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.

Recognition, comorbidity, and outcome of DSM-IV bipolar I and II disorders in psychiatric care

This study is part of an ongoing collaborative bipolar research project, the Jorvi Bipolar Study (JoBS). The JoBS is run by 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. It is a prospective, naturalistic cohort study of secondary level care psychiatric in- and outpatients with a new episode of bipolar disorder (BD). The second report also included 269 major depressive disorder (MDD) patients from the Vantaa Depression Study (VDS). The VDS was carried out in collaboration with the Department of Psychiatry of the Peijas Medical Care District. Using the Mood Disorder Questionnaire (MDQ), all in- and outpatients at the Department of Psychiatry at Jorvi Hospital who currently had a possible new phase of DSM-IV BD were sought. Altogether, 1630 psychiatric patients were screened, and 490 were interviewed using a semistructured interview (SCID-I/P). The patients included in the cohort (n=191) had at intake a current phase of BD. The patients were evaluated at intake and at 6- and 18-month interviews. Based on this study, BD is poorly recognized even in psychiatric settings. Of the BD patients with acute worsening of illness, 39% had never been correctly diagnosed. The classic presentations of BD with hospitalizations, manic episodes, and psychotic symptoms lead clinicians to correct diagnosis of BD I in psychiatric care. Time of follow-up elapsed in psychiatric care, but none of the clinical features, seemed to explain correct diagnosis of BD II, suggesting reliance on cross- sectional presentation of illness. Even though BD II was clearly less often correctly diagnosed than BD I, few other differences between the two types of BD were detected. BD I and II patients appeared to differ little in terms of clinical picture or comorbidity, and the prevalence of psychiatric comorbidity was strongly related to the current illness phase in both types. At the same time, the difference in outcome was clear. BD II patients spent about 40% more time depressed than BD I patients. Patterns of psychiatric comorbidity of BD and MDD differed somewhat qualitatively. Overall, MDD patients were likely to have more anxiety disorders and cluster A personality disorders, and bipolar patients to have more cluster B personality disorders. The adverse consequences of missing or delayed diagnosis are potentially serious. Thus, these findings strongly support the value of screening for BD in psychiatric settings, especially among the major depressive patients. Nevertheless, the diagnosis must be based on a clinical interview and follow-up of mood. Comorbidity, present in 59% of bipolar patients in a current phase, needs concomitant evaluation, follow-up, and treatment. To improve outcome in BD, treatment of bipolar depression is a major challenge for clinicians.

Depressive Disorders in Primary Health Care

The Vantaa Primary Care Depression Study (PC-VDS) is a naturalistic and prospective cohort study concerning primary care patients with depressive disorders. It forms a collaborative research project between the Department of Mental and Alcohol Research of the National Public Health Institute, and the Primary Health Care Organization of the City of Vantaa. The aim is to obtain a comprehensive view on clinically significant depression in primary care, and to compare depressive patients in primary care and in secondary level psychiatric care in terms of clinical characteristics. Consecutive patients (N=1111) in three primary care health centres were screened for depression with the PRIME-MD, and positive cases interviewed by telephone. Cases with current depressive symptoms were diagnosed face-to-face with the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I/P). A cohort of 137 patients with unipolar depressive disorders, comprising all patients with at least two depressive symptoms and clinically significant distress or disability, was recruited. The Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II), medical records, rating scales, interview and a retrospective life-chart were used to obtain comprehensive cross-sectional and retrospective longitudinal information. For investigation of suicidal behaviour the Scale for Suicidal Ideation (SSI), patient records and the interview were used. The methodology was designed to be comparable to The Vantaa Depression Study (VDS) conducted in secondary level psychiatric care. Comparison of major depressive disorder (MDD) patients aged 20-59 from primary care in PC-VDS (N=79) was conducted with new psychiatric outpatients (N =223) and inpatients (N =46) in VDS. The PC-VDS cohort was prospectively followed up at 3, 6 and 18 months. Altogether 123 patients (90%) completed the follow-up. Duration of the index episode and the timing of relapses or recurrences were examined using a life-chart. The retrospective investigation revealed current MDD in most (66%), and lifetime MDD in nearly all (90%) cases of clinically significant depressive syndromes. Two thirds of the “subsyndromal” cases had a history of major depressive episode (MDE), although they were currently either in partial remission or a potential prodromal phase. Recurrences and chronicity were common. The picture of depression was complicated by Axis I co-morbidity in 59%, Axis II in 52% and chronic Axis III disorders in 47%; only 12% had no co-morbidity. Within their lifetimes, one third (37%) had seriously considered suicide, and one sixth (17%) had attempted it. Suicidal behaviour clustered in patients with moderate to severe MDD, co-morbidity with personality disorders, and a history of treatment in psychiatric care. The majority had received treatment for depression, but suicidal ideation had mostly remained unrecognised. The comparison of patients with MDD in primary care to those in psychiatric care revealed that the majority of suicidal or psychotic patients were receiving psychiatric treatment, and the patients with the most severe symptoms and functional limitations were hospitalized. In other clinical aspects, patients with MDD in primary care were surprisingly similar to psychiatric outpatients. Mental health contacts earlier in the current MDE were common among primary care patients. The 18-month prospective investigation with a life-chart methodology verified the chronic and recurrent nature of depression in primary care. Only one-quarter of patients with MDD achieved and maintained full remission during the follow-up, while another quarter failed to remit at all. The remaining patients suffered either from residual symptoms or recurrences. While severity of depression was the strongest predictor of recovery, presence of co-morbid substance use disorders, chronic medical illness and cluster C personality disorders all contributed to an adverse outcome. In clinical decision making, beside severity of depression and co-morbidity, history of previous MDD should not be ignored by primary care doctors while depression there is usually severe enough to indicate at least follow-up, and concerning those with residual symptoms, evaluation of their current treatment. Moreover, recognition of suicidal behaviour among depressed patients should also be improved. In order to improve outcome of depression in primary care, the often chronic and recurrent nature of depression should be taken into account in organizing the care. According to literature management programs of a chronic disease, with enhancement of the role of case managers and greater integration of primary and specialist care, have been successful. Optimum ways of allocating resources between treatment providers as well as within health centres should be found.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

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.

Review of Diagnostic Screening Instruments for Alcohol and Other Drug Use and Other Psychiatric Disorders

In recent years there has been a growing recognition that many people with drug or alcohol problems are also experiencing a range of other psychiatric and psychological problems. The presence of concurrent psychiatric or psychological problems is likely to impact on the success of treatment services. These problems vary greatly, from undetected major psychiatric illnesses that meet internationally accepted diagnostic criteria such as those outlined in the Diagnostic and Statistical Manual (DSM-IV) of the American Psychiatric Association (1994), to less defined feelings of low mood and anxiety that do not meet diagnostic criteria but nevertheless impact on an individual’s sense of wellbeing and affect their quality of life. Similarly, the presence of a substance misuse problem among those suffering from a major psychiatric illness, often goes undetected. For example, the use of illicit drugs such as cannabis and amphetamine is higher among those individuals suffering from schizophrenia (Hall, 1992) and the misuse of alcohol in people suffering from schizophrenia is well documented (e.g., Gorelick et al., 1990; Searles et al., 1990; Soyka et al., 1993). High rates of alcohol misuse have also been reported in a number of groups including women presenting for treatment with a primary eating disorder (Holderness, Brooks Gunn, & Warren, 1994), individuals suffering from post-traumatic stress disorder (Seidel, Gusman and Aubueg, 1994), and those suffering from anxiety and depression. Despite considerable evidence of high levels of co-morbidity, drug and alcohol treatment agencies and mainstream psychiatric services often fail to identify and respond to concurrent psychiatric or drug and alcohol problems, respectively. The original review was conducted as a first step in providing clinicians with information on screening and diagnostic instruments that may be used to assess previously unidentified co-morbidity. The current revision was conducted to extend the original review by updating psychometric findings on measures in the original review, and incorporating other frequently used measures that were not previously included. The current revision has included information regarding special populations, specifically Indigenous Australians, older persons and adolescents. The objectives were to: ● update the original review of AOD and psychiatric screening/diagnostic instruments, ● recommend when these instruments should be used, by whom and how they should be interpreted, ● identify limitations and provide recommendations for further research, ● refer the reader to pertinent Internet sites for further information and/or purchasing of assessment instruments.

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.

Mental Disorders among Burn Patients

ABSTRACT Mental disorders and suicide attempts among acute burn patients were investigated in a collaborative research project between National Institute for Health and Welfare and Departments of Psychiatry and Plastic surgery in University of Helsinki in Finland. This project was realized in two parts. The first cohort of burn patients consisted of all burn patients admitted to the Helsinki Burn Centre during 1989 97. In this retrospective cohort, 5.7% (N=46) of the total of 811 burn patients had attempted suicide. The burn severity of suicide attempters was markedly higher than in the other burn patients. Suicide attempters were more often unemployed or on disability pension and had psychiatric history before the injury. The second sample was a prospective cohort of all acute consecutive burn patients admitted to the Helsinki Burn Centre during 18 months in 2006- 2007. All subjects (N=107) of the cohort were interviewed with the Structured Clinical Interview for DSM-IV for Axis I and II mental disorders (SCID-I and SCID-II) at baseline and then 86 % of all (N=92) with SCID-I at the end of six-month follow-up. Most (61%) patients had at least one lifetime mental disorder before burn; 47 % substance-related, 10% psychotic and 23% personality disorders. The overall prevalence of Axis I mental disorders increased significantly from the month prior to burn to acute care but decreased significantly from acute care to six months. However, more than one half (55%) of the cohort suffered from some mental disorder during follow-up. Less than one half of the burn patients with estimated need for psychiatric care received psychiatric care. Burn severity independently and strongly predicted risk for mental disorders during follow-up and pre-burn psychiatric history, severe burns and estimated need for psychiatric care significantly predicted psychiatric care received. The proportion of patients with self-inflicted burns is not high but mental disorders are common among burn patients. Mental disorders may predispose to burns. After burn injury, more than half of the patients suffer from mental disorders and a strong relationship exists between burn severity and some post-burn mental disorders. A minority of the patients with unequivocal need for psychiatric care actually receive it. Psychiatric consultations and care follow mainly the course of acute burn treatment.