Childhood-onset mitochondrial diseases : with emphasis on molecular diagnostics of neurological disorders

Childhood-onset mitochondrial diseases comprise a heterogeneous group of disorders, which may manifest with almost any symptom and affect any tissue or organ. Due to challenging diagnostics, most children still lack a specific aetiological diagnosis. The aim of this thesis was to find molecular causes for childhood-onset mitochondrial disorders in Finland. We identified the underlying cause for 25 children, and found three new diseases, which had not been diagnosed in Finland before. These diseases caused severe progressive infantile-onset encephalomyopathies, and were due to defects in mitochondrial DNA (mtDNA) maintenance. Furthermore, the thesis provides the molecular background of Finnish patients with ‘leukoencephalopathy with brain stem and spinal cord involvement and elevated brain lactate’ (LBSL). A new phenotype was identified to be due to mutations in Twinkle, resembling ‘infantile onset spinocerebellar ataxia’ (IOSCA). These mutations caused mtDNA depletion in the liver, thus confirming the essential role of Twinkle in mtDNA maintenance, and expanding the molecular background of mtDNA depletion syndromes. The major aetiology for infantile mitochondrial myopathy in Finland was discovered to be due to mutations in thymidine kinase 2 (TK2). A novel mutation with Finnish ancestry was identified, and a genotype-phenotype correlation with mutation-specific distribution of tissue involvement was found, thus proving that deficient TK2 may cause multi-tissue depletion and impair neuronal function. This work established the molecular diagnosis and advanced the knowledge of phenotypes among paediatric patients with polymerase gamma (POLG) mutations. The patients showed severe early-onset encephalopathy with intractable epilepsy. POLG mutations are not a prevalent cause of children’s ataxias, although ataxia is a major presenting symptom among adults. Our findings indicate that POLG mutations should be investigated even if typical MRI, histochemical or biochemical abnormalities are lacking. LBSL patients showed considerable variation in phenotype despite identical mutations. A common, most likely European, ancestry, and a relative high carrier frequency of these mutations in Finland were discovered; suggesting that LBSL may be a quite common leukoencephalopathy in other populations as well. The results suggest that MRI findings are so unique that the diagnosis of LBSL is possible to make without genetic studies. This thesis work has resulted in identification of new mitochondrial disorders in Finland, enhancing the understanding of the clinical variability and the importance of tissue-specificity of these disorders. In addition to providing specific diagnosis to the patients, these findings give light to the underlying pathogenetic mechanisms of childhood-onset mitochondrial disorders.

Genetic background of bipolar disorder and related cognitive impairments

Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.

Suicidal behaviour in bipolar disorder

The Jorvi Bipolar Study (JoBS) is a collaborative ongoing bipolar research project between the Department of Mental Health and Alcohol Research of the National Public Health Institute, Helsinki, and the Department of Psychiatry, Jorvi Hospital, Helsinki University Central Hospital (HUCH), Espoo, Finland. The JoBS is a prospective, naturalistic cohort study of secondary level care psychiatric out-and inpatients with a new episode of Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) bipolar disorder (BD). Altogether, 1630 patients (aged 18-59) years were screened using the Mood Disorder Questionnaire (MDQ) for a possible new episode of DSM-IV BD. 490 patients were interviewed with semi-structured interview [the Structured Clinical Interview for DSM-IV Disorders, research version with Psychotic Screen (SCID-I/P)]. 191 patients with new episode of DSM-IV BD were included in the bipolar cohort study. Psychiatric comorbidity was evaluated using semi-structured interviews. At 6- and 18-month follow-up, the interviews were repeated and life-chart methodology was used to integrate all available information about nature and duration of all different phases. Suicidal behaviour was examined both at intake and follow-up by psychometric scale [Scale for Suicidal Ideation (SSI)], interviewer s questions and medical and psychiatric records. The aim of this thesis was to evaluate prevalence of suicidal behaviour and incidence of suicide attempts, and examine the wide range of risk factors for attempted suicide both, at intake and follow-up, in representative secondary-level sample of psychiatric in- and outpatients with BD. In this study suicidal behaviour was common among psychiatric patients with BD. During the episode when patients were included into cohort study (index episode), 20% of the patients had attempted suicide and 61% had suicidal ideation. Severity of depressive episode and hopelessness were independent risk factors for suicidal ideation, whereas hopelessness, comorbid personality disorder and previous suicide attempt predicted suicide attempts during the index episode. There were no differences in prevalence of suicidal behaviour between bipolar I and II disorder; the risk factors were overlapping but not identical. During the index episode, suicide attempts took place during depressive, mixed and depressive mixed phases. Furthermore, there were marked differences regarding level of suicidal ideation during different phases, with the highest levels during the mixed phases of the illness. Hopelessness was independently associated with suicidal behaviour during the depressive phase. A subjective rating of severity of depression (Beck Depression Inventory) and younger age predicted suicide attempts during mixed phases. During the 18-month follow-up 20% of patients attempted suicide. Previous suicide attempts, hopelessness, depressive phase at index episode and younger age at intake were independent risk factors for suicide attempts during follow-up. Taken altogether, 55% patients attempted suicide before index episode, during index episode or during follow-up. The incidence of suicide attempts was 37-fold during combined mixed and depressive mixed states and 18-fold during major depressive phase as compared with other phases. Prior suicide attempt and time spent in combined mixed phases - mixed and depressive mixed - and depressive phases independently predicted the suicide attempt during follow-up. More than half of the patients have attempted suicide during their lifetime, a finding which highlights the public health importance of suicidal behaviour in bipolar disorder. Clinically, it is crucial to recognize BD and manage the mixed and depressive phases of bipolar patients fast and effectively, as time spent in depressive and mixed phases involves a remarkably high risk of suicide attempts.

Health-Related Quality of Life and Mental Disorders in Finland

Quality of life (QoL) and Health-related quality of life (HRQoL) are becoming one of the key outcomes of health care due to increased respect for the subjective valuations and well-being of patients and an increasing part of the ageing population living with chronic, non-fatal conditions. Preference-based HRQoL measures enable estimation of health utility, which can be useful for rational rationing, evidence-based medicine and health policy. This study aimed to compare the individual severity and public health burden of major chronic conditions in Finland, including and focusing on reliably diagnosed psychiatric conditions. The study is based on the Health 2000 survey, a representative general population survey of 8028 Finns aged 30 and over. Depressive, anxiety and alcohol use disorders were diagnosed with the Composite International Diagnostic Interview (M-CIDI). HRQoL was measured with the 15D and the EQ-5D, with 83% response rate. This study found that people with psychiatric disorders had the lowest 15D HRQoL scores at all ages, in comparison to other main groups of chronic conditions. Considering 29 individual conditions, three of the four most severe (on 15D) were psychiatric disorders; the most severe was Parkinson s disease. Of the psychiatric disorders, chronic conditions that have sometimes been considered relatively mild - dysthymia, agoraphobia, generalized anxiety disorder and social phobia - were found to be the most severe. This was explained both by the severity of the impact of these disorders on mental health domains of HRQoL, and also by the fact that decreases were widespread on most dimensions of HRQoL. Considering the public health burden of conditions, musculoskeletal disorders were associated with the largest burden, followed by psychiatric disorders. Psychiatric disorders were associated with the largest burden at younger ages. Of individual conditions, the largest burden found was for depressive disorders, followed by urinary incontinence and arthrosis of the hip and knee. The public health burden increased greatly with age, so the ageing of the Finnish population will mean that the disease burden caused by chronic conditions will increase by a quarter up to year 2040, if morbidity patterns do not change. Investigating alcohol consumption and HRQoL revealed that although abstainers had poorer HRQoL than moderate drinkers, this was mainly due to many abstainers being former drinkers and having the poorest HRQoL. Moderate drinkers did not have significantly better HRQoL than abstainers who were not former drinkers. Psychiatric disorders are associated with a large part of the non-fatal disease burden in Finland. In particular anxiety disorders appear to be more severe and have a larger public health burden than previously thought.

Genetic Mechanisms Underlying Autism Spectrum Disorders

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.

Identifying Genetic Risk Factors in Canine Autoimmune Disorders

Autoimmune diseases are more common in dogs than in humans and are already threatening the future of some highly predisposed dog breeds. Susceptibility to autoimmune diseases is controlled by environmental and genetic factors, especially the major histocompatibility complex (MHC) gene region. Dogs show a similar physiology, disease presentation and clinical response as humans, making them an excellent disease model for autoimmune diseases common to both species. The genetic background of canine autoimmune disorders is largely unknown, but recent annotation of the dog genome and subsequent development of new genomic tools offer a unique opportunity to map novel autoimmune genes in various breeds. Many autoimmune disorders show breed-specific enrichment, supporting a strong genetic background. Furthermore, the presence of hundreds of breeds as genetic isolates facilitates gene mapping in complex autoimmune disorders. Identification of novel predisposing genes establishes breeds as models and may reveal novel candidate genes for the corresponding human disorders. Genetic studies will eventually shed light on common biological functions and interactions between genes and the environment. This study aimed to identify genetic risk factors in various autoimmune disorders, including systemic lupus erythematosus (SLE)-related diseases, comprising immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis arteritis (SMRA) as well as Addison s disease (AD) in Nova Scotia Duck Tolling Retrievers (NSDTRs) and chronic superficial keratitis (CSK) in German Shepherd dogs (GSDs). We used two different approaches to identify genetic risk factors. Firstly, a candidate gene approach was applied to test the potential association of MHC class II, also known as a dog leukocyte antigen (DLA) in canine species. Secondly, a genome-wide association study (GWAS) was performed to identify novel risk loci for SLE-related disease and AD in NSDTRs. We identified DLA risk haplotypes for an IMRD subphenotype of SLE-related disease, AD and CSK, but not in SMRA, and show that the MHC class II gene region is a major genetic risk factor in canine autoimmune diseases. An elevated risk was found for IMRD in dogs that carried the DLA-DRB1*00601/DQA1*005011/DQB1*02001 haplotype (OR = 2.0, 99% CI = 1.03-3.95, p = 0.01) and for ANA-positive IMRD dogs (OR = 2.3, 99% CI = 1.07-5.04, p-value 0.007). We also found that DLA-DRB1*01502/DQA*00601/DQB1*02301 haplotype was significantly associated with AD in NSDTRs (OR = 2.1, CI = 1.0-4.4, P = 0.044) and the DLA-DRB1*01501/DQA1*00601/DQB1*00301 haplotype with the CSK in GSDs (OR=2.67, CI=1.17-6.44, p= 0.02). In addition, we found that homozygosity for the risk haplotype increases the risk for each disease phenotype and that an overall homozygosity for the DLA region predisposes to CSK and AD. Our results have enabled the development of genetic tests to improve breeding practices by avoiding the production of puppies homozygous for risk haplotypes. We also performed the first successful GWAS for a complex disease in dogs. With less than 100 cases and 100 controls, we identified five risk loci for SLE-related disease and AD and found strong candidate genes involved in a novel T-cell activation pathway. We show that an inbred dog population has fewer risk factors, but each of them has a stronger genetic risk. Ongoing studies aim to identify the causative mutations and bring new knowledge to help diagnostics, treatment and understanding of the aetiology of SLE-related diseases.

Cognitive Functioning in Alcohol and Other Substance Use Disorders in Young Adulthood : A Genetic Epidemiological Study

Alcohol and other substance use disorders (SUDs) result in great costs and suffering for individuals and families and constitute a notable public health burden. A multitude of factors, ranging from biological to societal, are associated with elevated risk of SUDs, but at the level of individuals, one of the best predictors is a family history of SUDs. Genetically informative twin and family studies have consistently indicated this familial risk to be mainly genetic. In addition, behavioral and temperamental factors such as early initiation of substance use and aggressiveness are associated with the development of SUDs. These familial, behavioral and temperamental risk factors often co-occur, but their relative importance is not well known. People with SUDs have also been found to differ from healthy controls in various domains of cognitive functioning, with poorer verbal ability being among the most consistent findings. However, representative population-based samples have rarely been used in neuropsychological studies of SUDs. In addition, both SUDs and cognitive abilities are influenced by genetic factors, but whether the co-variation of these traits might be partly explained by overlapping genetic influences has not been studied. Problematic substance use also often co-occurs with low educational level, but it is not known whether these outcomes share part of their underlying genetic influences. In addition, educational level may moderate the genetic etiology of alcohol problems, but gene-environment interactions between these phenomena have also not been widely studied. The incidence of SUDs peaks in young adulthood rendering epidemiological studies in this age group informative. This thesis investigated cognitive functioning and other correlates of SUDs in young adulthood in two representative population-based samples of young Finnish adults, one of which consisted of monozygotic and dizygotic twin pairs enabling genetically informative analyses. Using data from the population-based Mental Health in Early Adulthood in Finland (MEAF) study (n=605), the lifetime prevalence of DSM-IV any substance dependence or abuse among persons aged 21—35 years was found to be approximately 14%, with a majority of the diagnoses being alcohol use disorders. Several correlates representing the domains of behavioral and affective factors, parental factors, early initiation of substance use, and educational factors were individually associated with SUDs. The associations between behavioral and affective factors (attention or behavior problems at school, aggression, anxiousness) and SUDs were found to be largely independent of factors from other domains, whereas daily smoking and low education were still associated with SUDs after adjustment for behavioral and affective factors. Using a wide array of neuropsychological tests in the MEAF sample and in a subsample (n=602) of the population-based FinnTwin16 (FT16) study, consistent evidence of poorer verbal cognitive ability related to SUDs was found. In addition, participants with SUDs performed worse than those without disorders in a task assessing psychomotor processing speed in the MEAF sample, whereas no evidence of more specific cognitive deficits was found in either sample. Biometrical structural equation models of the twin data suggested that both alcohol problems and verbal ability had moderate heritabilities (0.54—0.72), and that their covariation could be explained by correlated genetic influences (genetic correlations -0.20 to -0.31). The relationship between educational level and alcohol problems, studied in the full epidemiological FT16 sample (n=4,858), was found to reflect both genetic correlation and gene-environment interaction. The co-occurrence of low education and alcohol problems was influenced by overlapping genetic factors. In addition, higher educational level was associated with increased relative importance of genetic influences on alcohol problems, whereas environmental influences played a more important role in young adults with lower education. In conclusion, SUDs, especially alcohol abuse and dependence, are common among young Finnish adults. Behavioral and affective factors are robustly related to SUDs independently of many other factors, and compared to healthy peers, young adults who have had SUDs during their life exhibit significantly poorer verbal cognitive ability, and possibly less efficient psychomotor processing. Genetic differences between individuals explain a notable proportion of individual differences in risk of alcohol dependence, verbal ability, and educational level, and the co-occurrence of alcohol problems with poorer verbal cognition and low education is influenced by shared genetic backgrounds. Finally, various environmental factors related to educational level in young adulthood moderate the relative importance of genetic factors influencing the risk of alcohol problems, possibly reflecting differences in social control mechanisms related to educational level.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.