**2.4.1 Real-time PCR**

The real-time PCR have been run on ABI7300 real times machines according to earlier publications (Cinek et al., 2006). Primers were first designed for main type of virus and thereafter for subtypes (serotypes) and optimalisation was performed for each of the reactions.

#### **2.4.2 Sequencing**

Sequencing for enterovirus, picornavirus and E.coli as well as other bacterial species will be done as earlier published (Tapia et al., 2011; Witsø et al., 2006, 2007; Muinck et al., 2011). Deep sequencing will be performed on at 454 machines at the Centre for Ethological and Evolutional Sciences (CEES), Institute of Biology, University of Oslo according to the manufacture instructions.

#### **2.4.3 Questionnaires**

Data from questionnaires will be used to test association between viral RNA/DNA in stool and symptoms reported by the parents (coughing, diarrhea, vomiting), and will be used to search for risk factors of viral infection, such as breastfeeding, number of siblings and socioeconomic status.

#### **2.5 Identification of eggs from enterobius vermicularis**

Parents of children participating in MIDIA have been asked to collect tape samples touching the anal region on three following mornings. They have then sent the samples in specially designed containers for tape sampling to the central laboratory in Oslo. Here all the tape samples have been examined by two scientists at different times. A child has been regarded as positive if down to one egg have been identified on one of the tapes.

### **3. Results**

346 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

autoantibodies on at least two consecutive time periods (3-6 months apart) is defined as islet autoimmunity for the purpose of data analysis, and will be used as the first outcome (optimal cut-off values for the autoantibodies has been defined after participation by the Hormone laboratory in international autoantibody standardisation workshops; DASPs). Clinical diagnosis of T1D will also be used as outcome, and analysis will be performed

A questionnaire summarising weekly diaries were filled out when the children were 3, 6, 9 and 12 months old, and annually thereafter. The questionnaires include for examples detailed information about dietary habits of the mother and the child (detailed information about diet for the mother as long as she breast-fed and intake of specific food items, etc.). Since few studies have been conducted on children's diet in Norway, new dietary questions had to be developed for both the MIDIA and the MoBa study. Validation of various aspects of dietary habits of pregnant women has recently been undertaken within the MoBa study. Blood samples and questionnaires from MIDIA can be used to assess validity of relevant information in childhood (Brantsæter et al., 2007a, 2007b, 2008, 2009; Willett, 1998; Serdula et

The distribution of fatty acids in the plasma phospholipid fraction as well as Vit D and Vit E will be analyzed in plasma samples from the same aliquots at a commercial laboratory in Oslo (AS Vitas; http://www.vitas.no/) using solid phase extraction and gas

The real-time PCR have been run on ABI7300 real times machines according to earlier publications (Cinek et al., 2006). Primers were first designed for main type of virus and thereafter for subtypes (serotypes) and optimalisation was performed for each of the

Sequencing for enterovirus, picornavirus and E.coli as well as other bacterial species will be done as earlier published (Tapia et al., 2011; Witsø et al., 2006, 2007; Muinck et al., 2011). Deep sequencing will be performed on at 454 machines at the Centre for Ethological and Evolutional Sciences (CEES), Institute of Biology, University of Oslo according to the

Data from questionnaires will be used to test association between viral RNA/DNA in stool and symptoms reported by the parents (coughing, diarrhea, vomiting), and will be used to search for risk factors of viral infection, such as breastfeeding, number of siblings and

when a sufficient number of children have developed either autoimmunity or T1D.

**2.3 Measurement of nutrition-related factors ("exposures")** 

**2.3.2 Biomarkers: Fatty acids, vitamin D and E** 

**2.4 Measurement of exposure to virus (viral infections)** 

**2.3.1 Questionnaires** 

al., 2001).

reactions.

chromatography.

**2.4.1 Real-time PCR** 

**2.4.2 Sequencing** 

manufacture instructions.

**2.4.3 Questionnaires** 

socioeconomic status.

#### **3.1 Psychosocial effects of risk information**

#### **3.1.1 Effects of genetic risk information on mental health variables among MIDIA mothers**

In the study of mothers who had participated in both MIDIA and MoBa (N=166 for those having a child with high genetic risk for T1D and N=7,224 for those who had been told that their child did not have the high-risk genotype) there were no sosiodemographic characteristics differences between those who had got the risk-information and those who had let their child be genotyped in MIDIA, but had been told that their child did not carry the high-risk genotype. Information on genetic risk in newborns was found to have no significant impact on maternal symptoms of anxiety and depression, self-esteem, satisfaction with life, or serious worry about their child. Mental health before birth was strongly associated with mental health after birth, see Table 1. Maternal symptoms of anxiety and depression were assed using a short version of SCL-25, including 4 question for anxiety and 4 for depression (Aas et al., 2010). The five-item Satisfaction With Life (SWLS) was developed to measure the cognitive component of subjective well-being. The short-form of the Rosenberg Self-Esteem Scale (RSES) used in the MoBa study includes four items. Maternal worry about their child was one of the items in an 11-item checklist of life events experienced during the last year, given in the 6 month questionnaire. The question was phased "Have you been serious worried that there is something wrong with your child?" Responses were coded as "yes" or "no". A dichotous variable was constructed to indicate the presence of maternal T1D. The variable was based on health questions from both The Medical Birth Registry of Norway (MBRN) and the MoBa questionnaires. The results from the linear regression analyses of the association between genetic risk information and change in maternal mental health are shown as unstandardized (B) and standard (β) coefficients in Table 1. The upper part of the table shows the results from the regression analysis with symptoms of anxiety and depression (SCL) as the dependent variable. The estimated regression coefficient (B=-0.001, p=0.95) for child's genetic risk indicate no effect of genetic information on changes in maternal mental health from baseline to postdisclosure. The maternal T1D status had neither any effect (B=0.040, p=0.409). However, as expected, the baseline anxiety/depression score was strongly associated with postdisclosure scores (B=0.536, p<0.001).

#### **3.1.2 How often do parents think on that they have a child with high genetic risk for T1D**

Although 5% of mothers and 2% of fathers did think of their child's high genetic risk for T1D when they filled out the 3 month questionnaire, usually just 2 week after they had got the information, very few parents continued to think often about their child's high genetic risk for T1D. To answer the questionnaire with the same questions each time needs that you sometimes think about having a high-risk child for T1D, see Figure 1 and 2.

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 349

3 mns 6 mns 9 mns 1 yr 2 yrs 3 yrs 4 yrs 5 yrs 6 yrs 7 yrs 8 yrs 9 yrs

Never % Sometimes % Often % Daily % Not answered %

3 mns 6 mns 9 mns 1 yr 2 yrs 3 yrs 4 yrs 5 yrs 6 yrs 7 yrs 8 yrs 9 yrs

Fig. 2. Fathers thoughts about having a child with high genetic risk of T1D

Fig. 1. Mothers thoughts about having a child with high genetic risk of T1D

0,0

0,0

10,0

20,0

30,0

40,0

50,0

60,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0


Table 1.

#### **3.2 Nutrition-related factors**

Cohort design was used for assessing whether BMI before pregnancy and weight gain during pregnancy predicted the risk of islet autoimmunity in 885 children who were followed with serial blood samples and questionnaires. 36 of the children developed autoimmunity, of whom 10 developed Type 1 Diabetes. Both maternal BMI before pregnancy and weight gain < or = 15 kg predicted increased risk for islet autoimmunity, significant hazard ratio at 2.5 for both situations (Rasmussen et al., 2009).

#### **3.3 Virus in stool samples**

Among 911 children, were stool samples were available, 27 had developed autoimmunity in two or more consecutive samples (case children) in December 2008. In the pilot study based on these cases two control children per case were matched by follow-up time, day of birth, and county of residence. The frequency of human enterovirus RNA in stool samples from cases before seroconversion (43 of 339, 12.7%) did not differ from the frequency in control subjects (94 of 692, 13.2%) (Tapia et al., 2011a). There was neither any difference in the prevalence of human parechovirus when cases and controls were compared: 13.0% and 11.1%, respectively (Tapia et al., 2011b). None of the 3,803 samples analysed were positive for rodent parechovirus-Ljungan virus (Tapia et al., 2008, Tapia et al., 2010). Indicating that Ljungan virus is rare among Norwegian children, and in contract to what have been reported earlier does not seem to be involved in T1D susceptibility.

Cohort design was used for assessing whether BMI before pregnancy and weight gain during pregnancy predicted the risk of islet autoimmunity in 885 children who were followed with serial blood samples and questionnaires. 36 of the children developed autoimmunity, of whom 10 developed Type 1 Diabetes. Both maternal BMI before pregnancy and weight gain < or = 15 kg predicted increased risk for islet autoimmunity,

Among 911 children, were stool samples were available, 27 had developed autoimmunity in two or more consecutive samples (case children) in December 2008. In the pilot study based on these cases two control children per case were matched by follow-up time, day of birth, and county of residence. The frequency of human enterovirus RNA in stool samples from cases before seroconversion (43 of 339, 12.7%) did not differ from the frequency in control subjects (94 of 692, 13.2%) (Tapia et al., 2011a). There was neither any difference in the prevalence of human parechovirus when cases and controls were compared: 13.0% and 11.1%, respectively (Tapia et al., 2011b). None of the 3,803 samples analysed were positive for rodent parechovirus-Ljungan virus (Tapia et al., 2008, Tapia et al., 2010). Indicating that Ljungan virus is rare among Norwegian children, and in contract to what have been

significant hazard ratio at 2.5 for both situations (Rasmussen et al., 2009).

reported earlier does not seem to be involved in T1D susceptibility.

Table 1.

**3.2 Nutrition-related factors** 

**3.3 Virus in stool samples** 

Fig. 1. Mothers thoughts about having a child with high genetic risk of T1D

Fig. 2. Fathers thoughts about having a child with high genetic risk of T1D

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 351

haven received good enough information about MIDIA before she and her husband had consented to participate. The Directorate for Health and Social Affairs then immediately decided that recruitment to MIDIA had to be stopped. Some days later it was, however, decided that new evaluation of the project had to take place according to the Norwegian Biotechnology Law, which tells that genotyping of children under the age of 18 years can only take place if there are a clear health benefit for a certain disease to get knowledge about genetics. During the fall of 2007 both the Biotechnology Board, the Ethical Committee for the Norwegian Medical Association, the National Committee for Medical Ethics as well as several experts contacted by the Directorate of Social and Health Affairs evaluated the MIDIA project. All these boards had earlier evaluated the MIDIA study; e.g. during the time of recruitment to the study. In addition the Health Department had clearly told that children who also had developed autoantibodies in MIDIA could get health insurance. The last aspect was based on the Biotechnology Law, which Norway has had since 1994, where it is clearly told that genetic risk for a disease cannot be used by the health insurance companies. The Directorate of Social and Health Affairs found, however, genotyping in MIDIA illegal December 10, 2007. A few days after the Norwegian Data Inspectorate said in newspapers that all data already collected from participants in MIDIA had to be thrown away. All ended luckily up with voting in the Norwegian Parliament in June 2008. As long as the Medical Regional Committee and the Norwegian Data Inspectorate approved the MIDIA study ones more, and all parents of children who already had been identified as high-risk children, gave a new informed consent , research in MIDIA could continue. In this respect Norway is different from Sweden, Finland, Germany and five states in USA were no similar Biotechnology Law

Has given problems with genotyping of 350,000 children for the TEDDY study.

2. Are not the parents able to give informed consent of behalf of their child?

The Norwegian Biotechnology Law tells: "Genetic testing of a child under the age of 18 years is not allowed if circumstances cannot be detected that can reduce or prevent health disadvantages for the child." Since the law came in 1994 it had only counted for clinical practice, the MIDIA project had been run for 6 ½ year before it was stopped December 10, 2007. In the work performed before the law got in use, science was never mentioned.

1. Do important scientific T1D projects involving genotyping of children have to be performed elsewhere in the word? Should not Norway as one of the riches countries in

4. Is it not so that if clear health benefit has been shown, it is no longer research but part of

The year after recruitment to MIDIA was stopped funding was given from the Norwegian Research Council to the study "Nutritional Intervention to Prevent Type 1 Diabetes". The project was based on that the incidence of T1D is increasing, particularly in very young children. The hypothesis was that the Decrease of omega-3 fatty acids in the diet has contributed to this increase. One case-control study from Norway reported that children with T1D less often than the control children had a mother who had taken cod liver oil during pregnancy, while a newer study from Norway indicated a protective effect of cod liver oil during infancy (Stene et al., 2000; Stene & Joner, 2003). In the longitudinal, observational study, the Diabetes Autoimmunity Study in the Young (DAISY), conducted in

general recommendation for public health or part of the health care system?

**4.1.2 Ethics and data protection in human biomarker studies** 

Important questions in this context are:

the word has a certain responsibility?

3. How should health benefit be defined?
