Dr Kathryn N Parkinson
email: kathryn.parkinson@ncl.ac.uk

Mark S Pearce

John J Reilly

Robert F Drewett

Charlotte M Wright

Ann S Le Couteur

Ashley J Adamson

Anne Dale

Caroline L Relton

Paul Mcardle

Cohort Profile: The Gateshead Millennium Study

Keywords: childhood, obesity, eating behaviour, dietary intake, physical activity

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

How did the study come about?

The Gateshead Millennium Baby Study (GMBS) originated from the observation that slower than expected weight gain in infancy, traditionally known as failure to thrive, but more recently as 'weight faltering', had never been satisfactorily explained. There were methodological problems associated with much previous research. The first was the use of attained weight criteria to identify slow weight gain in infancy, which confounds poor postnatal weight gain with poor prenatal weight gain. The second was the use of referred samples of children, leading to selection biases. The third was the use of retrospective accounts from parents after poor weight gain had already been identified. The GMBS was thus originally designed to investigate the antecedents of weight faltering in a population based prospective study which addressed the main methodological problems of previous research.

What does it cover?

The original GMBS set out to examine infant feeding behaviour and relate it to subsequent weight gain and weight faltering. It also investigated risk factors for iron deficiency in infancy. Relevant maternal characteristics including feeding style, eating attitudes and depression were also examined. More recently the title of the study has been changed to Gateshead Millennium Study (GMS) to reflect the age of the cohort. The study has continued to focus on feeding and growth, with three additional areas of interest emerging.

First, it investigated aspects of developmental psychopathology using measurements principally taken at 5-6 years of age. A recent UK prevalence survey in 5-19 year olds reported that mental health disorders are common, affecting approximately 8% and that sub-threshold symptoms were even more common. 1 Eating disorders comprise an important social and medical problem developing over late childhood.

Accurate figures on the prevalence of eating disorders have not been easy to establish.

Those in a recent comprehensive review suggest a prevalence of 0.28% for anorexia nervosa, 1% for bulimia nervosa, 1.5% for night eating syndrome and 2.3% for binge eating disorder. [2][3][4] A much higher proportion of young women report less specifically diagnosable problems such as occasional binge eating or self induced vomiting or score high on tests of abnormal eating attitudes. 5 There is also evidence that subthreshold eating disorders are commonly associated with depression in adolescents, and there is a strong link between restrictive eating and aggression, attempted suicide and substance misuse. 6,7 Most longitudinal studies of disordered eating have started in the teenage years, but the strongest predictor of later disordered eating in these studies has always been disordered eating at the start of the study. 8 There is some evidence for continuity between earlier feeding problems and later eating disorder symptoms 9 and we are examining this systematically in this study. The GMS also investigated the child's emotional and behavioural development, and social communication skills. A topic of particular interest has been the development and prevalence of repetitive behaviours, which are key behaviours in autistic spectrum disorder but are also found in typically developing children. These repetitive behaviours can range from repetitive motor mannerisms, intense special interests or hobbies to more unusual sensory interests in the sight, taste, sound, smell or texture of objects or people. 10 The GMS provided an opportunity to investigate prospectively the relationship between various symptoms of psychopathology in typically developing children, and to identify early risk and protective factors for both eating problems and mental health disorders. The second new area of interest was the high and rising rates of childhood obesity. [11][12][13] It was known that body mass index (BMI; weight/height 2 ) tracks through later childhood into adulthood 14 and that teenage and adult obesity are associated with increased health risks. [15][16][17][18] It was also suggested that low birth weight and subsequent underweight in infancy are related to an increased risk of a number of chronic diseases in adulthood, including obesity. 19 However, detailed normative data are scarce and little was known about how lean and fat mass vary between individual children and how far early weight gain is associated with early childhood fatness. In addition, risk factors for early adiposity, such as dietary intake, physical activity and sedentary behaviour, needed to be investigated. Finally, there was the potential for intervention to reduce new cases of obesity in childhood. Evidence suggested that family-based interventions offered potential for success in prevention of obesity, [START_REF] Mulvihill | The management of obesity and overweight: An analysis of reviews of diet, physical activity and behavioural approaches, 1st Edition[END_REF] yet little information is available on what the content and approach of interventions should be and which would be acceptable to families. The GMS was able to investigate these issues when the cohort was aged 6-8 years.

The third new area of interest was the mechanisms by which early life exposures may influence health in later life. It has been postulated that nutrition during pregnancy as well as early feeding behaviours may alter mitotically heritable markings on DNA, so called epigenetic markings, which can influence body composition and other phenotypic characteristics in children through altering gene expression. 21,22 Epigenetics provide a biologically plausible mechanism to explain developmental programming and this area is currently the focus of much research activity. The GMS

F o r R e v i e w O n l y -6 -
was able to investigate the association between early life exposure and epigenetic patterns in childhood when the cohort was aged 6-8 years.

Who is in the sample?

The GMBS recruited participants shortly after birth in Gateshead, an urban district in north east England. All infants born to mothers resident in Gateshead in 34 prespecified weeks between June 1999 and May 2000 were eligible. From the 1252 eligible mothers, 1011 mothers joined the study; 116 (12%) were under 20 years of age, 518 (51%) were over 20 and under 30 and 377 (37%) were 30 or over. A total of 1029 infants were recruited (18 sets of twins). Full details of recruitment are reported elsewhere. 23 The current GMS sample consists of any traceable survivor of the original cohort whose family have not asked to be withdrawn.

How often have they been followed up?

The cohort was recruited by study researchers and baseline data were collected in an interview shortly after the baby was born. This was followed up by data collected by midwives at hospital discharge and six days, and by health visitors at ten days and three months. Parents were sent postal questionnaires at 6 weeks, and 4, 8, 12 months. Families were invited to a clinic appointment at 13 months where the child and mother were measured for height and weight. A case-control study of children who failed to thrive had two lunchtime meals directly observed at 13-21 months.

There were further parent completed postal questionnaires at 30 months and 5-6 years.

A data sweep at age 6-8 years collected information from parents at home visits, and for the first time since 13 months children were assessed directly at school and/or home visits. At the 6-8 year data sweep parents were followed up with qualitative

F o r R e v i e w O n l y -7 -
focus groups and one-to-one interviews. At 8-10 years data were collected data from parents postally, and from children directly at school.

What has been measured?

Descriptions of the data collected are shown in Table 1.

[Insert Table 1 about here] Throughout infancy and early childhood detailed information was collected using questionnaires completed by the child's carer, usually the mother. Characteristics assessed included growth, feeding behaviour, temperament, illness and social behaviour in children. Information about parent characteristics including eating attitudes and depression was also collected. The GMBS mothers were given a special edition of the Personal Child Health Record (PCHR) which is issued routinely to all new mothers. The special edition PCHR included extra forms for health staff and parents to record details about the child on the study's behalf; in particular mothers were asked to get their baby weighed regularly and to transcribe the weights onto the questionnaires they received and send them to the study office. The children and parents were also measured directly at clinics run by study nurses at 13 months.

School-entry child height and weight measurements were retrieved from NHS records.

When the children were aged 5-6 years a postal questionnaire collected information about the child's eating behaviour and maternal approach to feeding their child.

Information was also collected on the child's social communication skills, repetitive When the children were aged 6-8 years information collected by parental questionnaire included the child's eating behaviour, the child's food and physical activity environment, parental eating characteristics and food intake, and socioeconomic status. The children completed questionnaires on their food knowledge and eating behaviour. The child's dietary intake was observed, and physical activity levels and sedentary behaviour were measured using accelerometry. The children's and mothers' anthropometric measurements were taken as well as saliva samples for the purposes of DNA extraction and subsequent epigenetic analysis. After the quantitative data collection had finished, for parents who expressed an interest, views on childhood obesity were sought using focus groups and one-to-one interviews to explore knowledge, perceptions of childhood overweight and potential strategies for intervention.

A data sweep when the children were 8-10 years old took direct measures of children's habitual physical activity and sedentary behaviour (using accelerometry) and anthropometric measurements. Additionally, questionnaire data were collected from the children on their home environment, sports club activities and eating attitudes, and from parents on the children's home environment.

What is attrition like?

The children have received a birthday card in the mail every year since birth with a letter including a reply slip for parents to return if they no longer wish to participate in the research. Unless parents have let the study know that they would prefer not to be contacted again, every effort is made to trace, contact and recruit the child for followup data sweeps regardless of whether they remain in the area of birth or have relocated. At the start of the 8-10 year data sweep two children were known to have died and the parents of 161 children had asked to leave the study leaving a sample of 866 children eligible to be contacted.

Non-participation for individual data sweeps has been substantial, but different families have participated in different data sweeps and consequently the study has retraced and collected data from 697 families over the last 3 years.

The original sample was comparable to the northern region of England in terms of socio-economic deprivation apart from slight under-representation of the most affluent quintile, assessed using the Townsend deprivation index from the 1991 census (Table 2). Overall, non-participation has been higher in the least affluent families than in the most affluent. Thus only 15% of families were in the most affluent quintile at birth, but 70% of them participated up to 6-8 years; 19% were in the least affluent quintile but only 56% participated at 6-8 years. This means that by 6-8 years the distribution across all the deprivation quintiles was fairly even (Table 2) and the current sample is representative of the north of England. Non-participation patterns according to maternal qualification are similar to the Townsend deprivation index, with non-participation being higher in less educated mothers, particularly for postal data sweeps (Table 2).

[Insert

What has it found? Key findings and publications

Weight loss, weight gain and feeding in the first year of life

Postnatal weight loss was less than previous studies have shown. At five days, the mean weight loss was 50g, only just over 1% below birth weight, and 3% of babies were 10% below, none showing any signs of serious illness. One third had already regained their birth weight. 24 More frequent feeding in the first week was related to higher weight gain at 6 weeks of age for breast-feeders, but not for bottle-feeders.

Babies who were fed by both breast and bottles were less likely to continue to be fed by breast at 6 weeks of age than those who received only breast milk. 25,26 Nearly one quarter of the babies started eating solids before 3 months of age.

Characteristics associated with early weaning were fast weight gain to 6 weeks, lower socioeconomic status, parents' perceptions that their baby was hungry, and being bottle fed. Babies weaned before 3 months, compared with those weaned after 4 months of age, had increased risk of diarrhoea. 27 Appetite at 6 weeks and 12 months was positively related to weight gain to 12 months, and encouraging a child to eat was associated with poorer weight gain.

Children's appetite appeared to be related to how well or poorly they eat and grow. 28 Mothers' characteristics such as their eating behaviour, mood, and social characteristics were not strongly associated with children's weight gain over the first year. Postnatal depression was associated with slower weight gain and increased rates of weight faltering in the infants up to 4 months, especially if they came from deprived families, but by 12 months they were no different from the remainder of the cohort. 29 Data from this cohort as well as the Avon Longitudinal Study of Parents and Children (ALSPAC) have been used to assess how the growth of UK infants compares to newly published WHO standards. Analysis showed that in both cohorts infants were heavier than infants in the WHO growth study from about the age of 6 months but showed very similar linear growth. 30

Mealtime energy intake and feeding behaviour

Video observations were used to identify types of feeding behaviour in children aged 13-21 months. Two meals were observed for each of 30 children who had shown weight faltering in the first year and 57 controls. Energy intake and weight of food eaten were also measured. The children who gained less weight were offered and accepted food at the same rate as children with normal weight gain but had a lower energy intake. 31

Diagnosis of borderline iron deficiency

This therapeutic trial included 462 infants whose parents agreed to have blood taken and were successfully bled during the 13 month assessment. It explored how successful five different blood markers were at identifying iron deficiency, as indicated by a clear response to treatment. Low total and mean cell haemoglobins proved good predictors of a response to treatment, but about half the children who responded had haemoglobins within the normal range. The three other markers were only associated with a response when two or more were abnormal. Using the above definition 13% of the children tested could be defined as truly iron deficient. These children did not generally show differences in social factors or growth, apart from a moderate association with breast feeding for over 4 months and a greatly increased risk for a small minority of children from ultra orthodox Jewish families. 32

Eating behaviour and repetitive behaviours in toddlers and young children

Eating problems were perceived as common in toddlers and in the majority were associated with normal growth, although weight faltering was more common in such children. Excessive milk-drinking may have been a cause of low appetite at meal times, but was not associated with poor growth. 33 Repetitive behaviours are an essential part of the diagnosis of autism but are also commonly seen in typically developing children. Repetitive behaviours data from this cohort, pooled with data from another local cohort, showed that 2 year old children frequently engage in a range of different types of repetitive behaviours (with boys showing higher scores than girls). These behaviours can be summarised within four subscales: unusual sensory interests; repetitive motor movements; rigidity/adherence to routine; and preoccupations with restricted patterns of interests. These study results support the proposal that repetitive behaviours measured on subscales that closely resemble the repetitive behaviour subtypes within the ICD-10 criteria for autism, represent a continuum of functioning that extends to the typically developing child population. 10 Subsequent analysis of new pooled data looking at the continuity of repetitive behaviours from 2 years to 6 years shows that the predominant repetitive

F o r R e v i e w O n l y -13 -
behaviours change over time and that although the total rates of repetitive behaviours reduce over time, they do not disappear. 34

Surveillance of physical activity in the UK

Public health surveillance of physical activity in children in the UK has depended on a parent-reported physical activity questionnaire which has not been validated. Physical activity measured objectively using accelerometry in 6-7 year olds showed that levels of habitual physical activity in children are likely to be substantially lower than those reported in UK Health Surveys. 35

Origins and perceptions of childhood adiposity

Parents' ability to identify whether their child was overweight was limited when compared to current standards for overweight or obesity. Parents neither used nor trusted current standards of childhood overweight and instead used alternative approaches which heavily relied on extreme cases as a reference point. [START_REF] Jones | Millennium Study core team. Parental perceptions of body size and childhood adiposity at 6-8 years in the Gateshead Millennium Study[END_REF]37 What are the main strengths and weaknesses? Gateshead has a stable population, but even so, maintaining up-to-date contact details and keeping the families involved has been difficult. Direct contact has achieved higher participation than postal contact. For example, 636 families returned postal questionnaires at 12 months whereas 847 attended a clinic run by study nurses at 13 months. Using direct contact for retracing and data collection, the study has managed to maintain a cohort of approximately 700 children.

F o r R e v i e w O n l y -14 -
Gateshead has a higher rate of deprivation than the rest of the northern region of England and this was reflected in the lower proportion of the most affluent families recruited to the study. As is common for community based studies, there has been higher rates of attrition among the most deprived families. 38,39 However, due to higher retention of the most affluent families compared to the least affluent families, the current sample is socially diverse and representative of the north of England.

The cohort has the benefit of a wealth of data on maternal characteristics and the child's early eating patterns, developmental psychopathology, dietary intake at 6-8 years, a comprehensive set of body composition measurements, lifestyle behaviours and physical activity at 6-8 and 8-10 years, and DNA samples. It will provide crucial information for investigation of both physical and mental health. Of obvious importance is the examination of the role of fast weight gain in infancy and childhood lifestyle behaviours in the development of later obesity and other chronic disease outcomes in later life. A cohort of this size is only adequately powered to examine variation within the normal range and between major subgroups, but since our current main area of interest, childhood obesity, is now so common, the study is likely to be adequately powered to examine important risk factors. General child health and education ** Mother's approach to feeding her child 50 Child's eating behaviour 51 Repetitive Behaviour Questionnaire 48,49 Common symptoms of psychopathology 52 Social communication problems 53 Parental employment (self-coded method) 54 Socio-economic status Child's eating behaviour ** Child's risk factors for eating disorders [START_REF] Stice | Risk factors for the emergence of childhood eating disturbances: A five-year prospective study[END_REF] Child's food environment 56 Child physical activity environment ** Parental concern about child's obesity in adulthood 57 Parental perception of child's body shape 58,59 Parental perception of child's activity 60 $ Parent's attitudes and knowledge about food [61][62][63] Maternal eating behaviour 44 Parent's physical activity 64 Parent food frequency 65 Family health status ** Parental employment (self-coded method) 54 Socio-economic status Attitudes and knowledge about food 62 Restrained eating 66 Body image 59 Habitual physical activity (accelerometry) 67 Habitual sedentary behaviour (accelerometry) 67 Habitual sedentary behaviour (accelerometry) 67 Sports activity and how they feel physically 70,71 Sedentary opportunities in the home 69 Eating patterns [START_REF] Maloney | Reliability testing of a children's version of the Eating Attitudes Test[END_REF]

Can

Key messages

The Gateshead Millennium Study recruited a socially diverse birth cohort in 1999/2000. The current sample is representative of the north of England.

The cohort provides a rich dataset on children's early eating characteristics, dietary intake, physical activity, and developmental psychopathology.

The study will allow examination of the development of childhood obesity, and the development of preventative strategies.

What does it cover?

The original GMBS set out to examine infant feeding behaviour and relate it to and that sub-threshold symptoms were even more common. 1 Eating disorders comprise an important social and medical problem developing over late childhood.

Accurate figures on the prevalence of eating disorders have not been easy to establish.

Those in a recent comprehensive review suggest a prevalence of 0.28% for anorexia nervosa, 1% for bulimia nervosa, 1.5% for night eating syndrome and 2.3% for binge eating disorder. [2][3][4] A much higher proportion of young women report less specifically diagnosable problems such as occasional binge eating or self induced vomiting or score high on tests of abnormal eating attitudes. 5 There is also evidence that subthreshold eating disorders are commonly associated with depression in adolescents, and there is a strong link between restrictive eating and aggression, attempted suicide and substance misuse. 6,7 Most longitudinal studies of disordered eating have started in the teenage years, but the strongest predictor of later disordered eating in these studies has always been disordered eating at the start of the study. 8 There is some evidence for continuity between earlier feeding problems and later eating disorder symptoms 9 and we are examining this systematically in this study. The GMS also investigated the child's emotional and behavioural development, and social communication skills. A topic of particular interest has been the development and prevalence of repetitive behaviours, which are key behaviours in autistic spectrum disorder but are also found in typically developing children. These repetitive behaviours can range from repetitive motor mannerisms, intense special interests or hobbies to more unusual sensory interests in the sight, taste, sound, smell or texture of objects or people. 10 The GMS provided an opportunity to investigate prospectively the relationship between various symptoms of psychopathology in typically developing children, and to identify early risk and protective factors for both eating problems and mental health disorders.

Deleted: it was possible to collect information from parents about the child's emot

F o r R e v i e w O n l y -5 -
The second new area of interest was the high and rising rates of childhood obesity. [11][12][13] It was known that body mass index (BMI; weight/height 2 ) tracks through later childhood into adulthood 14 and that teenage and adult obesity are associated with increased health risks. [15][16][17][18] It was also suggested that low birth weight and subsequent underweight in infancy are related to an increased risk of a number of chronic diseases in adulthood, including obesity. 19 However, detailed normative data are scarce and little was known about how lean and fat mass vary between individual children and how far early weight gain is associated with early childhood fatness. In addition, risk factors for early adiposity, such as dietary intake, physical activity and sedentary behaviour, needed to be investigated. Finally, there was the potential for intervention to reduce new cases of obesity in childhood. Evidence suggested that family-based interventions offered potential for success in prevention of obesity, 20 yet little information is available on what the content and approach of interventions should be and which would be acceptable to families. The GMS was able to investigate these issues when the cohort was aged 6-8 years.

The third new area of interest was the mechanisms by which early life exposures may influence health in later life. It has been postulated that nutrition during pregnancy as well as early feeding behaviours may alter mitotically heritable markings on DNA, so called epigenetic markings, which can influence body composition and other phenotypic characteristics in children through altering gene expression. 21,22 Epigenetics provide a biologically plausible mechanism to explain developmental programming and this area is currently the focus of much research activity. The GMS Deleted: s, behaviours (including repetitive behaviours) and social communication skills as well as eating behaviours. This information in combination with the early infant and maternal behaviour measures allows us to study the continuities and discontinuities of particular behaviours (such as repetitive behaviours) in a normally developing sample of children, identify early predictors of later onset disorders and consider prospectively potential risk and protective factors for child mental health problems 1 including persistent disordered eating behaviours in later childhood. The incidence of eating disorders is reported to rise steeply over the peri-pubertal period, so most longitudinal studies have started in the teenage years. However, in longitudinal studies the strongest predictor of later disordered eating has always been disordered eating at the start of the study. 2 There is evidence that there are early infant predictors of later eating disorder symptoms. 3 It has also been shown that some problems such as persistent binge eating are specifically associated with the development of obesity. 4 Eating problems are common in early childhood but may pose particular management problems for certain at risk groups such as those with autism/autism spectrum disorders 5 was able to investigate the association between early life exposure and epigenetic patterns in childhood when the cohort was aged 6-8 years.

Who is in the sample?

The GMBS recruited participants shortly after birth in Gateshead, an urban district in north east England. All infants born to mothers resident in Gateshead in 34 prespecified weeks between June 1999 and May 2000 were eligible. From the 1252 eligible mothers, 1011 mothers joined the study; 116 (12%) were under 20 years of age, 518 (51%) were over 20 and under 30 and 377 (37%) were 30 or over. A total of 1029 infants were recruited (18 sets of twins). Full details of recruitment are reported elsewhere. 23 The current GMS sample consists of any traceable survivor of the original cohort whose family have not asked to be withdrawn.

How often have they been followed up?

The cohort was recruited by study researchers and baseline data were collected in an interview shortly after the baby was born. This was followed up by data collected by midwives at hospital discharge and six days, and by health visitors at ten days and three months. Parents were sent postal questionnaires at 6 weeks, and 4, 8, 12 months. Families were invited to a clinic appointment at 13 months where the child and mother were measured for height and weight. A case-control study of children who failed to thrive had two lunchtime meals directly observed at 13-21 months.

There were further parent completed postal questionnaires at 30 months and 5-6 years.

A data sweep at age 6-8 years collected information from parents at home visits, and for the first time since 13 months children were assessed directly at school and/or home visits. At the 6-8 year data sweep parents were followed up with qualitative

What has been measured?

Descriptions of the data collected are shown in Table 1.

[Insert Table 1 about here]

Throughout infancy and early childhood detailed information was collected using questionnaires completed by the child's carer, usually the mother. Characteristics assessed included growth, feeding behaviour, temperament, illness and social behaviour in children. Information about parent characteristics including eating attitudes and depression was also collected. The GMBS mothers were given a special edition of the Personal Child Health Record (PCHR) which is issued routinely to all new mothers. The special edition PCHR included extra forms for health staff and parents to record details about the child on the study's behalf; in particular mothers were asked to get their baby weighed regularly and to transcribe the weights onto the questionnaires they received and send them to the study office. The children and parents were also measured directly at clinics run by study nurses at 13 months.

School-entry child height and weight measurements were retrieved from NHS records.

When the children were aged 5-6 years a postal questionnaire collected information about the child's eating behaviour and maternal approach to feeding their child.

Information was also collected on the child's social communication skills, repetitive What is attrition like?

The children have received a birthday card in the mail every year since birth with a letter including a reply slip for parents to return if they no longer wish to participate in The original sample was comparable to the northern region of England in terms of socio-economic deprivation apart from slight under-representation of the most affluent quintile, assessed using the Townsend deprivation index from the 1991 census (Table 2). Overall, non-participation has been higher in the least affluent families than in the most affluent. Thus only 15% of families were in the most affluent quintile at birth, but 70% of them participated up to 6-8 years; 19% were in the least affluent quintile but only 56% participated at 6-8 years. This means that by 6-8 years the distribution across all the deprivation quintiles was fairly even (Table 2)

and the current sample is representative of the north of England. Non-participation patterns according to maternal qualification are similar to the Townsend deprivation index, with non-participation being higher in less educated mothers, particularly for postal data sweeps (Table 2).

[Insert What has it found? Key findings and publications

Weight loss, weight gain and feeding in the first year of life Postnatal weight loss was less than previous studies have shown. At five days, the mean weight loss was 50g, only just over 1% below birth weight, and 3% of babies were 10% below, none showing any signs of serious illness. One third had already regained their birth weight. 24 More frequent feeding in the first week was related to higher weight gain at 6 weeks of age for breast-feeders, but not for bottle-feeders.

Babies who were fed by both breast and bottles were less likely to continue to be fed by breast at 6 weeks of age than those who received only breast milk. 25,26 Nearly one quarter of the babies started eating solids before 3 months of age.

Characteristics associated with early weaning were fast weight gain to 6 weeks, lower socioeconomic status, parents' perceptions that their baby was hungry, and being bottle fed. Babies weaned before 3 months, compared with those weaned after 4 months of age, had increased risk of diarrhoea. 27 Appetite at 6 weeks and 12 months was positively related to weight gain to 12 months, and encouraging a child to eat was associated with poorer weight gain.

Children's appetite appeared to be related to how well or poorly they eat and grow. 28 Mothers' characteristics such as their eating behaviour, mood, and social characteristics were not strongly associated with children's weight gain over the first showed very similar linear growth. 30 Mealtime energy intake and feeding behaviour responded had haemoglobins within the normal range. The three other markers were only associated with a response when two or more were abnormal. Using the above definition 13% of the children tested could be defined as truly iron deficient. These children did not generally show differences in social factors or growth, apart from a moderate association with breast feeding for over 4 months and a greatly increased risk for a small minority of children from ultra orthodox Jewish families. 32 Eating behaviour and repetitive behaviours in toddlers and young children

Deleted: ¶ [Insert
Eating problems were perceived as common in toddlers and in the majority were associated with normal growth, although weight faltering was more common in such children. Excessive milk-drinking may have been a cause of low appetite at meal times, but was not associated with poor growth. 33 Repetitive behaviours are an essential part of the diagnosis of autism but are also commonly seen in typically developing children. Repetitive behaviours data from this cohort, pooled with data from another local cohort, showed that 2 year old children frequently engage in a range of different types of repetitive behaviours (with boys showing higher scores than girls). These behaviours can be summarised within four subscales: unusual sensory interests; repetitive motor movements; rigidity/adherence to routine; and preoccupations with restricted patterns of interests. These study results support the proposal that repetitive behaviours measured on subscales that closely resemble the repetitive behaviour subtypes within the ICD-10 criteria for autism, represent a continuum of functioning that extends to the typically developing child population. 10 Subsequent analysis of new pooled data looking at the continuity of repetitive behaviours from 2 years to 6 years shows that the predominant repetitive behaviours change over time and that although the total rates of repetitive behaviours reduce over time, they do not disappear. 34

Surveillance of physical activity in the UK

Public health surveillance of physical activity in children in the UK has depended on a parent-reported physical activity questionnaire which has not been validated. Physical activity measured objectively using accelerometry in 6-7 year olds showed that levels of habitual physical activity in children are likely to be substantially lower than those reported in UK Health Surveys. 35 Origins and perceptions of childhood adiposity

Parents' ability to identify whether their child was overweight was limited when compared to current standards for overweight or obesity. Parents neither used nor trusted current standards of childhood overweight and instead used alternative approaches which heavily relied on extreme cases as a reference point. [START_REF] Jones | Millennium Study core team. Parental perceptions of body size and childhood adiposity at 6-8 years in the Gateshead Millennium Study[END_REF]37 What are the main strengths and weaknesses?

Gateshead has a stable population, but even so, maintaining up-to-date contact details and keeping the families involved has been difficult. Direct contact has achieved higher participation than postal contact. For example, 636 families returned postal questionnaires at 12 months whereas 847 attended a clinic run by study nurses at 13 months. Using direct contact for retracing and data collection, the study has managed to maintain a cohort of approximately 700 children. Gateshead has a higher rate of deprivation than the rest of the northern region of England and this was reflected in the lower proportion of the most affluent families recruited to the study. As is common for community based studies, there has been higher rates of attrition among the most deprived families. 38,39 However, due to higher retention of the most affluent families compared to the least affluent families, the current sample is socially diverse and representative of the north of England.

The cohort has the benefit of a wealth of data on maternal characteristics and the child's early eating patterns, developmental psychopathology, dietary intake at 6-8 Child's eating behaviour ** Child's risk factors for eating disorders [START_REF] Stice | Risk factors for the emergence of childhood eating disturbances: A five-year prospective study[END_REF] Child's food environment 56 Child physical activity environment ** Parental concern about child's obesity in adulthood 57 Parental perception of child's body shape 58,59 Parental perception of child's activity 60 $ Parent's attitudes and knowledge about food [61][62][63] Maternal eating behaviour 44 Parent's physical activity 64 Parent food frequency 65 Family health status ** Parental employment (self-coded method) 54 Socio-economic status Attitudes and knowledge about food 62 Restrained eating 66 Body image 59 Habitual physical activity (accelerometry) 67 Habitual sedentary behaviour (accelerometry) 67 Body measurements; height, weight, waist, skinfolds (biceps, triceps, subscapular and supra-iliac), bone frame (knee, wrist, shoulders, hips and elbow) and bioelectrical impedance DNA biomarkers (saliva sample) 67 Habitual sedentary behaviour (accelerometry) 67 Sports activity and how they feel physically 70,71 Sedentary opportunities in the home 69 Eating patterns [START_REF] Maloney | Reliability testing of a children's version of the Eating Attitudes Test[END_REF] and appetite * Body image 59 Body measurements; height, weight, waist, and bioelectrical impedance * Influenced by previous work [START_REF] Foster | Infant Feeding 1995: A survey of feeding practices in the United Kingdom[END_REF]

  symptoms related to psychopathology, especially anxiety, emotional and conduct related symptoms.

  of the GMS children is ongoing and they are being flagged with the UK National Health Service Central Register.

  I get hold of the data? Where can I find out more? Most of the data are currently being actively analysed by the existing study team, but we welcome suggestions for collaboration. Potential collaborators should contact Professor Ashley Adamson at the Institute of Health and Society at Newcastle University. The study has a website at http://www.research.ncl.ac.uk/gms/. contributed to protocol development, and was responsible for co-ordination of the study and writing the manuscript. Dr Mark Pearce contributed to protocol development and writing the manuscript, and was responsible for the analytical framework and supervision of data analysis. Dr Anne Dale maintained liaison with Gateshead Children's Services, and contributed to protocol development and writing the manuscript. Dr Robert Drewett contributed to protocol development and writing the manuscript.

  contributed to protocol development and writing the manuscript. Professor Charlotte Wright founded the GMBS cohort and was responsible for execution of the GMBS study, and contributed to protocol development and writing the manuscript. Professor Ann Le Couteur was responsible for execution of the 5-6 year datasweep, and contributed to protocol development and writing the manuscript. Dr Caroline Relton was responsible for execution of the epigenetic element of the study and contributed to the writing the manuscript.Professor Ashley Adamson had primary responsibility for execution of the GMS datasweeps, and contributed to protocol development and writing the manuscript.

3 -

 3 Word count: 3315 (main body of paper, ie excluding title page, contributor's list, references and tables)How did the study come about?The Gateshead Millennium Baby Study (GMBS) originated from the observation that slower than expected weight gain in infancy, traditionally known as failure to thrive, but more recently as 'weight faltering', had never been satisfactorily explained. There were methodological problems associated with much previous research. The first was the use of attained weight criteria to identify slow weight gain in infancy, which confounds poor postnatal weight gain with poor prenatal weight gain. The second was the use of referred samples of children, leading to selection biases. The third was the use of retrospective accounts from parents after poor weight gain had already been identified. The GMBS was thus originally designed to investigate the antecedents of weight faltering in a population based prospective study which addressed the main methodological problems of previous research.

  subsequent weight gain and weight faltering. It also investigated risk factors for iron deficiency in infancy. Relevant maternal characteristics including feeding style, eating attitudes and depression were also examined. More recently the title of the study has been changed to Gateshead Millennium Study (GMS) to reflect the age of the cohort. The study has continued to focus on feeding and growth, with three additional areas of interest emerging.First, it investigated aspects of developmental psychopathology using measurements principally taken at 5-6 years of age. A recent UK prevalence survey in 5-19 year olds reported that mental health disorders are common, affecting approximately 8% Deleted: behaviour Deleted: during the early feeding period and relate it to subsequent weight gain and weight faltering.Deleted: It also investigated risk factors for iron deficiency in infancy.

  The study recruited 1029 infants and f Deleted: At the most recent completed follow-up, 619 children took part. ¶ one-to-one interviews. At 8-10 years data were collected data from parents postally, and from children directly at school.

Deleted: with parentsDeleted:

  The latest data sweep at 8-10 years is collecting Deleted: on objectively measured habitual physical activity and sedentary behaviour, body measurements and eating attitudes. symptoms related to psychopathology, especially anxiety, emotional and conduct related symptoms.When the children were aged 6-8 years information collected by parental questionnaire included the child's eating behaviour, the child's food and physical activity environment, parental eating characteristics and food intake, and socioeconomic status. The children completed questionnaires on their food knowledge and eating behaviour. The child's dietary intake was observed, and physical activity levels and sedentary behaviour were measured using accelerometry. The children's and mothers' anthropometric measurements were taken as well as saliva samples for the purposes of DNA extraction and subsequent epigenetic analysis. After the quantitative data collection had finished, for parents who expressed an interest, views on childhood obesity were sought using focus groups and one-to-one interviews to explore knowledge, perceptions of childhood overweight and potential strategies for intervention.A data sweep when the children were 8-10 years old took direct measures of children's habitual physical activity and sedentary behaviour (using accelerometry) and anthropometric measurements. Additionally, questionnaire data were collected from the children on their home environment, sports club activities and eating attitudes, and from parents on the children's home environment.

  The children's and mother's anthropometric measurements were taken as well as saliva samples for the purposes of DNA extraction and subsequent epigenetic analysis.Deleted: Direct measures of the Deleted: , objectively measured habitual Deleted: also collected. Unless parents have let the study know that they would prefer not to be contacted again, every effort is made to trace, contact and recruit the child for followup data sweeps regardless of whether they remain in the area of birth or have relocated. At the start of the 8-10 year data sweep two children were known to have died and the parents of 161 children had asked to leave the study leaving a sample of 866 children eligible to be contacted.Non-participation for individual data sweeps has been substantial, but different families have participated in different data sweeps and consequently the study has retraced and collected data from 697 families over the last 3 years.

  of the GMS children is ongoing and they are being flagged with the UK National Health Service Central Register.

  Video observations were used to identify types of feeding behaviour in children aged 13-21 months. Two meals were observed for each of 30 children who had shown weight faltering in the first year and 57 controls. Energy intake and weight of food eaten were also measured. The children who gained less weight were offered and accepted food at the same rate as children with normal weight gain but had a lower energy intake.31 Diagnosis of borderline iron deficiencyThis therapeutic trial included 462 infants whose parents agreed to have blood taken and were successfully bled during the 13 month assessment. It explored how successful five different blood markers were at identifying iron deficiency, as indicated by a clear response to treatment. Low total and mean cell haemoglobins proved good predictors of a response to treatment, but about half the children who Deleted: who had shown weight faltering in the first year and controls Deleted: E Deleted: , Deleted: and length of time of the meal Deleted: were Deleted: gaining Deleted: seemed to be Deleted: eat Deleted: Deleted: amount of food Deleted: in fact Deleted: . They were also less likely to sit in a high chair throughout the meal

53 Deleted

 53 or developed for the GMS ** Items developed for the GMS *** Non-validated, based on previous work † Items relevant to six week infants used $ Interviewer led Deleted: General questions ** ¶ Deleted: Parental employment (selfcoded method) 44 ¶ Socio-economic status ** ¶ Child's physical activity by parent-recall diary 53 ¶ Parent food frequency 54 ¶ Deleted: Parent's physical activity $ ¶ Deleted: bony Deleted: In progress Deleted: ¶ Child's physical activity by parent-recall diary

Table 2

 2

	about here]

Table 1 . Summary of data collected since birth (n=1029 children)

 1

	Cohort age	Where	By whom	Number of	Data collected
				participants
				(%)
	After birth	Hospital/home	Mother	1029 (100%)	Birth weight
					Milk feeding behaviour *
					Mode of milk feeding
					Socio-economic status**
	Day 3	Hospital	Midwife	633 (62%)	Feeding ratings**
	Day 6	Hospital/home	Midwife	801 (78%)	Feeding ratings**
	Day 10	Home	Health visitor 944 (92%)	Feeding ratings**
	6 weeks	Home	Parent	832 (80%	Milk feeding behaviour *
					Cessation of breast feeding, weaning etc*
					Illness**
					Infant temperament 40 †
					Weights (from Personal Child Health Record (PCHR))
	3 months	Home	Health visitor 792 (77%)	Feeding ratings**
					Maternal depression 41
	4 months	Home	Parent	762 (74%)	Milk feeding behaviour *
					Cessation of breast feeding, weaning etc*
					Illness **
					Accidents **
					Adverse family life events 42, 43
					Weights (from PCHR)
	8 months	Home	Parent	675 (65%)	Solid feeding behaviour *
					Cessation of breast feeding, weaning etc*
					Illness **
					Accidents **
					Infant temperament 40
					Weights (from PCHR)
	12 months	Home	Parent	636 (61%)	Solid feeding behaviour *
					Cessation of breast feeding
					General behaviour **
					Illness **
					Accidents**
					Maternal eating behaviour 44
					Maternal childhood 45
					Weights (from PCHR)
	13 months	Clinic	Study nurse	847 (82%)	Child's height and weight (measured)
					Blood for full blood count, ferritin, zinc protoporphyrin
					Mother's height and weight (measured)
	30 months	Home	Parent	492 (47%)	General feeding **
					Food preferences 46 47
					Drinks **
					General behaviour **
					Repetitive Behaviour Questionnaire 48, 49
					Difficulties with child **
					Illness **
					Weights (from PCHR)
	4-5 years	School	School nurse	724 (70%)	Child's height and weight (measured)
	5-6 years	Home	Parent	506 (49%)	Child's height and weight (measured)
					Mother's height and weight (measured)
					Child's appetite, mealtimes, problems **

Table 2 . Socio-economic characteristics of mothers (based on data collected at birth)

 2 Percentages do not add up to 100 in all cases due to rounding * Based on Townsend deprivation index from 1991 census, using enumeration districts as the unit of analysis with the Northern Region of England as the population for comparison for the calculation of the quintiles Institute of Ageing and Health g , Newcastle University, Sir James Spence Institute of Child Health, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK Northumberland, Tyne & Wear NHS Trust h , Child and Adolescent Mental Health,

	and appetite *
	Body image 59
	Body measurements; height, weight, waist, and bioelectrical
	impedance

Tel: (0191) 2223828 Fax: (0191) 2225581 Email: kathryn.parkinson@ncl.ac.uk

  Table 2 about here]

	Deleted: Of the 1270 births eligible for
	recruitment, 1029 (82%) were recruited
	to 1011 mothers (18 sets of twins). The
	recruited sample
	Deleted: see

Table 2

 2 Postnatal depression was associated with slower weight gain and increased rates of weight faltering in the infants up to 4 months, especially if they came from deprived families, but by 12 months they were no different from the remainder of the cohort.29 Data from this cohort as well as the Avon Longitudinal Study of Parents and Children (ALSPAC) have been used to assess how the growth of UK infants compares to newly published WHO standards. Analysis showed that in both cohorts infants were heavier than infants in the WHO growth study from about the age of 6 months but

	about here] ¶
	Deleted: Postal questionnaire data were
	collected on 832 infants at 6 weeks
	(80%), 762 at 4 months (74%), 675 at 8
	months (65%), 636 at 12 months (61%),
	492 (47%) at 30 months and 506 (49%) at
	5-6 years. Child and mother weights and
	heights at 13 months were obtained for
	848 families (82%) by GMBS research
	nurses. Child weights and heights were
	retrieved from NHS records at school
	entry for 724 children (70%). Further
	postal questionnaire data were collected
	on 506 children at 5-6 years (49%). ¶
	¶
	At the start of the 6-8 year data sweep in
	2006 two children were known to have
	died and the parents of 99 children had
	asked to leave the study leaving an
	eligible sample of 928 children. From
	these 928 children and their families, data
	were collected from 619 children (66%)
	at school and/or home visits. 488 (79%)
	of mothers and 543 (88%) of children
	provided saliva samples for DNA
	extraction. At 6-8 years there were still
	substantial numbers in all quintiles of
	deprivation (see Table 2). ¶
	The children have received a birthday
	card in the mail every year since birth
	with a letter including a reply slip for
	parents to return if they no longer wish to
	participate in the research. Unless
	parents have let the study know that they
	would prefer not to be contacted again,
	every effort is made to trace, contact and
	recruit the child for follow-up data
	sweeps regardless of whether they remain
	in the area of birth or have relocated
	elsewhere.
	Deleted: Post natal w
	Deleted: in infants ¶
	Deleted: 5
	Deleted: and a
	Deleted: Only 26 babies lost more
	than 10% of their weight, none showing
	any signs of serious illness.
	Deleted: ¶
	¶
	Early feeding patterns and weaning ¶
	Deleted: and only 10% started after the
	then recommended age of 4 months
	although few problems were reported by
	parents.
	Deleted: age
	Deleted: Appetite and weight gain ¶
	Deleted: appears
	Deleted: Influence of maternal
	characteristics on weight gain ¶

Table 1 .

 1 years, a comprehensive set of body composition measurements, lifestyle behaviours and physical activity at 6-8 and 8-10 years, and DNA samples. It will provide crucial information for investigation of both physical and mental health. Of obvious importance is the examination of the role of fast weight gain in infancy and childhood lifestyle behaviours in the development of later obesity and other chronic disease outcomes in later life. A cohort of this size is only adequately powered to examine variation within the normal range and between major subgroups, but since our current main area of interest, childhood obesity, is now so common, the study is likely to be adequately powered to examine important risk factors. Most of the data are currently being actively analysed by the existing study team, but we welcome suggestions for collaboration. Potential collaborators should contact Professor Ashley Adamson at the Institute of Health and Society at Newcastle University. The study has a website at http://www.research.ncl.ac.uk/gms/. Professor Ashley Adamson had primary responsibility for execution of the GMS datasweeps, and contributed to protocol development and writing the manuscript. Maloney MJ, McGuire JB, Daniels SR. Reliability testing of a children's version of the Eating Attitudes Test. J Am Acad Child Psychiatry. 1988;27:541-3. 73. Foster K, Lader D, Cheesbrough S. Infant Feeding 1995: A survey of feeding practices in the United Kingdom. London: Stationery Office: 1997. Summary of data collected since birth (n=1029 children)

	Can I get hold of the data? Where can I find out more?
	Formatted: Normal, Line spacing:
	Double

Table 2 .

 2 Socio-economic characteristics of mothers (based on data collected at birth) Percentages do not add up to 100 in all cases due to rounding * Based on Townsend deprivation index from 1991 census, using enumeration districts as the unit of analysis with the Northern Region of England as the population for comparison for the calculation of the quintiles

															... [1]
															Formatted Table
		Baseline		12 months	13 month		30 months	5-6 years		6-8 years		8-10 years
						health check
		N=1011		N=624		N=830		N=482		N=495		N=605		N=576
		No.	%	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
	Townsend quintile*
	1 (most affluent)	156	15	129	21	137	17	110	23	102	21	109	18	103	18
	2	204	20	146	23	178	21	111	23	113	23	129	21	124	22
	3	227	23	140	22	189	23	107	22	115	23	135	22	128	22
	4	226	22	121	19	177	21	88	18	85	17	118	20	114	20
	5 (least affluent)	192	19	83	13	143	17	61	13	75	15	108	18	101	18
	Missing	6	1	5	1	6	1	5	1	5	1	6	1	6	1
	Qualification
	A levels or above	243	24	186	30	213	26	150	31	159	32	175	29	167	29
	GCSEs or equivalent	495	49	325	52	415	50	255	53	246	50	295	49	273	47
	NVQ's or none	194	19	83	13	144	17	55	11	61	12	98	16	99	17
	Missing	79	8	30	5	58	7	22	5	29	6	37	6	37	6

Acknowledgements

The cohort was first established with funding from the Henry Smith Charity and Sport Aiding Research in Kids (SPARKS) and followed up with grants from Gateshead NHS Trust R&D, Northern and Yorkshire NHS R&D, Northumberland, Tyne and Wear NHS Trust, the National Prevention Research Initiative (incorporating funding from British Heart Foundation; Cancer Research UK; Department of Health; Diabetes UK; Economic and Social Research Council; Food Standards Agency; Medical Research Council; Research and Development Office for the Northern Ireland Health and Social Services; Chief Scientist Office, Scottish Government Health Directorates; Welsh Assembly Government and World Cancer Research Fund), the Children's Foundation and the Scottish Government Health Directorates Chief Scientist Office. We acknowledge the support of an External Reference Group in conducting the study. We appreciate the support of Gateshead Health NHS Foundation Trust, Gateshead Education Authority and local schools. We warmly thank the research team for their effort. Thanks are especially due to the Gateshead Millennium Study families and children for their participation in the study. Acknowledgements The cohort was first established with funding from the Henry Smith Charity and Sport Aiding Research in Kids (SPARKS) and followed up with grants from Gateshead NHS Trust R&D, Northern and Yorkshire NHS R&D, Northumberland, Tyne and Wear NHS Trust, the National Prevention Research Initiative (incorporating funding from British Heart Foundation; Cancer Research UK; Department of Health; Diabetes UK; Economic and Social Research Council; Food Standards Agency; Medical Research Council; Research and Development Office for the Northern Ireland Health and Social Services; Chief Scientist Office, Scottish Government Health Directorates; Welsh Assembly Government and World Cancer Research Fund), the Children's Foundation and the Scottish Government Health Directorates Chief Scientist Office. We acknowledge the support of an External Reference Group in conducting the study. We appreciate the support of Gateshead Health NHS Foundation Trust, Gateshead Education Authority and local schools. We warmly thank the research team for their effort. Thanks are especially due to the Gateshead Millennium Study families and children for their participation in the study. Contributor's list Dr Kathryn Parkinson contributed to protocol development, and was responsible for co-ordination of the study and writing the manuscript. Dr Mark Pearce contributed to protocol development and writing the manuscript, and was responsible for the analytical framework and supervision of data analysis. Dr Anne Dale maintained liaison with Gateshead Children's Services, and contributed to protocol development and writing the manuscript. Dr Robert Drewett contributed to protocol development and writing the manuscript.

Data collected

Conflict of interest

The authors declare no conflict of interest.

Conflict of interest

The authors declare no conflict of interest. Child's appetite, mealtimes, problems ** General child health and education ** Mother's approach to feeding her child 50 Child's eating behaviour 51 Repetitive Behaviour Questionnaire 48,49 Common symptoms of psychopathology 52 Social communication problems 53 Parental employment (self-coded method) 54 Socio-economic status **


Alistair T Pagnamenta

Hameed Khan

Susan Walker

Dianne Gerrelli

Kirsty Wing

Maria Clara Bonaglia

Roberto Giorda

Tom Berney

Elisa Mani

Massimo Molteni

Maria Clara Bonaglia

Dalila Pinto

Ann Le Couteur

Joachim Hallmayer

James S Sutcliffe

Peter Szatmari

Andrew D Paterson

Stephen W Scherer

Veronica J Vieland

Professor Anthony P Monaco
email: anthony.monaco@well.ox.ac.uk

Rare familial 16q21 microdeletions under a linkage peak implicate cadherin 8 (CDH8) in susceptibility to autism and learning disability

Keywords: CDH8, CNV, autistic, in situ, synapse

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Autism Spectrum Disorder (ASD) is a clinically heterogeneous condition characterised by impairments in social communication and by a pattern of repetitive behaviours. There is strong evidence of genetic heritability. Learning disability (LD) is typically observed in up to 70% of cases, depending on ascertainment [START_REF] Fombonne | The Changing Epidemiology of Autism[END_REF], whilst epilepsy is reported in over 20%. [START_REF] Rossi | EEG features and epilepsy in patients with autism[END_REF][START_REF] Volkmar | Seizure disorders in autism[END_REF] An accumulating body of evidence suggests that ASD may result from aberrant synaptic connections. [START_REF] Bourgeron | A synaptic trek to autism[END_REF] For instance, rare variants involving neuroligin and neurexin genes, which encode proteins that interact across the synaptic cleft, have been implicated in autism susceptibility. [START_REF] Jamain | Mutations of the Xlinked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism[END_REF][START_REF] Szatmari | Mapping autism risk loci using genetic linkage and chromosomal rearrangements[END_REF] In addition, disruption of SHANK3, a gene that encodes a postsynaptic scaffolding protein that interacts with the neuroligins, has also been found at low frequency in some [START_REF] Moessner | Contribution of SHANK3 mutations to autism spectrum disorder[END_REF][START_REF] Durand | Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders[END_REF], but not all ASD cohorts [START_REF] Sykes | Copy number variation and association analysis of SHANK3 as a candidate gene for autism in the IMGSAC collection[END_REF]. Linkage analysis has traditionally been used to search for genetic loci involved in autism susceptibility. The large number of loci described to date [10] in part reflects the complex genetic architecture underlying the condition. However, it is likely that subtle differences exist between clinical cohorts in terms of ascertainment strategies, inclusion/exclusion criteria, as well as the population backgrounds from which subjects are taken. In the presence of locus heterogeneity, these factors, together with simple sampling variability, can lead to very different mixtures of genetic subtypes across studies. Such differences may also act to confound replication.

To address these issues, a reanalysis of the Autism Genome Project (AGP) consortium's linkage data has recently been undertaken, using the PPL analytical framework. [11][12][13] This study identified a novel susceptibility locus on 16q21 coming from the low IQ ASD subgroup. We were interested to note that this linkage peak overlaps a rare deletion found in an individual with autism and learning disability, detected as part of our recent genome-wide CNV scans. [START_REF] Szatmari | Mapping autism risk loci using genetic linkage and chromosomal rearrangements[END_REF]14] Although 1.64 Mb in size, this microdeletion involves a single gene, cadherin 8 (CDH8). In this study, we describe the genetic characterization of this rare microdeletion, further clinical evaluation and segregation analysis in this large nuclear family. The inheritance pattern seen, together with an absence of similar microdeletions in over 5 000 control subjects and the hypothesized involvement of other cadherin genes in ASD and related neurodevelopmental disorders [15][START_REF] Morrow | Identifying autism loci and genes by tracing recent shared ancestry[END_REF][START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF] led us to conclude that this rare microdeletion may be acting as an autism predisposition factor in this family. To extend these findings, we also assessed the expression pattern of this gene in the developing human brain, describe a second family with an overlapping microdeletion disrupting CDH8 and sequence additional individuals with ASD.

METHODS

Clinical details for family 3099

The Autism Diagnostic Interview-Revised (ADI-R) [START_REF] Lord | Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders[END_REF] and the Autism Diagnostic Observation Schedule (ADOS) [START_REF] Lord | The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism[END_REF] were administered when subjects were between 12 and 15 years of age. All three affected children met criteria for autism on the ADI and the ADOS. All three had word and phrase delay and in one affected child (3099_006) there is a history of regression. This child lost many skills, in particular language, over the course of 6 months, and took a year to regain them. Vineland Adaptive Behaviour Scales [START_REF] Sparrow | The Vineland Adaptive Behavior Scales -Interview Edition, Survey Form Manual[END_REF] scores were below 50 on all domains of socialization, communication and daily living skills for all three children with autism. Cognitive testing with the Ravens [START_REF] Raven | Raven manual: Section 3. Standard progressive matrices[END_REF] was performed on two of the three boys and provided IQ scores in the 70-75 range. The eldest affected son (3099_006) was assessed as having intellectual disability by clinical judgment and was in treatment for language delay and learning disabilities. There was no evidence of epilepsy or associated medical problems at the time of assessment, but all three had a head circumference at the 90 th to 97 th percentile, in spite of heights in the normal range. The mother and one unaffected sibling (3099_009) also have large head circumferences (>95 th centile). The mother had three previous miscarriages (around 12 weeks gestation) and was phenotypically normal. There were no reports of neurodevelopmental disorder in her extended family. The father, who is separated from the family, was reported to have a normal developmental history and personality, but to have developed a psychiatric disorder as an adult. His first cousin is reported to have had autism. There was no other history of developmental disability, mental illness or epilepsy in the immediate family, however the mother (3099_002) and youngest son (3099_009) have osteoarthritis.

Clinical details for family 09

The proband (09_003) was evaluated at 20 years, 11 months of age as part of a study on learning disability. On the ADI-R, he had a score of 6 on social interaction, 6 on communication, and 0 on repetitive behaviours. On module 4 of the ADOS, he scored 3, 1 and 2 on the social, communication and play sections respectively. Therefore on neither measure does he qualify for a diagnosis of autism or ASD. However his IQ was below 45 on the WAIS-R and scores on the Vineland Adaptive Behaviour Scales showed significant impairment on all scales. He was reported to have had language delay as a child. There was no history of epilepsy or other comorbid medical problems. However, peripheral palsy of the right 7° facial nerve and obesity (BMI of 31.9 kg/m 2 ) were reported at age 11 and 14 years, respectively. The father scored in the borderline range on the IQ tests (66 for verbal IQ and 77 for performance IQ). The younger brother demonstrated typical IQ scores, but was reported to have been in treatment for language delay and learning disabilities between the ages of 7 and 11.

CNV characterization and segregation analysis

In order to validate the microdeletion in family 3099 and carry out segregation analysis, long-range PCR was performed using the BIO-X-ACT long DNA polymerase kit (BIOLINE, London, UK) using the manufacturer's suggested protocol. Primers GCTATCCAGTAGGAAGTGAAACA and AATGAGTATAAGAATCAAAGATGTGA were designed following visual inspection of 1M-single SNP array data from a recent genome-wide CNV scan[14], within BeadStudio (Illumina, San Diego, CA). The 3 023 bp deletion-spanning amplicon was purified using exonuclease I (NEB, Ipswich, MA, USA) and SAP (USB, Cleveland, OH) and then sequenced using BigDye v3.1 (Applied Biosystems, Foster City, CA). We note that this CNV was initially detected based on Affymetrix 10K SNP data and reported as a de novo event (see Supplemental Table 3 in Szatmari et al [START_REF] Szatmari | Mapping autism risk loci using genetic linkage and chromosomal rearrangements[END_REF]). Higher resolution data from the current Illumina 1M scan indicates that it is in fact inherited from the mother.

For family 09, a combination of high resolution array-based Comparative Genomic Hybridisation (aCGH), using NA10851 (male) and NA15510 (female) as control DNAs, and quantitative PCR experiments, was first performed to help resolve the microdeletion breakpoints (data not shown). Long-range PCR across the deletion was then carried out using primers CCACATCCTTTCACACATGAGAA and TAGCTGCTTTCCCACATATCAT. The 4 610 bp deletion-spanning amplicon was then purified and sequenced as described above.

Sanger sequencing CDH8 for 3099_006, 3099_007 and 3099_008

PCR was used to amplify all coding exons (1-11) and the 5'-UTR of CDH8. PCR products were purified using the ChargeSwitch® PCR Clean-Up Kit (Invitrogen) and then sequenced using the BigDye Terminator kit (v3.1), according to manufacturers' recommendations. To search for novel variants, we compared results against dbSNP version 131. [START_REF] Sherry | dbSNP: the NCBI database of genetic variation[END_REF] Primer sequences are available in Supplementary Table 1. Further information about thermocycling conditions is available on request.

CNV controls

We used data from 5 023 control subjects of European ancestry (2 416 from the PopGen study[14], controls from the Ontario Ottawa Heart Control study [START_REF] Stewart | Kinesin family member 6 variant Trp719Arg does not associate with angiographically defined coronary artery disease in the Ottawa Heart Genomics Study[END_REF] or HapMap controls genotyped on the Affymetrix 6.0 array; 1 287 from the SAGE control project genotyped on the Illumina 1M platform [14,[START_REF] Bierut | A genome-wide association study of alcohol dependence[END_REF]; and 1 320 from the CHOP paediatric control study genotyped on the Illumina 550k array. [START_REF] Shaikh | Highresolution mapping and analysis of copy number variations in the human genome: a data resource for clinical and research applications[END_REF] RT-PCR

Reverse transcriptase (RT)-PCR was performed using the OneStep-RT PCR kit (Qiagen) according to the manufacturer's protocol. Exact-match primers were designed to the first coding exon and the 3'-UTR of each of the isoforms. For the shorter isoform, primer sequences corresponding to the coding exon and 3'-UTR were as follows:

- for 15 min, followed by 40 cycles of 94ºC for 30 sec, 52ºC for 30 sec, and 72ºC for 2 min, with a final extension at 72ºC for 10 min.

In situ hybridization

In situ hybridization was carried out on human fetal tissue (9 weeks gestation) as described by Wilkinson. [START_REF] Wilkinson | In-situ hybridization: A practical approach[END_REF] Probe regions comprising the 3'-UTR region of the short CDH8 isoform were amplified from whole brain cDNA using primers GAAAACCCGGCCAAGTAAAT and CAGATTTCAATATTCACTTCCTACAA.

Probe regions comprising the 3'-UTR region of the long CDH8 isoform were amplified using primers TCTACTCTGTTGGTGAAAGTGACA and TGTCTGTGGTGGTCAGGTAAA. Further details are provided in Supplementary Information.

RESULTS

Microdeletions involving CDH8 within a low IQ ASD linkage region

A recent study reanalysing the AGP's linkage data using the PPL statistical framework, has identified a new susceptibility locus on chromosome 16q13-21 in the low IQ ASD subset. This reaches a maximum PPL of 95.95% at rs1476307.

[13] This means there is a 95.95% chance that this region contains an autism susceptibility gene, based on the available data. The ~6 Mb region under the linkage peak, particularly in chromosome band 16q21 (distal to the peak of linkage) is relatively gene-poor. We have discovered two large inherited microdeletions within this linkage peak and overlapping the CDH8 gene, in two independent families (figure 1).

Family 3099 is of European ancestry and comes from the IMGSAC study cohort. [START_REF] Imgsac | A genomewide screen for autism: strong evidence for linkage to chromosomes 2q, 7q, and 16p[END_REF] Subject 3099_008 was included in a recent CNV scan of 996 individuals with ASD. [14] This individual was found to carry an inherited heterozygous 1.64 Mb microdeletion involving the whole CDH8 gene, but no other genes. This microdeletion was transmitted from the unaffected mother to the proband and his two brothers, all of whom presented with both LD and ASD. The microdeletion was not transmitted to the four unaffected siblings (figure 2A). Analysis of a combination of chromosome 16 SNPs and microsatellites indicates that the non-deleted paternal copy of CDH8 was also shared identical-by-descent in all three affected children, however Sanger sequencing uncovered no novel exonic variants in CDH8 on this chromosome. We validated the microdeletion using long-range PCR and then by Sanger sequencing the breakpoint junction fragment (figure 2B). The absence of any sequence similarities flanking the breakpoints suggests that this chr16:60 025 584-61 667 839 microdeletion is likely to be a rare, potentially "private" mutation. A combination of SNPs and microsatellite data from this family, generated in previous studies [START_REF] Szatmari | Mapping autism risk loci using genetic linkage and chromosomal rearrangements[END_REF][START_REF] Imgsac | A genomewide screen for autism: strong evidence for linkage to chromosomes 2q, 7q, and 16p[END_REF], determined that the linkage signal from this family alone, reached the maximum possible for a pedigree of this size (Maximum LOD = 1.7, maximising model = recessive).

Family 09 was discovered during an independent aCGH genome-wide screen of a heterogeneous cohort of 80 Italian children with generalized learning disability, based on Agilent 44k technology. All subjects were negative for karyotype and telomere-FISH abnormalities, as well as for fragile-X. The family comprises parents and two sons, with the deletion being transmitted from the father to both sons (figure 2A). Neither child received a diagnosis of ASD, however the proband scored in the very low range for IQ with substantial impairment, while the brother showed normal IQ but was reported to have had a history of language delay and treatment for learning disability. The father also scored in the borderline range for IQ. The distal breakpoint of this 1.82 Mb deletion is situated within the largest (120 kb) CDH8 intron and all but one of the coding exons (2-11) are removed. No other genes are disrupted due to the large gene desert proximal to CDH8 (figure 1). Analysis of chromosome 16 microsatellite markers showed that the proband and his sibling did not share their non-deleted maternal CDH8 haplotype (data not shown). The sequence surrounding the observed chr16:58 724 527-60 547 472 deletion revealed a 7bp tandem duplication nearby which may have occurred at the same time as the larger deletion (figure 2C).

CNV analysis of controls

There were no similar CNVs disrupting the CDH8 gene detected in 5 023 control subjects from published high-resolution (550k and above) SNP array data. We note that the Database of Genomic Variants (http://projects.tcag.ca/variation/) reports a duplication of 23.7 kb in a single population control sample (NA18852), involving a single coding exon of CDH8. [START_REF] Wang | PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data[END_REF] This CNV was detected using PennCNV analysis and involves just 4

SNPs from the Illumina 550k SNP array (Supplementary Figure 1). However, numerous other studies (some using higher resolution platforms [START_REF] Conrad | Origins and functional impact of copy number variation in the human genome[END_REF]) have also examined this same DNA sample and do not report the CNV, suggesting that it may be a false-positive. We therefore tested this HapMap sample using a combination of qPCR primer pairs and confirmed that there is a normal copy number at this locus (Supplementary Information).

Expression analysis

Although EBV-transformed peripheral blood lymphocytes were available for family 3099, we were unable to amplify CDH8 transcripts using RT-PCR (data not shown). This may be because this gene is not expressed in lymphocytes. Therefore we could not determine how strongly the remaining copy of CDH8 was expressed. However, using RT-PCR on a commercially-available RNA panel, expression of two known isoforms of CDH8 was confirmed in various parts of human the brain, particularly in the cortex (figures 3A,B). Both isoforms were also detected in fetal brain, although the long isoform was only just detectable (figures 3B).

A more complete, quantitative characterisation of CDH8 expression during early brain development was also carried out using in situ hybridization on sagittal brain sections from a 9 week old human embryo. These data showed expression of the shorter CDH8 isoform towards the front of the cerebral cortex (figure 3C), a similar pattern to other ASD candidate genes such as CNTNAP2 [START_REF] Abrahams | Genome-wide analyses of human perisylvian cerebral cortical patterning[END_REF] and CDH10. [START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF] The longer CDH8 isoform demonstrated a more posterior cortical expression at this early developmental stage (figure 3D).

DISCUSSION

To better account for different ASD subtypes and potential clinical-site-specific confounders, reanalysis of existing ASD linkage data has been carried out using the PPL statistical framework. In this study we describe two 16q21 microdeletions that are present within the novel linkage peak that was identified in the low IQ ASD subset.

The rarity of CNVs at this locus means it would be difficult to gain evidence of aetiological relevance using a case-control experimental design. For instance, in the AGP CNV study cohorts[14], deletions of this region were seen in 1/996 cases (i.e. the proband from family 3099, and 0/1 287 controls. One solution is to carry out global analysis on all rare CNVs present in a study cohort, to gather statistical support for enrichments of biological pathways. For example, analysis of rare CNVs in the AGP cohort implicated genes involved with cellular proliferation, projection, motility and GTPase/Ras signalling.

[14] Pathway analyses can also integrate other data sets such as information on mouse knockout phenotypes, as has been accomplished recently for CNVs detected in learning disability. [START_REF] Webber | Forging links between human mental retardation-associated CNVs and mouse gene knockout models[END_REF] Nevertheless, in larger pedigrees linkage analysis remains an additional way of supporting disease involvement. The size of the nuclear family 3099 and segregation pattern seen for the 16q21 deletion strongly suggests that this mutation plays an aetiological role. Given the evidence for overlapping aetiology between ASD and general intellectual disabilities and the detection of this linkage region specifically in the low IQ subset of the ASD sample, family 09 represents additional corroboration of CDH8 as a susceptibility gene for ASD and/or learning disability.

Although both deletions are large (>1.6 Mb), both disrupt a single gene in this relatively gene-sparse region of 16q21 (figure 1). This gene (CDH8) spans 383 kb of genomic sequence and encodes a classical type-II cadherin. Cadherins are calcium dependant cell adhesion molecules, many of which are expressed in the brain. A recent genome-wide association study implicated common variants between CDH9 and CDH10 on chromosome 5, in autism susceptibility. [START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF] In this same study, pathway analysis implicated the whole family of cadherins (CDH1-25) and this enrichment was enhanced when the cadherins were grouped with the three neurexins and the five closely-related CNTNAP genes. [START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF] A second study assessing both genome-wide linkage and association did not implicate the CDH9-CDH10 locus. [START_REF] Weiss | A genome-wide linkage and association scan reveals novel loci for autism[END_REF] Common variants near the CDH7 gene have reproducibly been linked to bipolar disorder. [START_REF] Soronen | Replication of GWAS of bipolar disorder: association of SNPs near CDH7 with bipolar disorder and visual processing[END_REF] De novo deletions overlapping CDH15, another member of this gene family, have been detected in 3 individuals with ASD or "autistic features". [START_REF] Willemsen | Identification of ANKRD11 and ZNF778 as candidate genes for autism and variable cognitive impairment in the novel 16q24.3 microdeletion syndrome[END_REF] Alterations in CDH15 have also been linked to LD and impaired cell-cell adhesion. [15]. Meanwhile in consanguineous kindreds, rare deletions within larger blocks of homozygosity-by-descent implicate protocadherin 10 in autism susceptibility. [START_REF] Morrow | Identifying autism loci and genes by tracing recent shared ancestry[END_REF] A cadherin-rich region on 13q21 has also been implicated in specific language impairment and previous autism studies. [START_REF] Bartlett | Examination of potential overlap in autism and language loci on chromosomes 2, 7, and 13 in two independent samples ascertained for specific language impairment[END_REF][START_REF] Bradford | Incorporating language phenotypes strengthens evidence of linkage to autism[END_REF] Finally, a de novo deletion in the affected member of a disconcordant monozygotic twin pair suggests CDH12 and CDH18 may be involved in schizophrenia. [START_REF] Singh | Autism meets schizophrenia via cadherin pathway[END_REF] In mice, knockout of the orthologous Cdh8 gene (~97% amino acid sequence identity to its human counterpart) results in abnormal synaptic transmission. [START_REF] Suzuki | Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation[END_REF] Neither deletion in this study appears to be fully penetrant. Across both families, a total of 5/7 individuals with CDH8 deletions were affected with autism and/or learning disability. This penetrance rate is similar to other recently described ASD-implicated CNVs, such as microdeletion of 15q13.3. [START_REF] Shinawi | A small recurrent deletion within 15q13.3 is associated with a range of neurodevelopmental phenotypes[END_REF] Although a normal IQ was observed for the brother carrying the deletion in family 09, he did have language delay, which can be considered part of the spectrum of learning disability.

It may be that other rare mutations such as CNVs, SNPs or indels elsewhere in the genome act to modulate the penetrance and expressivity of the CDH8 deletions in the two families described. For example, a recent study of individuals with developmental delay and the 16p12.1 microdeletion found a higher than expected rate of large secondary CNVs, suggesting a two-hit model. [START_REF] Girirajan | A recurrent 16p12.1 microdeletion supports a two-hit model for severe developmental delay[END_REF] Although there were no other rare CNVs with obvious aetiological relevance in the families described here, the resolution of our study is such that we cannot rule out smaller CNVs or other molecular features contributing to the phenotype in a similar fashion. It may be that CDH8 is itself just a risk factor for learning disability and this only leads to autism together with certain genetic backgrounds. The macrocephaly in family 3099 does not completely co-segregate with the deletion and so might suggest an additional risk factor interacting with the CDH8 deletion.

A recent study has identified DIAPH3 as a new autism susceptibility gene by virtue of rare non-synonymous variants lying in trans with a deletion. [START_REF] Vorstman | A double hit implicates DIAPH3 as an autism risk gene[END_REF] The sharing of nondeleted paternal CDH8 haplotypes in the three affected siblings in family 3099 made us consider the possibility that this 1.64 Mb deletion was also unmasking rare variants in the remaining copy of CDH8. Although sequence analysis did not detect any novel exonic CDH8 variants in family 3099, we cannot exclude the possibility of mutations in noncoding regions disrupting gene regulation. Variation in the non-deleted copy of CDH8 could potentially also explain the non-concordant phenotypes seen for the two boys with 16q21 deletions in family 09.

In situ analysis shows that the two CDH8 isoforms have a slightly different expression pattern, suggesting that they may potentially play distinct roles in early cortical development. The more anterior expression seen for the shorter CDH8 isoform somewhat resembles the pattern seen for CNTNAP2 and CDH10, other ASD susceptibility genes for which published in situ expression data is available at 20 weeks of gestation. [START_REF] Wang | Common genetic variants on 5p14.1 associate with autism spectrum disorders[END_REF][START_REF] Abrahams | Genome-wide analyses of human perisylvian cerebral cortical patterning[END_REF] However, although comparison between 9 and 20 week brain sections is difficult, we have shown that CDH8 is expressed within the germinal zone of cortex rather than throughout the entire cortex as seen for the other two ASD candidate genes. Recent studies on CNTNAP2 show that common ASD-associated variants in this gene influence brain morphology. [START_REF] Tan | Normal variation in fronto-occipital circuitry and cerebellar structure with an autism-associated polymorphism of CNTNAP2[END_REF] Unfortunately we were unable to obtain brain scans for affected individuals from our two families to assess whether CDH8 deletions had led to abnormal cortical folding. Arrows indicate cortical expression.

Figure 1

 1 Figure 1 Schematic from the UCSC genome browser. Figure shows the position of the two inherited deletions overlapping CDH8, in relation to the low-IQ ASD linkage peak from our recent analysis.[13] The Y axis indicates the PPL score. RefSeq gene coordinates are also plotted beneath the chromosome band track. The region shown corresponds to 54-65 Mb on 16q12.2-21 (NCBI build 36 coordinates).

Figure 2

 2 Figure 2 Deletions found in families 3099 and 09. (A) Pedigrees show the segregation pattern for these two deletions involving CDH8. Autism and learning disability indicated in black shading, learning disability alone indicated in gray; del, 16q21 deletion; wt, wild-type. N.B. Although individual 09_004 demonstrated typical IQ scores, he was reported to have been in treatment for language delay and learning disabilities between the ages of 7 and 11. (B) Electropherogram showing DNA sequence spanning the chr16:60 025 584-61 667 839 (NCBI build 36) deletion in family 3099. (C) Electropherogram showing DNA sequence spanning the chr16:58 724 527-60 547 472 deletion in family 09. The position of a 7 bp tandem duplication is indicated.

Figure 3

 3 Figure 3 Expression analysis of CDH8. (A) RT-PCR analysis detected a 1 760 bp amplicon corresponding to the short CDH8 isoform. (B) RT-PCR analysis detected a 2 514 bp product (boxed), corresponding to the longer CDH8 isoform. (C) in situ hybridization performed on sagittal sections through the head of a nine-week old human embryo, for the short CDH8 isoform. (D) in situ hybridization for the longer isoform.

  Although we did not detect any rare exonic CDH8 changes in 26 individuals with ASD (data not shown), taken from families who were contributing most to the original linkage signal[13], future studies should assess this locus for CNVs and rare sequencelevel variants in larger ASD cohorts and measure the functional effects of these changes.Until additional, nonsense point mutations or de novo disruptions to the CDH8 gene are detected in further autism cohorts, we cannot exclude the possibility that non-genic sequence motifs within this region might be acting to regulate other neighbouring genes or distant loci in trans. Nevertheless, the linkage seen at 16q21 in the low IQ ASD subgroup and the segregation pattern seen for the CDH8 deletion in family 3099 leads us to hypothesise that disruption to this gene may influence susceptibility to autism and/or learning disability. Disruption of CDH8 in two other individuals with learning disability, the absence of similar CNVs in controls, the expression of this gene in critical regions of the developing cortex and the role of other cadherin genes in neurodevelopmental disorders are consistent with this interpretation. this article (if accepted) to be published in Journal of Medical Genetics and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licenceforms).

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the families participating in the study and the international Autism Genome Project Consortium for sharing data and ideas. Funding for this work comes from the Simons Foundation, the Nancy Lurie Marks Family Foundation, the Wellcome Trust [075491/Z/04], the NIH [MH086117], a Telethon Grant [GGP06208A/B], Autism Speaks, Genome Canada/Ontario Genomics Institute, Canadian Institutes for Health Research, The Centre for Applied Genomics and the McLaughlin Centre. Tissue for the in situ work was provided by the MRC/Wellcome Trust Human Developmental Biology Resource. CDH8 in situ data can be accessed via the Human Developmental Biology Resource website at http://www.hdbr.org/

Competing interests None

Ethics approval Obtained from the Joint Ethics Committee (Newcastle & North Tyneside Health Authority/Universities of Newcastle upon Tyne/Northumbria) and the Ethics Committee at the "E. Medea" Scientific Institute. The HDBR has tissue bank ethics approval from the National Research Ethics Service.

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