankylosing spondylitis[END_REF].

It is likely that in addition to HLA-B27 there will be a small number of genes with moderate effect, and a large number of genes with small effect. A case in point is the finding of an association with AAU at a genetic on chromosome 9p [START_REF] Martin | A locus on chromosome 9p predisposes to a specific disease manifestation, acute anterior uveitis, in ankylosing spondylitis, a genetically complex, multisystem, inflammatory disease[END_REF]. There have also been candidate gene studies implicating several small effect genes in AAU [START_REF] Suhler | HLA-B27-associated uveitis: overview and current perspectives[END_REF].

But also HLA class II associations have been found. In 1995 Monowarul and colleagues described an association of AAU in spondylarthritis patients with HLA-DR8 40 . We found an allele frequency of 12.7% for HLA-DR8 in a cohort of AAU, an association not seen for ankylosing spondylitis [START_REF] Mackensen | The MHC Class II Haplotype HLA-DRB1*0801/DQA1*0401/DQB1*0402 is Associated with Acute Anterior Uveitis[END_REF] (Tammy Martin, unpublished data). DRB1*0102 was not frequent in this cohort of AAU. Thus, a similar allele frequency of DRB1*08 in our patients with isolated U was a surprising finding.

How HLA associations exactly promote immune mediated disease is not clear yet, but it is important to consider that the major histocompatibility complex on chromosome 6 contains an abundance of genes that encode products critical to the immune response. It is possible, that the HLA-DRB1*0102 positive haplotype is in linkage disequilibrium with genes on chromosome 6 that may be related to disease pathogenesis such as the TNF superfamily. TNF-857T, for example has been shown to be a genetic risk marker for AAU in HLA B27+ patients [START_REF] Kuo | Figurelegend: Figure 1: Histological kidney section of a patient with tubulointerstitial nephritis. A dense mixed inflammatory infiltrate expands the interstitium. Infiltrating cells are predominantly lymphocytes and plasma cells, but include eosinophils. Individual lymphocytes have infiltrated several tubules. (hematoxylin and eosin[END_REF]. HLA-class II is expressed on antigen presenting cells, the first line defense in innate as well as the crucial informant in cellular immunity. From the existing data we hypothesize that a specific class II subtype makes a subject more susceptible to a pathogenic immune response to a pathogen or to a normally harmless antigen. This would equally explain the sudden onset nature of the "TINU-type" uveitis as the "HLA-B27-type" AAU. So far this pathogen or antigen has not been discovered.

Conclusion

A significant number of patients with isolated, sudden onset, anterior, bilateral uveitis have the same HLA class II genotype as patients with TINU. HLA-DRB1*0102 might predispose to this subset of uveitis and provide a time-independent marker. It remains to be shown if the patients with the isolated uveitis really are subclinical TINU patients as renal biopsy generally is not performed or if there are other genes apart from DRB1*0102 leading to the U plus or minus renal disease. As a consequence, treating patients with this genotype with oral corticosteroids for subclinical renal disease might be considered, even when anterior uveitis is controlled with topical treatment alone. Further study should be done to determine if patients with this genotype have a distinct course of uveitis as indicated by prognosis or response to therapy.

♦

  number of alleles (%) ♦♦ number of individualsThere was no statistically significant difference in the frequency of minor laboratory or urinary changes between the U subgroup with the DRB1*0102 compared to those with other DRB1 types (MWU p = 0.1769). The number with urinalyses not obtained was lower in the subgroup with the DRB1*0102 (43%) as compared to those with other HLA DRB1 types (62%).

Table 2 .

 2 Demographic and Clinical Data

	U	TIN	TINU
	(n = 28)	(n =14)	(n = 18) 8

Table 3

 3

		: HLA-DRB1 Typing Results
	DRB1	TINU, 2n=36 8	U, 2n=56	TIN, 2n=28	control, 2n=2587 ♦♦
	*0101/05/07/08/11/13 3 (8.3) ♦	5 (8.9)	3 (10.7)	218 (8.4)
	*0102	12 (33.3)	6 (10.7)	0	16 (0.6)
	*0103	1 (2.8)	1 (1.8)	0	67 (2.6)
	*03	6 (16.7)	4 (7.1)	5 (17.9)	376 (14.5)
	*04	3 (8.3)	7 (12.5)	3 (10.7)	458 (17.7)
	*07	4 (11.1)	6 (10.7)	5 (17.9)	383 (14.8)
	*08	1 (2.8)	6 (10.7)	2 (7.1)	71 (2.7)
	*11	1 (2.8)	4 (7.1)	0	143 (5.5)
	*12	0	0	1 (3.6)	38 (1.5)
	*13	2 (5.5)	4 (7.1)	3 (10.7)	254 (9.8)
	*14	2 (5.5)	2 (3.6)	1 (3.6)	47 (1.8)
	*15	1 (2.8)	10 (17.9)	5 (17.9)	445 (17.2)
	*16	0	1 (1.8)	0	35 (1.4)

♦ number of alleles (%) ♦♦ http://www.ncbi.nlm.nih.gov/projects/gv/mhc/ihwg.cgi (dbMHC Europe cohort, accessed Nov.

[START_REF] Biester | Tubulointerstitial Nephritis and Uveitis in Siblings[END_REF] 2007)

Table 4 :

 4 Allele frequencies and gender distribution of DRB1*0102 and *08 in different age groups

Table 5 :

 5 Statistical analysis of HLA-DRB1 alleles for each patient group compared to controls.

	DRB1	TINU vs control*		U vs control	TIN vs control
	*0101/05/07/08/11/13	0.9840	♦		0.8937	0.9273
	*0102	<0.0001; 46.3 (25.8-83.1)	♦♦	<0.0001; 14.3 (6.9-29.8) 0.6764
	*0103	0.9438			0.7069	0.7938
	*03	0.9026			0.1716	0.8209
	*04	0.2126			0.4040	0.4740
	*07	0.7010			0.5066	0.8535
	*08	0.9903			0.0019; 4.0 (1.8-9.0)	0.4074
	*11	0.7256			0.8203	0.3888
	*12	0.9759			0.7292	0.8972
	*13	0.5665			0.6600	0.8741
	*14	0.3050			0.6438	0.4915
	*15	0.0390			0.8977	0.9271
	*16	0.4823			0.7822	0.5355
	Chi-square (2-sided with Yates);

♦ P value. ♦♦ P value; RR (95% CI); in bold, only shown for those with P < 0.005.

Mary Bauman Foundation; the Fund for Arthritis and Infectious Disease Research; The Mac Donald Family Foundation


Faye Borthwick

Anne-Marie Allen

Janice M W Taylor

Prof Annette Graham
email: ann.graham@gcal.ac.uk

Over-expression of STARD3 in human monocyte-macrophages induces an anti-atherogenic lipid phenotype

Keywords: Atherosclerosis, macrophage 'foam cell', STARD3/MLN64, lipid transport, cholesterol efflux Clinical Science Immediate

Introduction

Dysregulated macrophage cholesterol homeostasis lies at the heart of early and developing atheroma, principal cause of coronary heart disease. Macrophage 'foam cells', laden with cholesterol and cholesteryl ester, result from unregulated uptake of modified lipoproteins by macrophage scavenger receptors (CD36, CD68, SR-AI/AII), and influence both plaque stability and progression [START_REF] Gerrity | The role of the monocyte in atherogenesis[END_REF]. Removal of excess cholesterol from macrophage 'foam' cells is central to lesion regression and stabilisation, and can be orchestrated, at least in vitro, by ATP binding cassette (ABC) lipid transporters such as ABCA1, ABCG1 and ABCG4, and apolipoprotein acceptors, such as apoAI and apoE [START_REF] Schmitz | The molecular mechanisms of HDL and associated vesicular trafficking mechanisms to mediate cellular lipid homeostasis[END_REF]. Efficient intracellular cholesterol transport is pivotal in marshalling appropriate cholesterol homeostasis mechanisms, regulating sterol-responsive transcription factors, such as Liver X Receptors (LXR / ) and sterol regulatory element binding proteins (SREBPs), and controlling cholesterol content of organelles, lipid rafts and membranes, and storage as cytosolic droplets of cholesteryl ester [START_REF] Schmitz | The molecular mechanisms of HDL and associated vesicular trafficking mechanisms to mediate cellular lipid homeostasis[END_REF][START_REF] Soccio | Intracellular cholesterol transport[END_REF]. Despite this, the proteins involved in non-vesicular cholesterol transport mechanisms remain poorly understood.

The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a 210 amino acid, conserved 'helix-grip' fold, providing an adaptable binding site for lipids such as cholesterol, oxysterols, phospholipids and ceramides. In humans, 'START' domains are found in 15 distinct proteins (STARD1-STARD15), implicated in non-vesicular lipid transport, cell signalling and lipid metabolism [START_REF] Soccio | StAR-related Lipid Transfer (START) proteins: mediators of intracellular lipid metabolism[END_REF][START_REF] Alpy | Give lipids a START: the StAR-related lipid transfer (START) domain in mammals[END_REF]. The prototypic member of this family, steroidogenic acute regulatory protein (StAR, STARD1) delivers cholesterol from the outer to the inner mitochondrial membrane, to the P450 side chain cleavage enzyme involved in steroidogenesis [START_REF] Miller | Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter[END_REF]. Over-expression of StAR can also reduce macrophage lipid content, inflammatory status and arterial cholesterol levels [START_REF] Ning | Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages[END_REF][START_REF] Ning | StAR overexpression decreases serum and tissue lipids in apolipoprotein E-deficient mice[END_REF], and increase cholesterol efflux to apoAI via a mechanism dependent upon mitochondrial sterol 27hydroxylase (CYP27A1), LXR activation and increases in ABCA1 mRNA and protein [START_REF] Taylor | Over-expression of steroidogenic acute regulatory protein (StAR) increases macrophage cholesterol efflux to apolipoprotein AI[END_REF]. However, one 'down-side' of StAR over-expression in macrophages is induction of lipogenesis [START_REF] Taylor | Over-expression of steroidogenic acute regulatory protein (StAR) increases macrophage cholesterol efflux to apolipoprotein AI[END_REF], possibly mediated by LXR-dependent increases in SREBP-1c expression, a problem also associated with nonsterol LXR agonists.

The other member of the STARD1 subfamily, STARD3 (Metastatic Lymph Node 64; MLN64) is a 54kDa protein, co-amplified within chromosome band 17q12, a region containing potent oncogene ERBB2, in human breast carcinoma [START_REF] Alpy | The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol binding protein[END_REF][START_REF] Dressman | Gene expression profiling detects gene amplification and differentiates tumour types in breast cancer[END_REF][START_REF] Benusiglio | HapMap-based study of the 17Q21 ERBB2 amplicon in susceptibility to breast cancer[END_REF]. STARD3 is a late endosomal protein with two distinct, conserved, cholesterol-binding domains: a region of four transmembrane helices with three short intervening loops, called the 'MENTAL' domain (MLN64 N-terminal domain), and the C-terminal 'START' domain [START_REF] Alpy | Functional characterisation of the MENTAL domain[END_REF]; the MENTAL domain may maintain cholesterol at the late endosomal membrane, prior to its shuttle to cytoplasmic acceptor(s) via the START domain.

Our work shows that cholesterol loading represses STARD3 expression, implicating this protein in dysfunctional cholesterol pathologies [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF]. Here, we sought to establish a functional role for this endosomal cholesterol trafficking protein in macrophage lipid homeostasis, testing the hypothesis that enhanced expression of STARD3 may be useful in prevention of 'foam cell' formation.

Materials and Methods

Materials

Tissue culture reagents were purchased from Lonza (Wokingham, UK); other sources include pCMV.Script, STARD4 and STARD5 clones (Origene, Cambridge Biosciences, UK), STARD3 clone (Stratagene, UK), Amaxa monocyte-macrophage transfection reagent, NuPAGE gels and buffers (InVitrogen), antibodies (AbCAM, Cambridge, UK), primers and fluorescent probes (Eurogentec, Belgium); low-density lipoprotein (Athens Research, PA, USA), acetylated as previously [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF].

Peripheral human monocyte-macrophages were purchased from Lonza (Wokingham) and human heart aorta RNA, derived from four to seven human heart aortae, was purchased from Clontech (USA). STARD4, STARD5 and empty vector control (pCMV; 0.5 g DNA) were delivered to THP-1 monocytes (10 7 ) using Amaxa Human Monocyte Nucleofector® kit (VPA-1007). Transfection efficiency using protocol Y001 (78.5%) was determined using the proprietory pmaxGFP® vector provided by Amaxa, and flow cytometric analysis of 50,000 cells. Transfected human THP-1 monocytes (1.5x10 6 /well) were differentiated into macrophages by addition of phorbol 12-myristate 13-acetate (100nM). Cellular lipids, RNA and cell protein lysates were collected 72h posttransfection. Macrophage 'foam cell' prevention experiments were initiated 48h after transfection: macrophages were radiolabelled with [ 3 H]oleic acid (1µCi ml -1 ; 10 M) in the presence or absence of acetylated LDL (50µg ml -1 ) and with or without ACAT inhibitor (447C88, 10µM), for 24h. Wild type macrophages were incubated with progesterone (10 M; 24h) or U18666A (25 M; 24h) to inhibit endosomal trafficking, using ethanol vehicle (<0.1%, v/v).

Cellular viability was assessed by conversion of dimethylthiazolyl diphenyltetrazolium bromide (MTT) to formazan; the caspase-Glo® 3/7 assay system (Promega) was used to detect apoptosis.

Lipid analyses

Flux of [ 3 H]oleic acid (1 Ci ml -1 ; 10 M) into the cholesteryl ester pool, and of [ 14 C]acetate (1 Ci ml -1 ) into cholesterol, cholesteryl ester, fatty acid, triacylglycerol and phospholipid pools were assessed by t.l.c., as previously [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF][START_REF] Palmer | Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) AI from human macrophage foam cells[END_REF]. Efflux of [ 3 H]cholesterol (0.5 Ci ml -1 ) to apoA1 (10µg ml -1 ) and HDL (10µg ml -1 ) were measured as previously [START_REF] Alpy | Functional characterisation of the MENTAL domain[END_REF][START_REF] Palmer | Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) AI from human macrophage foam cells[END_REF]. Mass of macrophage total cholesterol, triacylglycerol and choline-containing phospholipids were measured using Infinity™ and Phospholipids-B colorimetric assays (Alpha Labs, UK) [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF][START_REF] Palmer | Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) AI from human macrophage foam cells[END_REF].

Gene and protein expression

Total RNA (Tri-Reagent, Sigma-Aldrich) was isolated from macrophages (above) and reverse transcribed to cDNA utilising MMLV reverse transcriptase (RT) enzyme (Bioline). Expression of STARD3, STARD4, STARD5, APOE, ABCA1, ABCG1, ABCG4, LXR and SREBP-1 and -2 mRNA, relative to the housekeeping gene, GAPDH, was performed by quantitative (real-time) PCR, using DNA engine Opticon 2 (MJ Research). The PCR reactions contained cDNA template, Q-PCR mix, molecular biology grade water and 100nM of each forward and reverse primers and fluorescent probe (FAM/TAMRA). Thermal cycling conditions were 15min at 95°C, followed by 40 cycles of 15s at 95°C and 20s at 60°C, with status at 50°C. Primers and probes details are given in Table 1. The comparative 2 -Ct method was utilized for quantitation of each gene relative to GAPDH mRNA.

Macrophage lysates were collected in RIPA buffer plus Complete TM protease cocktail inhibitor (Roche, UK). Protein lysates (15-30µg well -1 ) were separated by SDS-PAGE (NuPAGE, 10% w/v gels), transferred to PDVF membrane and probed using anti-STARD3 (1:1000), anti-GAPDH (1:1000) rabbit polyclonal antibodies, and an anti-ABCA1 murine monoclonal antibody (1:1000). Detection was achieved using appropriate secondary antibodies (1:1000) and ECL detection system [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF]; densitometry was performed using Scion Image software.

Statistical analyses

Numbers of independent experiments are indicated in legends to Figures; values are mean±SEM, with significance (p<0.05) determined by Student's t-test, or repeated measures ANOVA, followed by Dunnett's or Tukey-Kramer post t-tests, as appropriate; *p<0.05; **p<0.01 and ***p<0.001 for comparisons indicated.

Results

Comparison of the endogenous expression of STARD3 mRNA, relative to housekeeping gene GAPDH, in human THP-1 monocytes, THP-1 macrophages, human peripheral blood monocytemacrophages and human heart aortae, is shown in Table 2. Transient over-expression of STARD3 in human THP-1 monocyte-macrophages increased mRNA levels by 87-fold (24h; p<0.001), 20-fold (48h; p<0.001) and 18-fold (72h; p<0.05) (Figure 1a), which translated into 2.2-fold (p<0.05) increases in levels of STARD3 protein after 72h (Figure 2b), by comparison with empty vector control; all values normalised to housekeeping protein, GAPDH. Cell viability was not altered by STARD3 over-expression after 72h (empty vector control 25. No changes in steady state levels of mRNA encoding sterol-responsive transcription factors SREBP1 (not detectable), SREBP2 or LXR were noted, as judged by Q-PCR (Figure 1b). In comparison, over-expression of genes encoding cytosolic cholesterol transporters, STARD4 (Figure 1c) and STARD5 (Figure 1d), exerted distinct influences on gene expression of these transcription factors: STARD4 was associated with reduced (p<0.05) expression of SREBP2, while STARD5 overexpression was associated with marked increases (p<0.05) in both SREBP2 and LXR mRNA levels.

Over-expression of STARD3 was associated with significant (p<0.05) increases (20.7-fold) in ABCA1 mRNA (Figure 2a) and protein (2.6-fold) (Figure 2b); by contrast, no changes in ABCG1 and ABCG4 mRNA (Figure 2a) were noted between STARD3 over-expressing macrophages and control cells, following normalisation to GAPDH. These changes in gene expression strongly predicted functional increases in cholesterol efflux to apoAI, rather than to HDL, following STARD3 over-expression, and this proved correct, despite a small reduction in gene expression of the endogenous acceptor APOE: [ 3 H]cholesterol efflux to apoAI (10 g ml -1 ; 24h) was enhanced by 80% (p<0.05) (Figure 2c), whereas efflux to HDL (10 g ml -1 ; 24h) did not change significantly (% HDL-specific efflux: control 5.27±0.58% versus STARD3 6.9±2.5%; n=3; NS) By contrast, inhibition of endosomal trafficking using progesterone (10 M), inhibited apoAI-specific cholesterol efflux by 54% (n=3; p<0.01). Decreases in biosynthesis of cholesterol (25.5%; p<0.05) and cholesteryl ester (34.1%; p<0.01), fatty acid (27.3%; p<0.05), triacylglycerol (41.9%; p<0.01) and phospholipids (27.7%; p<0.05) from [ 14 C]acetate, were observed in STARD3 over-expressing macrophages (Figure 2d), compared with empty vector controls; no change in total cholesterol (control 26.1±3.91 g mg -1 protein versus STARD3, 27.2±3.15 g mg -1 ), triacylglycerol (83.7±10.9 g mg -1 protein versus STARD3 86.3±21.3 g mg -1 protein) or phospholipid mass (42.3± 3.96 g mg -1 protein versus STARD3 47.1 ±8.31 g mg -1 protein) occurred over the time scale investigated. Again, inhibition of endosomal trafficking using U18666A (25 M) or progesterone (10 M) induced the opposite effect, increasing incorporation of [ 14 C]acetate into each lipid pool (p<0.05) (Figure 2d).

Importantly, when STARD3 over-expressing cells were challenged with AcLDL (50 g ml -1 ; 24h), no significant increases in cholesterol esterification occurred, as judged by incorporation of [ 3 H]oleate into the cholesteryl ester pool; in fact, a small but significant (p<0.05) decrease was observed. By contrast, in untreated and empty vector control cells, cholesterol [ 3 H]esterification increased by around 2.2-fold (p<0.01) when treated with AcLDL under the same conditions (Figure 3); a change blocked by ACAT inhibitor, 447C88 (10 M). Equally, an apparent reduction in intensity of Oil-Red-O staining was observed in macrophages over-expressing STARD3, compared with empty vector control, after treatment with AcLDL (50 g ml -1 ; 24h) (Figure 3).

Discussion

The 'START' family of lipid trafficking may be involved in non-vesicular cholesterol transport, regulating sterol-responsive transcription factors, controlling the cholesterol content of organelles, lipid rafts and membranes, and storage of cholesterol as cytosolic droplets of cholesteryl ester, although the mechanisms remain poorly understood at present. Notably, our work demonstrates that over-expression of distinct cholesterol and oxysterol-binding proteins within the 'START' family of lipid trafficking proteins, STARD1 (StAR) [START_REF] Taylor | Over-expression of steroidogenic acute regulatory protein (StAR) increases macrophage cholesterol efflux to apolipoprotein AI[END_REF], STARD3, STARD4 and STARD5, exert different effects on gene expression of sterol-responsive transcription factors, SREBP2 and LXR . Over-expression of mitochondrial cholesterol transporter, StAR is associated with induction of lipogenesis [START_REF] Taylor | Over-expression of steroidogenic acute regulatory protein (StAR) increases macrophage cholesterol efflux to apolipoprotein AI[END_REF], possibly via LXR-dependent induction of SREBP-1c.

Transient over-expression of cytosolic STARD4 decreased SREBP2 mRNA levels by approximately half (Figure 1c), while over-expression of the gene encoding cytosolic STARD5 [START_REF] Rodriguez-Agudo | Localisation of the StarD5 cholesterol binding protein[END_REF] protein resulted in marked increases in SREBP2 and LXR message levels, compared with controls (Figure 1d). Over-expression of STARD4 [START_REF] Soccio | Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins[END_REF]. By contrast, STARD3 over-expression did not impact on either SREBP2 or LXR mRNA levels (Figure 1b) and repressed lipogenesis (Figure 2d). While some of the observed effects may be due to differences in gene expression and translation levels, despite the delivery of equivalent amounts of DNA to the same number of cells, but may also reflect the efficiency and/or directionality of cellular cholesterol transport achieved [START_REF] Soccio | Intracellular cholesterol transport[END_REF][START_REF] Soccio | StAR-related Lipid Transfer (START) proteins: mediators of intracellular lipid metabolism[END_REF].

Previously, we speculated that STARD3 may traffick endosomally derived cholesterol to the endoplasmic reticulum and/or the plasma membrane [START_REF] Borthwick | Differential regulation of the STARD1 subfamily of START lipid trafficking proteins in human macrophages[END_REF]; even in the absence of lipoprotein-derived cholesterol, cholesterol cycles between the plasma membrane and endosomes [START_REF] Holtta-Vuori | Endosomal cholesterol traffic: vesicular and nonvesicular mechanisms meet[END_REF]. Thus, STARD3 could help to increase the cholesterol content of the ER membrane, and retention of SREBPs by the SCAP/Insig-1(-2) complex, in agreement with the generalised repression of cholesterol, fatty acid and triacylglycerol biosynthesis observed here. Cholesterol delivery to the ER might also be expected to expand the pool available for esterification by Acyl CoA: Cholesterol AcylTransferase-1 (ACAT-1; SOAT1). Instead, expansion of the cholesteryl [ 3 H]oleate pool following exposure to acetylated LDL, was effectively blocked in cells over-expressing STARD3. One explanation for this data is that delivery of sterol to the endoplasmic reticulum was just sufficient to sequester SREBPs, but does not reach the threshold required to activate ACAT. In turn, this must imply that the bulk of the endosomal cholesterol trafficked by STARD3 be efficiently directed elsewhere, perhaps to the plasma membrane to facilitate cholesterol efflux (below), possibly via vesicular transport facilitated by the Rab family of small GTPases. Dissociation between cholesterol transport to membrane-bound SREBP transcription factors and the substrate pool available for cholesterol esterification is documented [START_REF] Du | Effects of 25-hydroxycholesterol on cholesterol esterification and SREBP processing are dissociable: implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum[END_REF][START_REF] Kristiana | Different kinetics of cholesterol delivery to components of the cholesterol homeostatic machinery: Implications for cholesterol trafficking to the endoplasmic reticulum[END_REF]: Kristiana et al (2008) noted clear differences in kinetics between endosomal delivery of LDL-derived cholesterol to SREBPs and ACAT in mutant Chinese Hamster Ovary cells with cholesterol trafficking defects (including Niemann Pick Type C), contending that different cholesterol pools and/or transport pathways supply SREBPs and ACAT within the ER [START_REF] Kristiana | Different kinetics of cholesterol delivery to components of the cholesterol homeostatic machinery: Implications for cholesterol trafficking to the endoplasmic reticulum[END_REF].

Our data also agrees well with deletion studies of the START domain of STARD3 in mice in vivo [START_REF] Kishida | Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism[END_REF]. Although relatively modest changes in lipid phenotype were observed, probably due to functional redundancies within the START family of cholesterol transfer proteins [START_REF] Kishida | Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism[END_REF][START_REF] Riegelhaupt | Targeted disruption of StARD4 leads to modest weight reduction and minor alterations in lipid metabolism[END_REF], significant increases in hepatic sterol ester were noted after feeding a high fat diet, together with reduced conversion of cholesterol to steroid hormones [START_REF] Kishida | Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism[END_REF]. Use of a dominant negative mutant of STARD3 ( START-STARD3) caused extensive cholesterol accumulation in CHO cells and COS-7 cells, accompanied by inhibition of late endosomal trafficking, similar to the phenotype caused by functional loss of Niemann Pick Type C1/2 proteins [START_REF] Zhang | MLN64 mediates mobilisation of lysosomal cholesterol to steroidogenic mitochondria[END_REF]. Moreover, in cholesterol-laden cells, STARD3 becomes trapped at the periphery of cholesterol-laden lysosomes, reflecting loss of dynamic cholesterol movement [START_REF] Zhang | MLN64 mediates mobilisation of lysosomal cholesterol to steroidogenic mitochondria[END_REF], and deletion of STARD3 is linked with disrupted actin-mediated dynamics of late endocytic organelles, suggesting that cholesterol binding or sensing by STARD3 in late endosomal membranes may govern actin-dependent fusion and degradative activity of that compartment [START_REF] Holtta-Vuori | MLN64 is involved in actin-mediated dynamics of late endocytic organelles[END_REF]. Hepatic over-expression of STARD3 in vivo is associated with increased conversion to bile acids [START_REF] Ren | Effect of increasing the expression of cholesterol transporters (StAR, MLN64 and SCP-2) on bile acid synthesis[END_REF], although in a separate study, apoptosis and hepatic toxicity were also reported, probably resulting from the grossly elevated and highly unphysiological levels of STARD3 utilised [START_REF] Tichauer | Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse[END_REF]; such changes were not observed in this study.

Over-expression of STARD3 in this study was also associated with increased expression of ABCA1 mRNA and protein. The latter suggests that STARD3, like NPC1, may facilitate trafficking of endosomal cholesterol and possibly ABCA1 protein, through this compartment to the plasma membrane, increasing pools of membrane cholesterol and/or transporter available for efflux to apoAI [START_REF] Rigamonti | Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages[END_REF]. The mechanism(s) by which STARD3 increases mRNA levels of ABCA1 are less obvious, as no induction of LXR was observed in our experiments, and Npc1 inactivation reduces Abca1 protein, but does not alter Abca1 mRNA levels in murine macrophages [START_REF] Wang | Differentiation regulation of ATP binding cassette protein A1 expression and ApoA-I lipidation by Niemann-Pick Type C1 in murine hepatocytes and macrophages[END_REF]. However, SREBPs (1 and 2) can exert repressive effects on ABCA1 expression, decreasing cholesterol efflux [START_REF] Zhou | Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism[END_REF][START_REF] Zeng | Sterol responsive element binding protein (SREBP) 2 down-regulates ATP binding cassette transporter A1 in vascular endothelial cells: a novel role of SREBP in regulating cholesterol metabolism[END_REF]. Thus, it is possible that sequestration of SREBPs at the endoplasmic reticulum, suggested by the coordinated loss of lipid biosynthesis observed here, may relieve inhibition of ABCA1 gene expression and increase cholesterol efflux to apoAI.

Alternatively, STARD3 expression could alter levels of ABCA1 mRNA by changing ratios of saturated to unsaturated fatty acids [START_REF] Schmitz | Transcriptional regulatory networks in lipid metabolism control ABCA1 expression[END_REF], or perhaps via its involvement in actin-mediated dynamics of late endosomes [32] trigger changes in actin-dependent gene expression [33].

In conclusion, STARD3 over-expression may be useful in limiting atherogenesis, by upregulating cholesterol efflux mechanisms, reducing cholesterol synthesis and blockade of cholesterol esterification: in vivo studies are now needed to establish this contention, using murine models of atheroma. 33. Castano, E., Philimonenko, V.V., Kahle, M., Fukalova, J., Kalendova, A., Yildirim, S., Dzijak, R., Dingova-Krasna, H. and Hozak, P. (2010) Actin complexes in the cell nucleus: new stones in an old field. Histochem. Cell Biol. 133: 607-626. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  4± 2.4 M formazan versus STARD3 29.2±6.4 M formazan; p=0.59; mean±SEM from six independent experiments), and levels of Clinical Science Immediate Publication. Published on 21 May 2010 as manuscript CS20100266 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100266 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited macrophage caspase 3/7 activity (empty vector control 6971±270 versus STARD3 7632±1905; p=0.509; mean±SEM from three independent experiments; data expressed as arbitrary fluorescent units) did not change when STARD3 over-expression was compared with the empty vector control.

  Science Immediate Publication. Published on 21 May 2010 as manuscript CS20100266 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100266 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited 32. Holtta-Vuori, M., Alpy, F., Tanuanpaa, K., Jokitalo, E., Mutka, A-L and Ikonen, E. (2005) MLN64 is involved in actin-mediated dynamics of late endocytic organelles. Mol Biol. Cell 16: 3873-3886.

Figure 1 a

 1 Figure 1 a) Transient over-expression of STARD3 compared with empty vector control (24-72h), in human THP-1 monocyte-macrophages and b) effect of STARD3 over-expression (72h) on levels of SREBP2 and LXR mRNA compared with over-expression of STARD4 (c) and STARD5 (d) on gene expression of the same transcription factors. Values are mean±SEM of three independent experiments; *p<0.05, **p<0.01 and ***p<0.001 compared with empty vector control.

Figure 2 a

 2 Figure 2a) Levels of ABCA1, ABCG1, ABCG4 and APOE mRNA in monocyte-macrophages over-expressing STARD3 (72h), compared with empty vector control, in three independent experiments. b) Levels of STARD3 and ABCA1 protein, compared with housekeeping protein, GAPDH, in THP-1 monocyte-macrophages (72h): values representative of three independent experiments. The effect of STARD3 over-expression on c) cholesterol efflux to apoAI (10 g ml -1 ), in five independent experiments, and d) biosynthesis of lipids from [ 14 C]acetate (1 Ci ml -1 ), compared with empty vector controls, in four independent experiments; endosomal inhibitors U18666A (25 M) and/or progesterone (10 M) are included as positive controls. All values are mean±SEM; significant differences (p<0.05) from the control incubation for each lipid are indicated as a: free cholesterol, b: cholesteryl esters, c: fatty acids, d: triacylglycerol and e: phospholipids.

Figure 3 10 Figure

 310 Figure 3Incorporation of [ 3 H]oleate (10 M; 1 Ci ml -1 ) into the cholesteryl ester pool, following incubation with acetylated LDL (50 g ml -1 ; 24h) in the presence or absence of ACAT inhibitor 447C88 (10 M) in wild type cells, and in cells transfected with either empty vector or STARD3. Values are mean±SEM of four independent experiments; *p<0.05, **p<0.01 compared with controls; †p<0.05 compared with acetylated LDL alone, and ‡ ‡p<0.01 compared with empty vector control treated with acetylated LDL. The bottom panel (A-D) illustrates Oil-Red-O staining in empty vector (A, C) and STARD3 over-expressing (B, D), incubated in the absence (A, B) or presence (C, D) of AcLDL (50 g ml -1 ; 24h).

Figure 3 Clinical

 3 Figure 2aFigure2c

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgments

We gratefully acknowledge the support of HEART RESEARCH UK Project Grant 2515/07/09. Table 1 Sequence of primers and probes used to quantify mRNA by polymerase chain reaction Gene Forward Primer Reverse Primer Probe Sequence


Quality management for the collection of biological samples in multicentre studies

Keywords: Quality management, Survey, Multicentre study, Cohort study, Biological specimen bank, Sample database

Large scale international multicentre studies require sophisticated quality management for the collection, processing and logistics of biological samples to ensure a maximum degree of standardisation across different environmental conditions and settings. This paper describes a quality management system for the collection of biological samples (QMS-BS) which was applied during IDEFICS, a large European multicentre study.

The application was evaluated by several criteria like response rates for the different types of biological samples, measures of sample quality, compliance with the QMS-BS and efficiency of the document and sample control and of the quality assurance system.

Response rates varied from 56.6% for venous blood collection to 90.1% for saliva collection. All sample types were associated with problems of sample quality (e.g. haemolysis of blood samples, lack of cooling for urine samples or desiccation of saliva samples). Overall compliance with the QMS-BS was good, with some exceptions mainly related to sample control. In conclusion the QMS-BS is a valuable tool for the management of biological sample collection in epidemiological multicentre studies.

Introduction

In epidemiological studies, biological samples have become an indispensable source of information [START_REF] Vineis | Molecular epidemiology[END_REF]. Different sample types are used to measure a broad range of biomarkers, for example hormones, lipids, glucose, protein, bulk and trace elements or genetic factors. In epidemiology these are used to determine exposure, susceptibility or effects [START_REF] Grandjean | Biomarkers in epidemiology[END_REF].

Several factors influence the observed concentration of a biomarker in the human body, some are inherent like age or sex, but others are controllable. These can affect the concentration of a biomarker either before, during or after sample collection. Before sample collection, fasting status, medication, drug intake, physical activity and diurnal and seasonal variation play an important role. For example glucose and fatty acids are known to be closely related to food intake and therefore fasting measurements are mostly used in epidemiological studies. The levels of most biomarkers vary during the day; some can even change considerably within minutes. Salivary morning cortisol for instance was found to change by approx. 10% within 30 minutes in adolescents [START_REF] Kelly | Levels and confounders of morning cortisol collected from adolescents in anaturalistic (school) setting[END_REF]. Deacon et al. [START_REF] Deacon | Posture influences melatonin concentrations in plasma and saliva in humans[END_REF] reported the effects of posture on melatonin concentrations in plasma and saliva. After sample collection, other variables affect the stability of a biomarker in vitro, e.g. temperature, time until processing or freezing and additives like anticoagulants or stabilising agents. As one example, stability tests for fatty acids showed that these are very unstable at room temperature without any treatment [START_REF] González-Gross | Sampling and processing of fresh blood samples within a European multicenter nutritional study: evaluation of biomarker stability during transport and storage[END_REF]. All of these factors are potential sources of bias, and thus require to be standardised. The most important influences on biological samples are summarised in Table 1. A comprehensive summary of factors influencing the quality of biological samples in molecular epidemiological studies is given in a review by Holland et al. [START_REF] Holland | Biological sample collection and processing for molecular epidemiological studies[END_REF].

The standardised collection of biological samples in epidemiological studies is a challenging task, especially in international multicentre studies. Environmental conditions, such as ambient temperature, distance between field study centres and laboratories, may largely vary thus affecting the quality of the biological samples.

Nevertheless, if data ought to be suitable for joint data analysis, standardised collection, processing, shipment, storage and analyses of biological samples is indispensable. A reliable quality management system can ensure standardisation across different environmental conditions and settings.

The International Organization for Standardization (ISO) created their quality management system (QMS) standards in 1987. These are applicable in different types of industries, for different types of activities or processes, e.g. design, production or service delivery. The ISO QMS standards certify processes, not the product itself. The Since the 1990s, the International Epidemiological Association (IEA) and many national associations have discussed quality criteria for epidemiological research. They have agreed on guidelines for Good Epidemiological Practice (GEP) which can be found on their respective websites [START_REF]Good Epidemiological Practice guidelines for proper conduct of epidemiological research[END_REF]. All of these guidelines promote the idea of quality assurance in epidemiological studies, which was elaborated by Rajaraman and Samet [START_REF] Rajaraman | Quality Control and Good Epidemiological Practice[END_REF]. A working group of the Department of Epidemiology at the INRS in France successfully implemented the ISO 9002 system in their department [START_REF] Moulin | Implementation of ISO 9002 for research in occupational epidemiology Accreditation and quality assurance[END_REF]. Their certified quality system includes procedures specific to the conduct of epidemiological studies in occupational epidemiology but detailed aspects of biomarker collection are not addressed by their quality system. An overview of quality aspects in molecular epidemiology was provided by Holland et al. [START_REF] Holland | Molecular epidemiology biomarkers-sample collection and processing considerations[END_REF].

Considering the work of Moulin et al. [START_REF] Moulin | Implementation of ISO 9002 for research in occupational epidemiology Accreditation and quality assurance[END_REF] and Holland et al. [START_REF] Holland | Molecular epidemiology biomarkers-sample collection and processing considerations[END_REF], we present a newly developed quality management system designed for the collection of biological samples in epidemiological studies (QMS-BS). The applicability of the QMS-BS was evaluated in the context of the IDEFICS study, a large European multi-centre study on childhood obesity, where several types of biological samples were collected. To evaluate the application of the QMS-BS, the following criteria were applied: sample response rates, measures of sample quality, compliance with the QMS-BS and efficiency of the document and sample control and of the quality assurance system.

Methods

Quality management system for biological samples (QMS-BS)

The conduct of an epidemiological study is suitable for the implementation of an ISO standard as shown by Moulin et al. [START_REF] Moulin | Implementation of ISO 9002 for research in occupational epidemiology Accreditation and quality assurance[END_REF]. Table 2 shows the embedding of the newly developed quality management system for the collection of biological samples (QMS-BS) into the ISO system, in analogy to the quality system for occupational epidemiology by Moulin. The key elements of the QMS-BS are described below.

Standard operating procedures

In clinical research, SOPs are defined as "detailed written instructions to achieve uniformity of the performance of a specific function" [START_REF]International Conference on Harmonization (ICH). Guidance for Industry, E6 Good Clinical Practice: Consolidated Guidance[END_REF]. The QMS-BS foresees SOPs that are study-specific and have to be defined for each type of biological sample on the following aspects: Collection: consent, time of collection, materials, devices, temperatures, and prerequisites like e.g. fasting status Processing: laboratory devices, calibration procedures, time and temperature ranges allowed for processing steps Shipping: conditions, intervals Storage: temperatures, sorting conditions ISO 15189:2007 specifies in detail the QMS requirements for medical laboratories and ensures both regular internal and external quality audits and participation in interlaboratory comparisons [START_REF]2007 Medical laboratories --Particular requirements for quality and competence[END_REF]. Since ISO 15189:2007 accredited laboratories have SOPs for all their procedures, the QMS-BS does not include SOPs on laboratory analyses.

For the QMS-BS an SOP template was developed (see Figure 1) which also includes a document log to record changes to previous versions. SOPs have to be accessible to study personnel at any time during their work. In a multicentre study, if the study personnel do not have sufficient skill of English language, all SOPs must be translated to the native languages. Possible errors introduced by translation are minimised by back translation.

Document and sample control

Identification and tracing of biological samples requires appropriate labelling of each sample aliquot. ID-numbers given to study subjects and samples should be pseudonymised in a way that does not allow for the identification of the donor but still clearly defines the sample and enables its retrieval at any time.

Depending on the storage needs, ID-labels should be frost-resistant for down to -80°C or even suitable for storage in liquid nitrogen. An SOP has to address how labels are correctly attached to the biosample tubes, as samples with missing ID-labels will usually be excluded from the study. Coding of IDs in barcodes minimises reading mistakes and simplifies tracing of samples with the help of scanners. There are 1D, 2D or 3D barcode systems and radio-frequency identification (RFID) tags. The choice should depend on the amount of information that needs to be coded and the space available on the ID-stickers. 3D-labels are smallest and contain most information. RFID tags have read and write capabilities and can store up to 2 KB. If biological specimens are stored in a biobank for later analysis, a biosample database should be used for efficient retrieval and documentation of storage conditions. If all samples are used up for laboratory analysis, it might be more economic to use simple delivery notes.

Purchases and subcontracting

Uniform sampling equipment and consumables are important means of standardisation in a multicentre study. Use of standard materials can be assured by central purchasing, but additional management and shipping costs have to be accounted for. Alternatively, all materials have to be clearly defined for local purchase. In this case, feedback on the realisation of the requirements should be collected prior to study commencement.

Several components of biological sample collection can be subject to subcontract, either if they cannot be accomplished by own personnel or as a means of standardisation to improve quality. Possible candidates for subcontracting are sample collection, processing or sample analysis. It has to be kept in mind that study elements which are subcontracted are usually more difficult to control by the study management concerning adherence to defined SOPs.

Process control

Before the beginning of the study, biological samples of interest must be identified along with all the steps for their collection, processing, storage, shipment and analyses.

In a multicentre study there are two main options for sample processing: it can either be done locally, followed by freezing and shipping on dry ice or centrally, after shipping on wet ice. To evaluate the best option several parameters have to be weighed against each other: shipping times and costs (if processing is done centrally, daily shipping is necessary) and the higher need for quality assurance if several local laboratories are involved. Either way a lot of conditions need to be defined including minimum and maximum resting times before centrifugation, analysis or freezing and temperatures at which samples should be kept at each stage.

Provisions should be taken for each sample type regarding packaging, shipping intervals and delivery documentation. Shipment of biological samples requires a tracking system to follow packages and a notification by the sender before shipping to ensure that the recipient can accept the package and properly store the samples upon receipt. In the European Community, human biosamples are not generally considered as potentially infectious material any more [START_REF]European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR)[END_REF][START_REF]Technical Instructions for the Safe Transport of Dangerous Goods by Air; Guidance document for Infectious Substances[END_REF]. Dry ice used for shipping however is subject to the dangerous goods regulations of the International Air Transport Association (IATA), which implies e.g. that only cargo planes are used for transportation.

SOPs for long-term storage of biological samples define storage temperatures, a system for sample retrieval, a surveillance system to detect equipment failures, an emergency plan, regular quality checks, and sample inventories.

Many laboratories have a QMS accredited according to ISO 15189. It is advisable to choose an accredited central laboratory to guarantee for uniform sample analysis. If shipment of samples from the survey centres to a central laboratory cannot be achieved within 48h, transportation, even on dry ice, is not a safe option and decentral sample analysis should be preferred. Depending on the stability of the markers of interest there might be a need to analyse certain parameters directly upon collection (point-of-care analysis).

Quality assurance

According to ISO standards a set of activities should be introduced to ensure that the defined SOPs are implemented appropriately: This includes training activities and pretesting of all procedures. Internal and external quality audits should be conducted to verify adherence to the defined SOPs. All means and results of quality assurance have to be documented and should be comprehensible for the study personnel.

Application of the QMS-BS in the IDEFICS-study

IDEFICS is an Integrated Project in the 6 th Framework Programme of the European Commission tackling the -Identification and prevention of dietaryand lifestyle-induced health effects in children and infants‖. [START_REF] Ahrens | Understanding and preventing childhood obesity and related disorders -IDEFICS: a European multilevel epidemiological approach[END_REF],188 pre-school and primary school children from eight European countries were examined during the baseline survey in 2007/2008. One major objective of the IDEFICS study was to assess the distribution of diet-and lifestyle-related health problems and to understand the causal pathways leading there.

The main emphasis of the study was put on three disorders: obesity/ overweight, insulin resistance and impaired bone health. Each of these health problems is associated with a set of biological markers or is even partly defined by them, as it is the case for insulin resistance. The background of the study, its research goals and instruments have been described elsewhere in detail [START_REF] Ahrens | Understanding and preventing childhood obesity and related disorders -IDEFICS: a European multilevel epidemiological approach[END_REF][START_REF] Bammann | IDEFICS: a multicenter European project on diet-and lifestyle-related disorders in children[END_REF] Sample collection in the IDEFICS study was conducted according to the newly introduced QMS-BS (see Table 3). Children in the baseline survey were asked to donate fasting venous blood (native and EDTA blood), morning urine and saliva samples. If venous blood could not be obtained, capillary blood was taken where possible.

A set of SOPs was developed and translated to all survey languages describing the collection, processing, storage and shipping of all types of biological samples in detail. Barcoded labels were used for sample tracking. Each biological sample was labelled with an unambiguous 10 digit identification number (ID) with the last two digits clearly defining the type of aliquot. A biosample database was developed to record detailed information on the preanalytical conditions (collection, processing and storage) of each sample (e.g. cryotube or vial) and its storage location (down to the position in the cryobox) so that retrieval of samples for further use or withdrawal of samples can be done easily. The database facilitates shipping of samples to the central laboratories by an automated generation of delivery notes. A central biobank of remaining samples was built up for long-term storage.

All sampling kits and processing material were purchased centrally. Biochemical analyses were carried out in a central laboratory accredited according to ISO 15189. DNA extraction, genotyping, RNA extraction and gene expression analysis were conducted at different central laboratories.

As blood lipids and blood glucose were markers of primary interest to assess the risk of study participants for metabolic syndrome, these were assessed on site by point-of-care analysis with the Cholestech LDX analyser [START_REF] Panz | Performance of the CardioChek PA and Cholestech LDX point-of-care analysers compared to clinical diagnostic laboratory methods for the measurement of lipids[END_REF]. The decision was made to use this relatively expensive method because it only uses one drop of venous or capillary blood; thus children who only agreed to give capillary blood could be included to maximize response rates for these key measures. The immediate feedback on these core variables also served as an incentive for the parents. Another drop of blood was sufficient for the fatty acid (FA) analysis of circulating lipids and was collected via a simple kit (Sigma-Aldrich cod. 11312-1KT). This method, developed by Marangoni et al. [START_REF] Marangoni | A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies[END_REF], avoids the complex procedures for collection, storage, shipment, and sample preparation involved with the conventional method of FA analysis.

Apart from point-of-care analyses, all biomarkers were analysed by central laboratories.

Markers analysed in blood samples comprised insulin, CRP and HbA1c, as well as hormones of energy/fat metabolism and markers of bone metabolism. Markers analysed in morning urine included glucose, albumin, and creatinine, as well as several minerals and cortisol. An overview of all biological markers assessed during the IDEFICS study is given in Table 4.

PAXgene tubes containing an RNA stabilising agent were used to collect blood for gene expression analysis in a subsample of children [START_REF] Rainen | Stabilization of mRNA expression in whole blood samples[END_REF]. In order to maximise response rates, sample collection of morning urine was performed at home by the parents, who received a collection kit and a detailed instruction sheet and DNA was obtained noninvasively from saliva samples. Collection procedures for saliva differed by the children's ability to spit the required amount of saliva: Oragene TM DNA collection kit OG 250 with saliva sponges were used for younger children who were usually not yet able to spit, Oragene TM DNA Self-Collection Kit OG 300 were used otherwise. The kits are user-friendly and provide a high amount of good quality DNA [START_REF] Rainen | Stabilization of mRNA expression in whole blood samples[END_REF]. Samples of all types were processed at the local survey centres and shipped to central laboratories at regular intervals.

All procedures were instructed at a central training which was mandatory for all survey centres. The whole set of instruments was tested during a pretest (Suling, in preparation). During the survey, a central telephone hotline was established for all questions regarding the biological samples. Survey site visits were conducted by a central quality control unit where the practical field work was inspected and deviations from SOPs were corrected directly if possible, or else remedial actions were initiated.

Results

Evaluation of applied QMS-BS during the IDEFICS baseline survey

Response rates for different biological sample types in the eight survey centres are listed in Table 5. Response rates varied for different types of biological samples with highest response rates for non-invasive sample types. Urine samples were obtained from 85.6% of the children in total, with the lowest response of 67.9% in centre 3. Saliva samples were collected from 90.2% of the children; again centre 3 had the lowest response rate of 76.7%. Two of the centres (centre 7 and 8) nearly reached completeness for this sample type. For blood sampling, it was attempted to collect venous blood; only children that were not willing to give venous blood were asked for capillary blood. In total, a remarkable 79.7% of the children gave capillary or venous blood and were thus eligible for point-of-care analysis. Centre 4 obtained a substantially lower rate than the other centres (61.6%). This centre also had some initial problems in obtaining fasting blood (7.3% of non-fasting blood samples compared to an average of 1.4%). Venous blood could be collected from on average 56.6% of children across all countries.

Response rates for venous blood ranged from 51% to 83% between the centres with the exception of centre 3 where venous blood was only collected from 7.7 % of the children. The site visit showed that the low response rate at centre 3 resulted from noncompliance with the study protocol: due to the specific situation in the study region children were primarily asked for capillary blood at the survey site and then given the additional opportunity for later venipuncture at another facility.

As part of process control various measures of sample quality were recorded accompanying the different steps of sample collection and a quality check was performed for each sample in the laboratory upon arrival (see Table 6). Collection of morning urine was documented by the parents on a collection sheet; this included recording of potential problems with sample quality. About 2.9% of the parents reported that the sample was not the first morning urine, 3.9% that the children went to the toilet at night and 6.9% stated that the urine was left uncooled for several hours. These rates varied among the centres: for 45% of the urine samples of centre 4 at least one of these problems was reported compared to only 0.3% of centre 3. The quality check of urine samples at the central laboratory on the other hand generally showed no problems (data not shown).

Venous blood samples were repeatedly associated with several problems as noted both by the study centres in the biosample database and by the central laboratory. According to the study centres an average of 5.4% of the samples were haemolytic with a maximum prevalence of 25.1% in centre 1. The central laboratory on the other hand categorised 10.3% of samples as haemolytic, where again centre 1 was of main concern with 30.8% of samples classified as haemolytic. High amounts of haemolytic samples were also reported for centre 2, 4 and 8 (13.8%, 8.2% and 12.1% respectively). It can thus be concluded that the assessment of haemolytic samples by the study centres was not a suitable tool to detect problematic samples for laboratory analyses. Coagulation of EDTA samples occurred in 0.7% of the checked samples, most of which came from centre 4. All centres had occasional problems to reach the required filling quantity for the aliquots. In total 4% of the samples were considered to be ‗short samples' by the central laboratory.

Saliva samples were least error-prone of all sample types; the small fraction of dry samples (0.7%) which arrived at the central laboratory (probably due to leakage) were re-hydrated and still extracted with a lower, but yet sufficient, yield of DNA.

Compliance with the QMS-BS varied between survey centres and components. All survey centres participated in the central training and conducted subsequent local trainings as foreseen in the quality assurance system. The survey centres implemented all standardised procedures according to SOPs as verified by the external quality audits during site visits. The only exception was the SOP for blood collection, were one centre (centre 3) did not follow the instructions to take venous blood preferentially as mentioned above. For some other components compliance was lower. Most notably, centres had difficulties using the biosample database. The software was considered to be too complex and data entry too time-consuming. This caused a delay in data entry which in turn resulted in problems in the central laboratory, where biochemical analyses could not be conducted for samples delivered without a corresponding database.

Moreover, due to the delay it was not possible to carry out direct quality control of sample processing with the biosample database as initially planned. Variables recorded in the database like time intervals, e.g. between collection of blood samples and their centrifugation represent important measures of quality assurance. As depicted in Table 6 especially one centre had problems to manage sample processing within the requested time span. 11.4% of the samples in centre 5 were centrifuged later than foreseen in the SOP, for all other centres this rate was below 1.5%.

Discussion

This paper introduces a quality management system for the collection of biological samples in epidemiological studies (QMS-BS). The QMS-BS was applied to a large multi-centre study, the IDEFICS-study with the goal to collect biological samples of a standardised quality across all study centres and to build up a large biobank of blood, saliva and urine samples from children from different European regions. Evaluation showed that high average response rates were reached for all sample types. Similar rates were achieved during the German KIGGS study, a health study on children and adolescents, where a response rate of 85% was reached for urine samples in the ‗Iodine Module' of the study [START_REF] Thamm | Iodine intake in Germany. Results of iodine monitoring in the German Health Interview and Examination Survey for Children and Adolescents (KiGGS), Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz[END_REF] and 52.6% for an environmental survey module which included venous blood sampling amongst others [START_REF] Schulz | German Environmental Survey for Children (GerES IV) in the German Health Interview and Examination Survey for Children and Adolescents (KiGGS). First results[END_REF]. Measures of sample quality revealed several problems which mainly occurred during sample collection and processing. Central purchase of consumables guaranteed comparability but was accompanied by high shipping costs; depending on the monetary value of the respective consumables up to 10% were added to their costs. Overall compliance with the QMS-BS was good in the study centres, although some exceptions were noted. These were related primarily to sample control. Study centres complained about the complexity of the biosample database, leading to a considerable delay in data entry. The database could thus not be used for quality control during the ongoing survey, only retrospectively. Nevertheless biobanking and sample retrieval would not have been possible without the biosample database.

Generally, not all differences between centres could be avoided by the QMS-BS. In particular, blood response rates and sample quality differed substantially between centres. Different response rates for invasive sample types may be related to national characteristics, whereas differences in sample quality were probably rather based on different survey logistics, e.g.allocated manpower or geographical distances between sample collection and processing. The influence of sample quality on the analyses of biological makers in the IDEFICS-study will be the topic of future investigations.

It should be noted that the introduction of a quality management system is not able to solve all problems during sample collection in epidemiological studies. Another key aspect is the complexity of the applied procedures. Decisions made for user-friendly solutions within IDEFICS were generally expensive but very successful like e.g. pointof-care analysis of the main biological markers for metabolic syndrome (glucose, total cholesterol, HDL, triglycerides), DNA colletion with saliva kits that didn't need to be processed or cooled, and FA test strips that only needed one drop of blood for the analysis of complete fatty acid profiles. These solutions generally reached high response rates and offered a standardised quality. For example, because point-of-care analysis was employed for blood glucose and lipid analyses, response rates for the respective markers were increased by about 23% (representing the children which only agreed to the collection of capillary blood).

Results of the evaluation will contribute to further optimisation of biological sample collection for the IDEFICS follow-up survey that starts in autumn 2009. The biosample database has been substantially simplified and a barcode scanner and laptop will be used for storage documentation. Moreover, procedures for serum collection will be changed to reduce the amount of haemolytic samples which was most likely caused by insufficient clotting times in combination with the completely native blood tubes that were used to allow for the removal of two drops of venous blood for point-of-care analysis and FA analysis before clotting started. To enhance compliance with the QMS-BS in the IDEFICS follow-up survey different options are currently discussed. Krockenberger et al. [START_REF] Krockenberger | Usage and usability of standard operating procedures (SOPs) among the coordination centers for clinical trials (KKS)[END_REF] investigated the adherence to SOPs in the context of clinical trials. They found that, for questions about the daily work, the staff was more likely to ask a colleague rather than to read the corresponding SOP (18.4% versus 13.8%). The authors suggest a computer-based information retrieval system for SOPs to increase the ease-of-use and usefulness of SOPs wich might also be an interesting option for the QMS-BS. Other options are centralised re-training sessions and/or special emphasis on selected aspects of biological samples collection during the external site visits.

Practical aspects of field work and sample collection are often neglected in scientific exchange. The QMS-BS fills this gap and represents a systematic approach to sample collection for application in epidemiological studies. Over all its application in IDEFICS helped to obtain a high quality standard for the biological samples collection in this European multicentre study.

  standards are regularly reviewed by the ISO, the last revision was done in 2008 and the series was called ISO 9001:2008 series [7].

Table 1 : Major sources of bias for biomarkers before, during and after sample collection Before sample collection During sample collection After sample collection

 1

	Fasting status	Posture of study subject	Centrifugation
	(food, drinking,	Sample type (e.g. venous	conditions
	medication, smoking)	blood vs. Capillary blood)	Haemolysis of blood
	Timing of collection	Collection materials (e.g.	samples
	(diurnal variation,	tourniquet, collection cups)	Storage temperature
	seasonal variation)	Use of additives	UV light
	Recent exercise	(e.g. anticoagulating agents,	(e.g. direct sunlight)
	State of health	stabilising agents)	Time before
	(e.g. infections, fever,	Insufficient volume in tube	processing/ freezing
	lipaemia, pregnancy)	Order of draw	Shipping conditions
		Sterility	(temperature and
			time)
	25.

Table 2 : Quality management system for the collection of biological samples (QMS-BS), adapted from the quality system for occupational epidemiology (Moulin, 1998)

 2

	ISO 9002 elements	QMS-BS

Table 4 : Overview of biological markers analysed in the IDEFICS-study

 4

	Sample type	Biological marker	Exposure
	Native blood	Point-of care analysis:	Metabolic syndrome,
		Blood glucose, Total cholesterol,	Insulin resistance
		HDL cholesterol Triglycerides
		Fatty acid profiles (collection kit)	Dietary patterns
	EDTA plasma	Fatty acid profiles (conventional	Dietary patterns
	RBC	methodology for validating the collection kit)
	EDTA blood	HbA1c	Diabetes
	Serum	Insulin	Insulin resistance
		C-reactive protein (CRP)	Inflammation
		Leptin	Markers of energy
		Adiponectin	metabolism
		Vit D	Bone metabolism
		Ca
		NTX-peptide
	Whole blood collected in	Quantity of RNA for selected genes	Gene expression
	RNA-stabilising
	PAXgene tubes
	Morning urine	Urinary glucose	Diabetes
		Urinary albumin	Metabolic syndrome
		Urinary creatinine	Reference marker
		Minerals (Na, K, Mg, P, Ca)	Dietary patterns
		Cortisol	Chronic stress
	Saliva	Selected candidate genes (sequencing	Genetic risk factors
		of tag-haplotypes)
	28.

Table 5 : Response rates for biological samples during the baseline survey of IDEFICS Study centre Total blood Venous blood Capillary blood

 5 a study subject was included if at least data on age, sex, height and weight was collected 29.

	Urine Saliva	Subjects
		included*

*

Table 6 : Measures of samples quality during IDEFICS baseline survey

 6

	Study centre	1	2	3	4	5	6	7	8 Total
	Morning urine
	Samples collected	1,946	1,419 1,615	1,498	1,596 1,858 2,550	1,405 13,887
	Problems with sample quality
	Any problem	499	12	5	679	60	176	37	221	1689
	% of urine samples 25.64% 0.85% 0.31% 45.33%	3.76% 9.47% 1.45% 15.73% 12.16%
	Problems with collection
	Not first morning	145	6	2	60	37	44	30	80	404
	urine
	% of urine samples 7.45% 0.42% 0.12% 4.01%	2.32% 2.37% 1.18% 5.69% 2.91%
	Child went to the toilet	95	2	2	202	15	123	2	97	538
	at night
	% of urine samples 4.88% 0.14% 0.12% 13.48%	0.94% 6.62% 0.08% 6.90% 3.87%
	Problems with processing
	Urine uncooled for	326	4	1	522	8	12	8	77	958
	several hours
	% of urine samples 16.75% 0.28% 0.06% 34.85%	0.50% 0.65% 0.31% 5.48% 6.90%
	Problems with storage					n/a
	Venous blood
	Samples collected	1,296	882	184	996	1,058 1,413 2,133	1,221	9,183
	Problems with collection
	Child not fasting for	2	13	0	71	33	18	17	29	112
	>8h
	% of blood samples 0.15% 1.47% 0.00% 7.13%	3.12% 1.27% 0.80% 2.38% 1.22%
	Problems with processing
	Samples haemolytic	399	122	3	82	61	35	94	148	944
	% of blood samples 30.79% 13.83% 1.63% 8.23%	5.77% 2.48% 4.41% 12.12% 10.28%

Acknowledgement

This work was done as part of the IDEFICS Study (www.idefics.eu). We gratefully acknowledge the financial support of the European Community within the Sixth RTD Framework Programme Contract No. 016181 (FOOD). The authors are indebted to the IDEFICS survey centres for their work in gathering the data. We also thank Wolfgang Ahrens for helpful suggestions on the manuscript.

Abbreviations

Production procedures

Standard procedures (SOPs) specific to the collection of biological samples

Document and data control Document and sample control

Purchasing

Purchases and subcontracting

Process control

Process control for biological samples:

-collection -processing -shipping -storage -laboratory analyses

Inspection and testing

Quality assurance for sample collection 26.