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.

On the environmental genetics of depressive symptoms : Focus on two serotonergic genes

Depression is a complex psychiatric disorder influenced by several genes, environmental factors, and their interplay. Serotonin receptor 2A (HTR2A) and tryptophan hydroxylase 1 (TPH1) genes have been implicated in vulnerability to depression and other psychiatric disorders, but the results have been inconsistent. The present study examined whether these two genes moderated the influence of different depressogenic environmental factors on subthreshold depressive symptoms (assessed on a modified version of Beck s Depression Inventory, BDI) and depression-related temperament, i.e., harm avoidance (assessed on the Temperament and Character Inventory, TCI). The environmental factors included measures of childhood and adolescence exposure, i.e., maternal nurturance and parental socioeconomic status, and adulthood social circumstances, i.e., perceived social support and urban/rural residence. The participants were two randomly selected subsamples (n = 1246, n = 341) from the longitudinal population-based Cardiovascular Risk in Young Finns study (n = 3596). Childhood environmental factors were assessed when the participants were 3 to 18 years of age, and three years after the baseline. Adulthood environmental factors and outcome measures were assessed 17 and 21 years later when the participants were 21 to 39 years of age. The T102C polymorphism of the HTR2A gene moderated the association between childhood maternal nurturance and adulthood depressive symptoms, such that exposure to high maternal nurturance predicted low depressive symptoms among individuals carrying the T/T or T/C genotypes, but not among those carrying the C/C genotype. Likewise, high parental SES predicted low adulthood harm avoidance in individuals carrying the T/T or T/C genotype, but not in C/C-genotype carriers. Individuals carrying the T/T or T/C genotype were also sensitive to urban/rural residence, such that they had lower depressive symptoms in urban than in rural areas, whereas those carrying the C/C genotype were not sensitive to urban/rural residence difference. HTR2A did not moderate the influence of social support. TheA779C/A218C haplotype of the TPH1 gene was not involved in the association between childhood environment and adulthood outcomes. However, individuals carrying A alleles of the TPH1 haplotype were more vulnerable to the lack of adulthood social support in terms of high depressive symptoms than their counterparts carrying no A alleles. Furthermore, individuals living in remote rural areas and carrying the A/A haplotype had higher depressive symptoms than those carrying other genotypes of the TPH1. The findings suggest that the HTR2A and TPH1 genes may be involved in the development of depression by influencing individual s sensitivity to depressogenic environmental influences.

High-density lipoprotein 17beta-estradiol fatty acyl esters and phytoestrogens : Impact on reverse cholesterol transport and women's cardiovascular health

Reverse cholesterol transport (RCT) is an important function of high-density lipoproteins (HDL) in the protection of atherosclerosis. RCT is the process by which HDL stimulates cholesterol removal from peripheral cells and transports it to the liver for excretion. Premenopausal women have a reduced risk for atherosclerosis compared to age-matched men and there exists a positive correlation for serum 17β-estradiol (E2) and HDL levels in premenopausal women supporting the role of E2 in atherosclerosis prevention. In premenopausal women, E2 associates with HDL as E2 fatty acyl esters. Discovery of the cellular targets, metabolism, and assessment of the macrophage cholesterol efflux potential of these HDL-associated E2 fatty acyl esters were the major objectives of this thesis (study I, III, and IV). Soy phytoestrogens, which are related to E2 in both structure and function, have been proposed to be protective against atherosclerosis but the evidence to support these claims is conflicting. Therefore, another objective of this thesis was to assess the ability of serum from postmenopausal women, treated with isoflavone supplements (compared to placebo), to promote macrophage cholesterol efflux (study II). The scope of this thesis was to cover the roles that HDL-associated E2 fatty acyl esters have in the cellular aspects of RCT and to determine if soy isoflavones can also influence RCT mechanisms. SR-BI was a pivotal cellular receptor, responsible for hepatic and macrophage uptake and macrophage cholesterol efflux potential of HDL-associated E2 fatty acyl esters. Functional SR-BI was also critical for proper LCAT esterification activity which could impact HDL-associated E2 fatty acyl ester assembly and its function. In hepatic cells, LDL receptors also contributed to HDL-associated E2 fatty acyl esters uptake and in macrophage cells, estrogen receptors (ERs) were necessary for both HDL-associated E2 ester-specific uptake and cholesterol efflux potential. HDL-containing E2 fatty acyl esters (E2-FAE) stimulated enhanced cholesterol efflux compared to male HDL (which are deficient in E2) demonstrating the importance of the E2 ester in this process. To support this, premenopausal female HDL, which naturally contains E2, showed greater macrophage cholesterol efflux compared to males. Additionally, hepatic and macrophage cells hydrolyzed the HDL-associated E2 fatty acyl ester into unesterified E2. This could have important biological ramifications because E2, not the esterified form, has potent cellular effects which may influence RCT mechanisms. Lastly, soy isoflavone supplementation in postmenopausal women did not modulate ABCA1-specific macrophage cholesterol efflux but did increase production of plasma pre-β HDL levels, a subclass of HDL. Therefore, the impact of isoflavones on RCT and cardiovascular health needs to be further investigated. Taken as a whole, HDL-associated E2 fatty acyl esters from premenopausal women and soy phytoestrogen treatment in postmenopausal women may be important factors that increase the efficiency of RCT through cellular lipoprotein-related processes and may have direct implications on the cardiovascular health of women.

Living on the edge : Population genetics of Finno-Ugric-speaking humans in North Eurasia

The major aim of this thesis was to examine the origins and distribution of uniparental and autosomal genetic variation among the Finno-Ugric-speaking human populations living in Boreal and Arctic regions of North Eurasia. In more detail, I aimed to disentangle the underlying molecular and population genetic factors which have produced the patterns of uniparental and autosomal genetic diversity in these populations. Among Finno-Ugrics the genetic amalgamation and clinal distribution of West and East Eurasian gene pools were observed within uniparental markers. This admixture indicates that North Eurasia was colonized through Central Asia/ South Siberia by human groups already carrying both West and East Eurasian lineages. The complex combination of founder effects, gene flow and genetic drift underlying the genetic diversity of the Finno-Ugric- speaking populations were emphasized by low haplotype diversity within and among uniparental and biparental markers. A high prevalence of lactase persistence allele among the North Eurasian Finno- Ugric agriculturalist populations was also shown indicating a local adaptation to subsistence change with lactose rich diet. Moreover, the haplotype background of lactase persistence allele among the Finno- Ugric-speakers strongly suggested that the lactase persistence T-13910 mutation was introduced independently more than once to the North Eurasian gene pool. A significant difference in genetic diversity, haplotype structure and LD distribution within the cytochrome P450 CYP2C and CYP2D regions revealed the unique gene pool of the Finno-Ugric Saami created mainly by population genetic processes compared to other Europeans and sub-Saharan Mandenka population. From all studied populations the Saami showed also significantly the highest allele frequency of a CYP2C19 gene mutation causing variable drug reactions. The diversity patterns observed within CYP2C and CYP2D regions emphasize the strong effect of demographic history shaping genetic diversity and LD especially among such small and constant size populations as the Finno-Ugric-speaking Saami. Moreover, the increased LD in Saami due to genetic drift and/or admixture was shown to offer an advantage for further attempts to identify alleles associated to common complex pharmacogenetic traits.

Molecular Genetics of Lactase Deficiencies

Congenital lactase deficiency (CLD) (MIM 223000) is a rare autosomal recessive gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. The CLD locus was previously assigned by linkage and linkage disequilibrium analyses on 2q21 in 19 Finnish families. In this study, the molecular background of this disorder is reported. The CLD locus was refined in 32 CLD patients in 24 families by using microsatellite and single nucleotide polymorphism (SNP) haplotypes. Mutation analyses were performed by direct sequencing. We identified 5 distinct mutations in the lactase (LCT) gene, encoding the enzyme that hydrolyzes lactose in the intestinal lumen. These findings facilitate genetic testing of CLD in clinical practice and enable genetic counseling. The present data also provide the basis for detailed characterization of the molecular pathogenesis of this disorder. Adult-type hypolactasia (MIM 223100) (lactase non-persistence, lactose intolerance) is an autosomal recessive gastrointestinal condition that is a result of a decline in the activity of lactase in the intestinal lumen after weaning. Adult-type hypolactasia is considered to be a normal phenomenon among mammals and symptoms are remarkably milder than experienced in CLD. Recently, a variant C/T-13910 was shown to associate with the adult-type hypolactasia trait, locating 13.9 kb upstream of the LCT gene. In this study, the functional significance of the C/T-13910 variant was determined by studying the LCT mRNA levels in intestinal biopsy samples in children and adults with different genotypes. RT-PCR followed by solid-phase minisequencing was applied to determine the relative expression levels of the LCT alleles using an informative SNP located in exon 1. In children, the C-13910 allele was observed to be downregulated after five years of age in parallel with lactase enzyme activity. The expression of the LCT mRNA in the intestinal mucosa in individuals with the T-13910 A-22018 alleles was 11.5 times higher than that found in individuals with the C-13910, G-22018 alleles. These findings suggest that the C/T-13910 associated with adult-type hypolactasia is associated with the transcriptional regulation of the LCT gene. The presence of the T-13910 A-22018 allele also showed significant elevation lactase activity. Galactose, the hydrolysing product of the milk sugar lactose, has been hypothesized to be poisonous to ovarian epithelial cells. Hence, consumption of dairy products and lactase persistence has been proposed to be a risk factor for ovarian carcinoma. To investigate whether lactase persistence is related to the risk of ovarian carcinoma the C/T-13910 genotype was determined in a cohort of 782 women with ovarian carcinoma 1331 individuals serving as controls. Lactase persistence did not associate significantly with the risk for ovarian carcinoma in the Finnish, in the Polish or in the Swedish populations. The findings do not support the hypothesis that lactase persistence increases the risk for ovarian carcinoma.

Molecular Genetics of Schizophrenia and Related Intermediate Phenotypes in a Founder Population

Schizophrenia, affecting about 1% of population worldwide, is a severe mental disorder characterized by positive and negative symptoms, such as psychosis and anhedonia, as well as cognitive deficits. At present, schizophrenia is considered a complex disorder of neurodevelopmental origin with both genetic and environmental factors contributing to its onset. Although a number of candidate genes for schizophrenia have been highlighted, only very few schizophrenia patients are likely to share identical genetic liability. This study is based on the nation-wide schizophrenia family sample of the National Institute for Health and Welfare, and represents one of the largest and most well-characterized familial series in the world. In the first part of this study, we investigated the roles of the DTNBP1, NRG1, and AKT1 genes in the background of schizophrenia in Finland. Although these genes are associated with schizophrenia liability in several populations, any significant association with clinical diagnostic information of schizophrenia remained absent in our sample of 441 schizophrenia families. In the second part of this study, we first replicated schizophrenia linkage on the long arm of chromosome 7 in 352 schizophrenia families. In the following association analysis, we utilized additional clinical disorder features and intermediate phenotypes – endophenotypes - in addition to diagnostic information from altogether 290 neuropsychologically assessed schizophrenia families. An intragenic short tandem repeat allele of the regional RELN gene, supposed to play a role in the background of several neurodevelopmental disorders, showed significant association with poorer cognitive functioning and more severe schizophrenia symptoms. Additionally, this risk allele was significantly more prevalent among the individuals affected with schizophrenia spectrum disorders. We have previously identified linkage of schizophrenia and its cognitive endophenotypes on the long arms of chromosomes 2, 4, and 5. In the last part of this study, we selected altogether 104 functionally relevant candidate genes from the linked regions. We detected several promising associations, of which especially interesting are the ERBB4 gene, showing association with the severity of schizophrenia symptoms and impairments in traits related to verbal abilities, and the GRIA1 gene, showing association with the severity of schizophrenia symptoms. Our results extend the previous evidence that the genetic risk for schizophrenia is at least partially mediated via the effects of the candidate genes and their combinations on relevant brain systems, resulting in alterations in different disorder domains, such as the cognitive deficits.

Insights into the molecular genetics of celiac disease : Applying family- and population-based strategies

Celiac disease, or gluten intolerance, is triggered by dietary glutens in genetically susceptible individuals and it affects approximately 1% of the Caucasian population. The best known genetic risk factors for celiac disease are HLA DQ2 and DQ8 heterodimers, which are necessary for the development of the disease. However, they alone are not sufficient for disease induction, other risk factors are required. This thesis investigated genetic factors for celiac disease, concentrating on susceptibility loci on chromosomes 5q31-q33, 19p13 and 2q12 previously reported in genome-wide linkage and association studies. In addition, a novel genotyping method for the detection of HLA DQ2 and DQ8 coding haplotypes was validated. This study was conducted using Finnish and Hungarian family materials, and Finnish, Hungarian and Italian case-control materials. Genetic linkage and association were analysed in these materials using candidate gene and fine-mapping approaches. The results confirmed linkage to celiac disease on the chromosomal regions 5q31-q33 and 19p13. Fine-mapping on chromosome 5q31-q33 revealed several modest associations in the region, and highlighted the need for further investigations to locate the causal risk variants. The MYO9B gene on chromosome 19p13 showed evidence for linkage and association particularly with dermatitis herpetiformis, the skin manifestation of celiac disease. This implies a potential difference in the genetic background of the intestinal and skin forms of the disease, although studies on larger samplesets are required. The IL18RAP locus on chromosome 2q12, shown to be associated with celiac disease in a previous genome-wide association study and a subsequent follow-up, showed association in the Hungarian population in this study. The expression of IL18RAP was further investigated in small intestinal tissue and in peripheral blood mononuclear cells. The results showed that IL18RAP is expressed in the relevant tissues. Two putative isoforms of IL18RAP were detected by Western blot analysis, and the results suggested that the ratios and total levels of these isoforms may contribute to the aetiology of celiac disease. A novel genotyping method for celiac disease-associated HLA haplotypes was also validated in this thesis. The method utilises single-nucleotide polymorphisms tagging these HLA haplotypes with high sensitivity and specificity. Our results suggest that this method is transferable between populations, and it is suitable for large-scale analysis. In conclusion, this doctorate study provides an insight into the roles of the 5q31-q33, MYO9B, IL18RAP and HLA loci in the susceptibility to celiac disease in the Finnish, Hungarian and Italian populations, highlighting the need for further studies at these genetic loci and examination of the function of the candidate genes.

Molecular genetics of primary open angle glaucoma and exfoliation syndrome

Glaucoma is the second leading cause of blindness worldwide. It is a group of optic neuropathies, characterized by progressive optic nerve degeneration, excavation of the optic disc due to apoptosis of retinal ganglion cells and corresponding visual field defects. Open angle glaucoma (OAG) is a subtype of glaucoma, classified according to the age of onset into juvenile and adult- forms with a cut-off point of 40 years of age. The prevalence of OAG is 1-2% of the population over 40 years and increases with age. During the last decade several candidate loci and three candidate genes, myocilin (MYOC), optineurin (OPTN) and WD40-repeat 36 (WDR36), for OAG have been identified. Exfoliation syndrome (XFS), age, elevated intraocular pressure and genetic predisposition are known risk factors for OAG. XFS is characterized by accumulation of grayish scales of fibrillogranular extracellular material in the anterior segment of the eye. XFS is overall the most common identifiable cause of glaucoma (exfoliation glaucoma, XFG). In the past year, three single nucleotide polymorphisms (SNPs) on the lysyl oxidase like 1 (LOXL1) gene have been associated with XFS and XFG in several populations. This thesis describes the first molecular genetic studies of OAG and XFS/XFG in the Finnish population. The role of the MYOC and OPTN genes and fourteen candidate loci was investigated in eight Finnish glaucoma families. Both candidate genes and loci were excluded in families, further confirming the heterogeneous nature of OAG. To investigate the genetic basis of glaucoma in a large Finnish family with juvenile and adult onset OAG, we analysed the MYOC gene in family members. Glaucoma associated mutation (Thr377Met) was identified in the MYOC gene segregating with the disease in the family. This finding has great significance for the family and encourages investigating the MYOC gene also in other Finnish OAG families. In order to identify the genetic susceptibility loci for XFS, we carried out a genome-wide scan in the extended Finnish XFS family. This scan produced promising candidate locus on chromosomal region 18q12.1-21.33 and several additional putative susceptibility loci for XFS. This locus on chromosome 18 provides a solid starting point for the fine-scale mapping studies, which are needed to identify variants conferring susceptibility to XFS in the region. A case-control and family-based association study and family-based linkage study was performed to evaluate whether SNPs in the LOXL1 gene contain a risk for XFS, XFG or POAG in the Finnish patients. A significant association between the LOXL1 gene SNPs and XFS and XFG was confirmed in the Finnish population. However, no association was detected with POAG. Probably also other genetic and environmental factors are involved in the pathogenesis of XFS and XFG.