Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

Risk factors for open angle glaucoma; a clinical and molecular genetic study

Glaucoma, optic neuropathy with excavation in the optic nerve head and corresponding visual field defect, is one of the leading causes for blindness worldwide. However, visual disability can often be avoided or delayed if the disease is diagnosed at an early stage. Therefore, recognising the risk factors for development and progression of glaucoma may prevent further damage. The purpose of the present study was to evaluate factors associated with visual disability caused by glaucoma and the genetic features of two risk factors, exfoliation syndrome (ES) and a positive family history of glaucoma. The present study material consisted of three study groups 1) deceased glaucoma patients from the Ekenäs practice 2) glaucoma families from the Ekenäs region and 3) population based families with and without exfoliation syndrome from Kökar Island. For the retrospective study, 106 patients with open angle glaucoma (OAG) were identified. At the last visit, 17 patients were visually impaired. Blindness induced by glaucoma was found in one or both eyes in 16 patients and in both eyes in six patients. The cumulative incidence of glaucoma caused blindness for one eye was 6% at 5 years, 9% at 10 years, and 15% at 15 years from initialising the treatment. The factors associated with blindness caused by glaucoma were an advanced stage of glaucoma at diagnosis, fluctuation in intraocular pressure during treatment, the presence of exfoliation syndrome, and poor patient compliance. A cross-sectional population based study performed in 1960-1962 on Kökar Island and the same population was followed until 2002. In total 965 subjects (530 over 50 years) have been examined at least once. The prevalence of exfoliation syndrome (ES) was 18% among subjects older than 50 years. Seventy-five of all 78 ES-positives belonged to the same extended pedigree. According to the segregation and family analysis, exfoliation syndrome seemed to be inherited as an autosomal dominant trait with reduced penetrance. The penetrance was more reduced for males, but the risk for glaucoma was higher in males than in females. To find the gene or genes associated with exfoliation syndrome, a genome wide scan was performed for 64 members (28 ES affected and 36 controls) of the Kökar pedigree. A promising result was found: the highest two-point LOD score of 3.45 (θ=0.04) in chromosome18q12.1-21.33. The presence of mutations in glaucoma genes TIGR/MYOC (myocilin) and OPTN (optineurin) was analysed in eight glaucoma families from the Ekenäs region. An inheritance pattern resembling autosomal dominant mode was detected in all these families. Primary open angle glaucoma or exfoliation glaucoma was found in 35% of 136 family members and 28% were suspected to have glaucoma. No mutations were detected in these families.

Molecular mediators linking stroke and carotid artery disease

Carotid artery disease is the most prevalent etiologic precursor of ischemic stroke, which is a major health hazard and the second most common cause of death in the world. If a patient presents with a symptomatic high-grade (>70%) stenosis in the internal carotid artery, the treatment of choice is carotid endarterectomy. However, the natural course of radiologically equivalent carotid lesions may be clinically quite diverse, and the reason for that is unknown. It would be of utmost importance to develop molecular markers that predict the symptomatic phenotype of an atherosclerotic carotid plaque (CP) and help to differentiate vulnerable lesions from stable ones. The aim of this study was to investigate the morphologic and molecular factors that associate with stroke-prone CPs. In addition to immunohistochemistry, DNA microarrays were utilized to identify molecular markers that would differentiate between symptomatic and asymptomatic CPs. Endothelial adhesion molecule expression (ICAM-1, VCAM-1, P-selectin, and E-selectin) did not differ between symptomatic and asymptomatic patients. Denudation of endothelial cells was associated with symptom-generating carotid lesions, but in studies on the mechanism of decay of endothelial cells, markers of apoptosis (TUNEL, activated caspase 3) were found to be decreased in the endothelium of symptomatic lesions. Furthermore, markers of endothelial apoptosis were directly associated with those of cell proliferation (Ki-67) in all plaques. FasL expression was significantly increased on the endothelium of symptomatic CPs. DNA microarray analysis revealed prominent induction of specific genes in symptomatic CPs, including those subserving iron and heme metabolism, namely HO-1, and hemoglobin scavenger receptor CD163. HO-1 and CD163 proteins were also increased in symptomatic CPs and associated with intraplaque iron deposits, which, however, did not correlate with symptom status itself. ADRP, the gene for adipophilin, was also overexpressed in symptomatic CPs. Adipophilin expression was markedly increased in ulcerated CPs and colocalized with extravasated red blood cells and cholesterol crystals. Taken together, the phenotypic characteristics and the numerous possible molecular mediators of the destabilization of carotid plaques provide potential platforms for future research. The denudation of the endothelial lining observed in symptomatic CPs may lead to direct thromboembolism and maintain harmful oxidative and inflammatory processes, predispose to plaque microhemorrhages, and contribute to lipid accumulation into the plaque, thereby making it vulnerable to rupture.

SPECT-aided diagnostics and genetic risk factors in Parkinson's disease

Hypokinesia, rigidity, tremor, and postural instability are the cardinal symptoms of Parkinson s disease (PD). Since these symptoms are not specific to PD the diagnosis may be uncertain in early PD. Etiology and pathogenesis of PD remain unclear. There is no neuroprotective therapy. Genetic findings are expected to reveal metabolic routes in PD pathogenesis and thereby eventually lead to therapeutic innovations. In this thesis, we first aimed to study the usefulness and accuracy of 123I-b-CIT SPECT in the diagnosis of PD in a consecutive clinic-based material including various movement disorders. We subsequently a genetic project to identify genetic risk factors for sporadic PD using a candidate gene approach in a case-control setting including 147 sporadic PD patients and 137 spouse controls. Dopamine transporter imaging by 123I-b-CIT SPECT could distinguish PD from essential tremor, drug-induced parkinsonism, dystonia and psychogenic parkinsonism. However, b-CIT uptake in Parkinson plus syndromes (PSP and multiple system atrophy) and dementia with Lewy bodies was not significantly different from PD. 123I-b-CIT SPECT could not reliably differentiate PD from vascular parkinsonism. 123I-b-CIT SPECT was 100% sensitive and specific in the diagnosis of PD in patients younger than 55 years but less specific in older patients, due to differential distribution of the above conditions in the younger and older age groups. 123I-b-CIT SPECT correlated with symptoms and detected bilateral nigrostriatal defect in patients whose PD was still in unilateral stage. Thus, in addition to as a differential diagnostic aid, 123I-b-CIT SPECT may be used to detect PD early, even pre-symptomatically in at-risk individuals. 123I-b-CIT SPECT was used to aid in the collection of patients to the genetic studies. In the genetic part of this thesis we found an association between PD and a polymorphic CAG-repeat in POLG1 gene encoding the catalytic subunit of mitochondrial polymerase gamma. The CAG-repeat encodes a polyglutamine tract (polyQ), the two most common lengths of which are 10Q (86-90%) and 11Q. In our Finnish material, the rarer non-10Q or non-11Q length variants (6Q-9Q, 12Q-14Q, 4R+9Q) were more frequent in patients than in spouse controls (10% vs. 3.5 %, p=0.003), or population controls (p=0.001). Therefore, we performed a replication study in 652 North American PD patients and 292 controls. Non-10/11Q alleles were more common in the US PD patients compared to the controls but the difference did not reach statistical significance (p=0.07). This larger data suggested our original definition of variant length allele might need reconsideration. Most previous studies on phenotypic effects of POLG1 polyQ have defined 10Q as the only normal allele. Non-10Q alleles were significantly more common in patients compared to the controls (17.3% vs. 12.3 %, p= 0.005). This association between non-10Q length variants and PD remained significant when compared to a larger set of 1541 literature controls (p=0.00005). In conclusion, POLG1 polyQ alleles other than 10Q may predispose to PD. We did not find association between PD and parkin or DJ-1, genes underlying autosomal recessive parkinsonism. The functional Val158Met polymorphism, which affects the catalytic effect of COMT enzyme, and another coding polymorphism in COMT were not associated with PD in our patient material. The APOE e2/3/4 polymorphism modifies risk for Alzheimer s disease and prognosis of for example brain trauma. APOE promoter and enhancer polymorphisms 219G/T and +113G/C, and APOE e3 haplotypes, have also been shown to modify the risk of Alzheimer s disease but not reported in PD. No association was found between PD and APOE e2/3/4 polymorphism, the promoter or enhancer polymorphisms, or the e3 haplotypes.

Prognostic molecular factors and algorithms in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas. As DLBCL is characterized by heterogeneous clinical and biological features, its prognosis varies. To date, the International Prognostic Index has been the strongest predictor of outcome for DLBCL patients. However, no biological characters of the disease are taken into account. Gene expression profiling studies have identified two major cell-of-origin phenotypes in DLBCL with different prognoses, the favourable germinal centre B-cell-like (GCB) and the unfavourable activated B-cell-like (ABC) phenotypes. However, results of the prognostic impact of the immunohistochemically defined GCB and non-GCB distinction are controversial. Furthermore, since the addition of the CD20 antibody rituximab to chemotherapy has been established as the standard treatment of DLBCL, all molecular markers need to be evaluated in the post-rituximab era. In this study, we aimed to evaluate the predictive value of immunohistochemically defined cell-of-origin classification in DLBCL patients. The GCB and non-GCB phenotypes were defined according to the Hans algorithm (CD10, BCL6 and MUM1/IRF4) among 90 immunochemotherapy- and 104 chemotherapy-treated DLBCL patients. In the chemotherapy group, we observed a significant difference in survival between GCB and non-GCB patients, with a good and a poor prognosis, respectively. However, in the rituximab group, no prognostic value of the GCB phenotype was observed. Likewise, among 29 high-risk de novo DLBCL patients receiving high-dose chemotherapy and autologous stem cell transplantation, the survival of non-GCB patients was improved, but no difference in outcome was seen between GCB and non-GCB subgroups. Since the results suggested that the Hans algorithm was not applicable in immunochemotherapy-treated DLBCL patients, we aimed to further focus on algorithms based on ABC markers. We examined the modified activated B-cell-like algorithm based (MUM1/IRF4 and FOXP1), as well as a previously reported Muris algorithm (BCL2, CD10 and MUM1/IRF4) among 88 DLBCL patients uniformly treated with immunochemotherapy. Both algorithms distinguished the unfavourable ABC-like subgroup with a significantly inferior failure-free survival relative to the GCB-like DLBCL patients. Similarly, the results of the individual predictive molecular markers transcription factor FOXP1 and anti-apoptotic protein BCL2 have been inconsistent and should be assessed in immunochemotherapy-treated DLBCL patients. The markers were evaluated in a cohort of 117 patients treated with rituximab and chemotherapy. FOXP1 expression could not distinguish between patients, with favourable and those with poor outcomes. In contrast, BCL2-negative DLBCL patients had significantly superior survival relative to BCL2-positive patients. Our results indicate that the immunohistochemically defined cell-of-origin classification in DLBCL has a prognostic impact in the immunochemotherapy era, when the identifying algorithms are based on ABC-associated markers. We also propose that BCL2 negativity is predictive of a favourable outcome. Further investigational efforts are, however, warranted to identify the molecular features of DLBCL that could enable individualized cancer therapy in routine patient care.

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.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

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.

Neurological manifestations and molecular genetics of acute intermittent porphyria in North Western Russia

Acute intermittent porphyria (AIP, MIM #176000) is an inherited metabolic disease due to a partial deficiency of the third enzyme, hydroxymethylbilane synthase (HMBS, EC: 4.3.1.8), in the haem biosynthesis. Neurological symptoms during an acute attack, which is the major manifestation of AIP, are variable and relatively rare, but may endanger a patient's life. In the present study, 12 Russian and two Finnish AIP patients with severe neurological manifestations during an acute attack were studied prospectively from 1995 to 2006. Autonomic neuropathy manifested as abdominal pain (88%), tachycardia (94%), hypertension (75%) and constipation (88%). The most common neurological sign was acute motor peripheral neuropathy (PNP, 81%) often associated with neuropathic sensory loss (54%) and CNS involvement (85%). Despite heterogeneity of the neurological manifestations in our patients with acute porphyria, the major pattern of PNP associated with abdominal pain, dysautonomia, CNS involvement and mild hepatopathy could be demonstrated. If more strict inclusion criteria for biochemical abnormalities (>10-fold increase in excretion of urinary PBG) are applied, neurological manifestations in an acute attack are probably more homogeneous than described previously, which suggests that some of the neurological patients described previously may not have acute porphyria but rather secondary porphyrinuria. Screening for acute porphyria using urinary PBG is useful in a selected group of neurological patients with acute PNP or encephalopathy and seizures associated with pain and dysautonomia. Clinical manifestations and the outcome of acute attacks were used as a basis for developing a 30-score scale of the severity of an acute attack. This scale can easily be used in clinical practice and to standardise the outcome of an attack. Degree of muscle weakness scored by MRC, prolonged mechanical ventilation, bulbar paralysis, impairment of consciousness and hyponatraemia were important signs of a poor prognosis. Arrhythmia was less important and autonomic dysfunction, severity of pain and mental symptoms did not affect the outcome. The delay in the diagnosis and repeated administrations of precipitating factors were the main cause of proceeding of an acute attack into pareses and severe CNS involvement and a fatal outcome in two patients. Nerve conduction studies and needle EMG were performed in eleven AIP patients during an acute attack and/or in remission. Nine patients had severe PNP and two patients had an acute encephalopathy but no clinically evident PNP. In addition to axonopathy, features suggestive of demyelination could be demonstrated in patients with severe PNP during an acute attack. PNP with a moderate muscle weakness was mainly pure axonal. Sensory involvement was common in acute PNP and could be subclinical. Decreased conduction velocities with normal amplitudes of evoked potentials during acute attacks with no clinically evident PNP indicated subclinical polyneuropathy. Reversible symmetrical lesions comparable with posterior reversible encephalopathy syndrome (PRES) were revealed in two patients' brain CT or MRI during an acute attack. In other five patients brain MRI during or soon after the symptoms was normal. The frequency of reversible brain oedema in AIP is probably under-estimated since it may be short-lasting and often indistinguishable on CT or MRI. In the present study, nine different mutations were identified in the HMBS gene in 11 unrelated Russian AIP patients from North Western Russia and their 32 relatives. AIP was diagnosed in nine symptom-free relatives. The majority of the mutations were family-specific and confirmed allelic heterogeneity also among Russian AIP patients. Three mutations, c.825+5G>C, c.825+3_825+6del and c.770T>C, were novel. Six mutations, c.77G>A (p.R26H), c.517C>T (p.R173W), c.583C>T (p.R195C), c.673C>T (p.R225X), c.739T>C (p.C247R) and c.748G>C (p.E250A), have previously been identified in AIP patients from Western and other Eastern European populations. The effects of novel mutations were studied by amplification and sequencing of the reverse-transcribed total RNA obtained from the patients' lymphoblastoid or fibroblast cell lines. The mutations c.825+5G>C and c.770T>C resulted in varyable amounts of abnormal transcripts, r.822_825del (p.C275fsX2) and [r.770u>c, r.652_771del, r.613_771del (p.L257P, p.G218_L257del, p.I205_L257del)]. All mutations demonstrated low residual activities (0.1-1.3 %) when expressed in COS-1 cells confirming the causality of the mutations and the enzymatic defect of the disease. The clinical outcome, prognosis and correlation between the HMBS genotype and phenotype were studied in 143 Finnish and Russian AIP patients with ten mutations (c.33G>T, c.97delA, InsAlu333, p.R149X, p.R167W, p.R173W, p.R173Q, p.R225G, p.R225X, c.1073delA) and more than six patients in each group. The patients were selected from the pool of 287 Finnish AIP patients presented in a Finnish Porphyria Register (1966-2003) and 23 Russian AIP patients (diagnosed 1995-2003). Patients with the p.R167W and p.R225G mutations showed lower penetrance (19% and 11%) and the recurrence rate (33% and 0%) in comparison to the patients with other mutations (range 36 to 67% and 0 to 66%, respectively), as well as milder biochemical abnormalities [urinary porphobilinogen 47±10 vs. 163±21 mol/L, p<0.001; uroporphyrin 130±40 vs. 942±183 nmol/L, p<0.001] suggesting a milder form of AIP in these patients. Erythrocyte HMBS activity did not correlate with the porphobilinogen excretion in remission or the clinical of the disease. In all AIP severity patients, normal PBG excretion predicted freedom from acute attacks. Urinary PBG excretion together with gender, age at the time of diagnosis and mutation type could predict the likelihood of acute attacks in AIP patients.