Molecular genetics of RECQL4 syndromes

RAPADILINO syndrome is an autosomally resessively inherited condition that belongs to a group of rare syndromes more common in Finland than in other parts of the world. RAPADILINO is characterized by pre- and postnatal growth retardation, radial ray defects, diarrhoea of unknown aetiology during chilhood, a facial resemblance with other patients and normal intelligence. In Finland, 15 patients with this condition have been found which compares with only five patients in other parts of the world. We found RECQL4 gene mutations in RAPADILINO patients and proved this syndrome to be allelic with a subgroup of Rothmund-Thomson syndrome (RTS). Later we found RECQL4 mutations in patients with Baller-Gerold syndrome (BGS). These three syndromes share clinical findings and differential diagnostics rely on poikiloderma and craniosynostosis not seen in RAPADILINO syndrome. We found five different mutations in the Finnish RAPADILINO patients. The g.2545delT mutation is the founder mutation in the Finnish population as all the patients are either homozygotes or compound heterozygotes for it. This mutation leads to the inframe skipping of exon seven from mRNA. The protein encoded by this mutant mRNA lacks the nuclear retention signal and thus leads to the mislocalization of the mutant protein. The genotype-phenotype correlation is not straightforward but it seems that RAPADILINO could be due to alteration in protein function and truncating mutations in both alleles are more common among RTS patients. RTS patients with RECQL4 mutations have an elevated risk for osteosarcoma, but their risk to develop other types of malignancies is not increased.Two Finnish RAPADILINO patients have been diagnosed with osteosarcoma, but in addition to this we have found an excess of lymphoma cases among the Finnish RAPADILINO patients. This difference between cancer types could be due to different mutations found in these syndromes. The mutation screening of the patients will help to differentiate patients who have RECQL4 mutations and thus the elevated cancer risk. Patients will benefit from the follow up since early detection of malignancies is important for the treatment.

Molecular genetics of X-linked cone-rod dystrophy and Åland Island eye disease

Inherited retinal diseases are the most common cause of vision loss among the working population in Western countries. It is estimated that ~1 of the people worldwide suffer from vision loss due to inherited retinal diseases. The severity of these diseases varies from partial vision loss to total blindness, and at the moment no effective cure exists. To date, nearly 200 mapped loci, including 140 cloned genes for inherited retinal diseases have been identified. By a rough estimation 50% of the retinal dystrophy genes still await discovery. In this thesis we aimed to study the genetic background of two inherited retinal diseases, X-linked cone-rod dystrophy and Åland Island eye disease. X-linked cone-rod dystrophy (CORDX) is characterized by progressive loss of visual function in school age or early adulthood. Affected males show reduced visual acuity, photophobia, myopia, color vision defects, central scotomas, and variable changes in fundus. The disease is genetically heterogeneous and two disease loci, CORDX1 and CORDX2, were known prior to the present thesis work. CORDX1, located on Xp21.1-11.4, is caused by mutations in the RPGR gene. CORDX2 is located on Xq27-28 but the causative gene is still unknown. Åland Island eye disease (AIED), originally described in a family living in Åland Islands, is a congenital retinal disease characterized by decreased visual acuity, fundus hypopigmentation, nystagmus, astigmatism, red color vision defect, myopia, and defective night vision. AIED shares similarities with another retinal disease, congenital stationary night blindness (CSNB2). Mutations in the L-type calcium channel α1F-subunit gene, CACNA1F, are known to cause CSNB2, as well as AIED-like disease. The disease locus of the original AIED family maps to the same genetic interval as the CACNA1F gene, but efforts to reveal CACNA1F mutations in patients of the original AIED family have been unsuccessful. The specific aims of this study were to map the disease gene in a large Finnish family with X-linked cone-rod dystrophy and to identify the disease-causing genes in the patients of the Finnish cone-rod dystrophy family and the original AIED family. With the help of linkage and haplotype analyses, we could localize the disease gene of the Finnish cone-rod dystrophy family to the Xp11.4-Xq13.1 region, and thus establish a new genetic X-linked cone-rod dystrophy locus, CORDX3. Mutation analyses of candidate genes revealed three novel CACNA1F gene mutations: IVS28-1 GCGTC>TGG in CORDX3 patients, a 425 bp deletion, comprising exon 30 and flanking intronic regions in AIED patients, and IVS16+2T>C in an additional Finnish patient with a CSNB2-like phenotype. All three novel mutations altered splice sites of the CACNA1F gene, and resulted in defective pre-mRNA splicing suggesting altered or absent channel function as a disease mechanism. The analyses of CACNA1F mRNA also revealed novel alternative wt splice variants, which may enhance channel diversity or regulate the overall expression level of the channel. The results of our studies may be utilized in genetic counseling of the families, and they provide a basis for studies on the pathogenesis of these diseases. In the future, the knowledge of the genetic defects may be used in the identification of specific therapies for the patients.

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.

Molecular Genetics of Age-related Macular Degeneration

Age-related macular degeneration (AMD; OMIM # 603075) is an eye disease of the elderly, signs of which appear after the age of 50. In the Western world it is a leading cause of permanent visual loss with a prevalence of 8.5% in persons under 54 years of age and of 37% in persons over 75 years of age. Early forms of AMD may be asymptomatic, but in the late forms usually a central scotoma in the visual field follows severely complicating daily tasks. Smoking, age, and genetic predisposition are known risk factors for AMD. Until recently no true susceptibility genes had been identified though the composition of drusen deposits, the hallmarks of AMD, has suggested that the complement system might play a role in the pathogenesis of AMD. When four groups reported in March 2005, that, on chromosome 1q32, a Y402H variant in the complement factor H (CFH) gene confers risk for AMD in independent Caucasian samples, a new period in the field of genetic research of AMD started. CFH is a key regulator of the complement system. Thus, it is logical to speculate, that it plays a role in the pathogenesis of AMD. We performed a case-control association study to analyse whether the CFH Y402H variant contain a risk for AMD in the Finnish population. Although the population of Finland represents a genetic isolate, the CFH Y402H polymorphism was associated with AMD also in our patient sample with similar risk allele frequencies as in the other Caucasian populations. We further evaluated the effects of this variant, but no association between lesion subtype (predominantly classic, minimally classic or occult lesion) or lesion size of neovascular AMD and the CFH Y402H variant was detected. Neither did the variant have an effect on the photodynamic therapy (PDT) outcome. The patients that respond to PDT carried the risk genotype as frequently as those who did not respond, and no difference was found in the number of PDT sessions needed in patients with or without the risk genotypes of CFH Y402H. Functional analyses, however, showed that the binding of C-reactive protein (CRP) to CFH was significantly reduced in patients with the risk genotype of Y402H. In the past two years, the LOC387715/ high-temperature requirement factor A1 (HTRA1) locus on 10q26 has also been repeatedly associated with AMD in several populations. The recent discovery of the LOC387715 protein on the mitochondrial outer membrane suggests that the LOC387715 gene, not HTRA1, is the true predisposing gene in this region, although its biological function is still unknown. In our Finnish patient material, patients with AMD carried the A69S risk genotype of LOC387715 more frequently than the controls. Also, for the first time, an interaction between the CFH Y402H and the LOC387715 A69S variants was found. The most recently detected susceptibilty gene of AMD, the complement component 3 (C3) gene, encodes the central component of the complement system, C3. In our Finnish sample, an additive gene effect for the C3 locus was detected, though weaker than the effects for the two main loci, CFH and LOC387715. Instead, the hemicentin-1 or the elongation of very long chain fatty acids-like 4 genes that have also been suggested as candidate genes for AMD did not carry a risk for AMD in the Finnish population. This was the first series of molecular genetic study of AMD in Finland. We showed that two common risk variants, CFH Y402H and LOC387715 A69S, represent a high risk of AMD also in the isolated Finnish population, and furthermore, that they had a statistical interaction. It was demonstrated that the CFH Y402H risk genotype affects the binding of CFH to CRP thus suggesting that complement indeed plays an important role in the pathogenesis of AMD.

Pharmacogenetics of SLCO1B1: Population genetics and effect on statins

Kohonneiden kolesterolipitoisuuksien alentamisessa käytettävien statiinien hyödyt sydän- ja verisuonisairauksien estossa on vahvasti osoitettu ja niiden käyttö on niin Suomessa kuin muuallakin maailmassa kasvanut voimakkaasti – Suomessa statiininkäyttäjiä on noin 600 000. Statiinilääkitys on pitkäaikaisessakin käytössä melko hyvin siedetty, mutta yleisimpinä haittavaikutuksina voi ilmetä lihasheikkoutta, -kipua ja -kramppeja, jotka voivat edetä jopa henkeä uhkaavaksi lihasvaurioksi. Lihashaittariski suurenee suhteessa statiiniannokseen ja plasman statiinipitoisuuksiin. Statiinien plasmapitoisuuksissa, tehossa ja haittavaikutusten ilmenemisessä on suuria potilaskohtaisia eroja. SLCO1B1-geenin koodaama OATP1B1-kuljetusproteiini kuljettaa monia elimistön omia aineita ja lääkeaineita verenkierrosta solukalvon läpi maksasoluun, mm. statiineja, joiden kolesterolia alentava vaikutus ja poistuminen elimistöstä tapahtuvat pääosin maksassa. Erään SLCO1B1-geenin nukleotidimuutoksen (c.521T>C) tiedetään heikentävän OATP1B1:n kuljetustehoa. Tässä väitöskirjatyössä selvitettiin SLCO1B1-geenin perinnöllistä muuntelua suomalaisilla ja eri väestöissä maailmanlaajuisesti. Lisäksi selvitettiin SLCO1B1:n muunnosten vaikutusta eri statiinien pitoisuuksiin (farmakokinetiikka) ja vaikutuksiin (farmakodynamiikka) sekä kolesteroliaineenvaihduntaan. Näihin tutkimuksiin valittiin SLCO1B1-genotyypin perusteella terveitä vapaaehtoisia koehenkilöitä, joille annettiin eri päivinä kerta-annos kutakin tutkittavaa statiinia: fluvastatiinia, pravastatiinia, simvastatiinia, rosuvastatiinia ja atorvastatiinia. Verinäytteistä määritettiin plasman statiinien ja niiden aineenvaihduntatuotteiden sekä kolesterolin ja sen muodostumista ja imeytymistä kuvaavien merkkiaineiden pitoisuuksia. Toiminnallisesti merkittävien SLCO1B1-geenimuunnosten esiintyvyydessä todettiin suuria eroja eri väestöjen välillä. Suomalaisilla SLCO1B1 c.521TC-genotyypin (geenimuunnos toisessa vastinkromosomissa) esiintyvyys oli noin 32 % ja SLCO1B1 c.521CC-genotyypin (geenimuunnos molemmissa vastinkromosomeissa) esiintyvyys noin 4 %. Globaalisti geenimuunnosten esiintyvyys korreloi maapallon leveyspiirien kanssa siten, että matalaan transportteriaktiivisuuteen johtavat muunnokset olivat yleisimpiä pohjoisessa ja korkeaan aktiivisuuteen johtavat päiväntasaajan lähellä asuvilla väestöillä. SLCO1B1-genotyypillä oli merkittävä vaikutus statiinien plasmapitoisuksiin lukuun ottamatta fluvastatiinia. Simvastatiinihapon plasmapitoisuudet olivat keskimäärin 220 %, atorvastatiinin 140 %, pravastatiinin 90 % ja rosuvastatiinin 70 % suuremmat c.521CC-genotyypin omaavilla koehenkilöillä verrattuna normaalin c.521TT-genotyypin omaaviin. Genotyypillä ei ollut merkittävää vaikutusta minkään statiinin tehoon tässä kerta-annostutkimuksessa, mutta geenimuunnoksen kantajilla perustason kolesterolisynteesinopeus oli suurempi. Tulokset osoittavat, että SLCO1B1 c.521T>C geenimuunnos on varsin yleinen suomalaisilla ja muilla ei-afrikkalaisilla väestöillä. Tämä geenimuunnos voi altistaa erityisesti simvastatiinin, mutta myös atorvastatiinin, pravastatiinin ja rosuvastatiinin, aiheuttamille lihashaitoille suurentamalla niiden plasmapitoisuuksia. SLCO1B1:n geenimuunnoksen testaamista voidaan tulevaisuudessa käyttää apuna valittaessa sopivaa statiinilääkitystä ja -annosta potilaalle, ja näin parantaa sekä statiinihoidon turvallisuutta että tehoa.

Calculating life : Nation, Nature, and the Nativisation of Nonhuman Genetics

In recent decades, nation-states have become major stakeholders in nonhuman genetic resource networks as a result of several international treaties. The most important of these is the juridically binding international Convention on Biological Diversity (CBD), signed at the Rio Earth Summit in 1992 by some 150 nations. This convention was a watershed for the identification of global rights related to genetic resources in recognising the sovereign power of signatory nations over their natural resources. The contracting parties are legally obliged to identify their native genetic material and to take legislative, administrative, and/or policy measures to foster research on genetic resources. In this process of global bioprospecting in the name of biodiversity conservation, the world's nonhuman genetic material is to be indexed according to nation and nationality. This globally legitimated process of native genetic identification inscribes national identity into nature and flesh. As a consequence, this new form of potential national biowealth forms also what could be called novel nonhuman genetic nationhoods. These national corporealities are produced in tactical and strategic encounters of the political and the scientific, in new spaces crafted through technical and institutional innovation, and between the national reconfiguration of the natural and cultural as framed by international political agreements. This work follows the creation of national genetic resources in one of the biodiversity-poor countries of the North, Finland. The thesis is an ethnographic work addressing the calculation of life: practices of identifying, evaluating, and collecting nonhuman life in national genetic programmes. The core of the thesis is about observations made within the Finnish Genetic Resources Programmes in 2004 2008, gathered via multi-sited ethnography and related methods derived from the anthropology of science. The thesis explores the problematic relations of the communal forms of human and nonhuman life in an increasingly technoscientific contemporaneity  the co-production and coexistence of human and nonhuman life in biopolitical formations called nations.

Uniparental DNA markers and forensic genetics in Finland

Over the years, a wide range of methods to verify identity have been developed. Molecular markers have been used for identification since the 1920s, commencing with blood types and culminating with the advent of DNA techniques in the 1980s. Identification is required by authorities in many occasions, e.g. in disputed paternity cases, identification of deceased, or crime investigation. To clarify maternal and paternal lineages, uniparental DNA markers in mtDNA and Y-chromosome can be utilized. These markers have several advantages: male specific Y-chromosome can be used to identify a male from a mixture of male and female cells, e.g. in rape cases. MtDNA is durable and has a high copy number, allowing analyses even from old or degraded samples. However, both markers are lineage-specific, not individualizing, and susceptible to genetic drift. Prior to the application of any DNA marker in forensic casework, it is of utmost importance to investigate its qualities and peculiarities in the target population. Earlier studies on the Finnish population have shown reduced variation in the Y-chromosome, but in mtDNA results have been ambiguous. The obtained results confirmed the low diversity in Y-chromosome in Finland. Detailed population analysis revealed large regional differences, and extremely reduced diversity especially in East Finland. Analysis of the qualities affecting Y-chromosomal short tandem repeat (Y-STR) variation and mutation frequencies, and search of new polymorphic markers resulted a set of Y-STRs with especially high diversity in Finland. Contrary to Y-chromosome, neither reduced diversity nor regional differences were found in mtDNA within Finland. In fact, mtDNA diversity was found similar to other European populations. The revealed peculiarities in the uniparental markers are a legacy of the Finnish population history. The obtained results challenge the traditional explanation which emphasizes relatively recent founder effects creating the observed east-west patterns. Uniparentally inherited markers, both mtDNA and Y-chromosome, are applicable for identification purposes in Finland. By adjusting the analysed Y marker set to meet the characteristics of Finnish population, Y-chromosomal diversity increases and the regional differentiation decreases, resulting increase in discrimination power and thus usefulness of Y-chromosomal analysis in forensic casework.