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.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Examination of Genetic Components Affecting Human Obesity-Related Quantitative Traits

Obesity increases the risk for several conditions, including type 2 diabetes mellitus, cardiovascular disease, hypertension, osteoarthirits and certain types of cancer. Twin- and family studies have shown that there is a major genetic component in the determination of body mass. In recent years several technological and scientific advance have been made in obesity research. For instance, novel replicated loci have been revealed by a number of genome wide association studies. This thesis aimed to investigate the association of genetic factors and obesity-related quantitative traits. The first study investigated the role of the lactase gene in anthropometric traits. We genetically defined lactose persistence by genotyping 31 720 individuals of European descent. We found that lactase persistence was significantly correlated with weight and body mass index but not with height. In the second study we performed the largest whole genome linkage scan for body mass index to date. The sample consisted of 4401 twin families and 10 535 individuals from six European countries. We found supporting evidence for two loci (3q29 and 7q36). We observed that the heritability estimate increased substantially when additional family members were removed from the analyses, which suggests reduced environmental variance in the twin sample. In the third study we assessed metabonomic, transcriptomic and genomic variation in a Finnish population cohort of 518 individuals. We formed gene expression networks to portray pathways and showed that a set of highly correlated genes of an inflammatory pathway associated with 80 serum metabolites (of 134 quantified measures). Strong association was found, for example, with several lipoprotein subclasses. We inferred causality by using genetic variation as anchors. The expression of the network genes was found to be dependent on the circulatory metabolite concentrations.

Methicillin Resistance In Staphylococci : Horizontal Transfer of Mobile Genetic Element (SCCmec) Between Staphylococcal Species

Regardless of the existence of antibiotics, infectious diseases are the leading causes of death in the world. Staphylococci cause many infections of varying severity, although they can also exist peacefully in many parts of the human body. Most often Staphylococcus aureus colonises the nose, and that colonisation is considered to be a risk factor for spread of this bacterium. S. aureus is considered to be the most important Staphylococcus species. It poses a challenge to the field of medicine, and one of the most problematic aspects is the drastic increase of the methicillin-resistant S. aureus (MRSA) strains in hospitals and community world-wide, including Finland. In addition, most of the clinical coagulase-negative staphylococcus (CNS) isolates express resistance to methicillin. Methicillin-resistance in S. aureus is caused by the mecA gene that encodes an extra penicillin-binding protein (PBP) 2a. The mecA gene is found in a mobile genomic island called staphylococcal chromosome cassette mec (SCCmec). The SCCmec consists of the mec gene and cassette chromosome recombinase (ccr)gene complexes. The areas of the SCCmec element outside the ccr and mec complex are known as the junkyard J regions. So far, eight types of SCCmec(SCCmec I- SCCmec VIII) and a number of variants have been described. The SCCmec island is an acquired element in S. aureus. Lately, it appears that CNS might be the storage place of the SCCmec that aid the S. aureus by providing it with the resistant elements. The SCCmec is known to exist only in the staphylococci. The aim of the present study was to investigate the horizontal transfer of SCCmec between the S. aureus and CNS. One specific aim was to study whether or not some methicillin-sensitive S. aureus (MSSA) strains are more inclined to receive the SCCmec than others. This was done by comparing the genetic background of clinical MSSA isolates in the health care facilities of the Helsinki and Uusimaa Hospital District in 2001 to the representatives of the epidemic MRSA (EMRSA) genotypes, which have been encountered in Finland during 1992-2004. Majority of the clinical MSSA strains were related to the EMRSA strains. This finding suggests that horizontal transfer of SCCmec from unknown donor(s) to several MSSA background genotypes has occurred in Finland. The molecular characteristics of representative clinical methicillin-resistant S. epidermidis (MRSE) isolates recovered in Finnish hospitals between 1990 and 1998 were also studied, examining their genetic relation to each other and to the internationally recognised MRSE clones as well, so as to ascertain the common traits between the SCCmec elements in MRSE and MRSA. The clinical MRSE strains were genetically related to each other; eleven PFGE types were associated with sequence type ST2 that has been identified world-wide. A single MRSE strain may possess two SCCmec types III and IV, which were recognised among the MRSA strains. Moreover, six months after the onset of an outbreak of MRSA possessing a SCCmec type V in a long-term care facility in Northern Finland (LTCF) in 2003, the SCCmec element of nasally carried methicillin-resistant staphylococci was studied. Among the residents of a LTCF, nasal carriage of MR-CNS was common with extreme diversity of SCCmec types. MRSE was the most prevalent CNS species. Horizontal transfer of SCCmec elements is speculated to be based on the sharing of SCCmec type V between MRSA and MRSE in the same person. Additionally, the SCCmec element of the clinical human S. sciuri isolates was studied. Some of the SCCmec regions were present in S. sciuri and the pls gene was common in it. This finding supports the hypothesis of genetic exchange happening between staphylococcal species. Evaluation of the epidemiology of methicillin-resistant staphylococcal colonisation is necessary in order to understand the apparent emergence of these strains and to develop appropriate control strategies. SCCmec typing is essential for understanding the emergence of MRSA strains from CNS, considering that the MR-CNS may represent the gene pool for the continuous creation of new SCCmec types from which MRSA might originate.

Mutation analysis of TGF-β signaling pathway genes among Finnish patients with primary pulmonary hypertension and hereditary hemorrhagic telangiectasia

Primary pulmonary hypertension (PPH), or according to the recent classification idiopathic pulmonary hypertension (IPAH), is a rare, progressive disease of pulmonary vasculature leading to pulmonary hypertension and right heart failure. Most of the patients are sporadic but in about 6% of cases the disease is familial (FPPH). In 2000 two different groups identified the gene predisposing to PPH. This gene, Bone morphogenetic protein receptor type 2 (BMPR2), encodes a subunit of transforming growth factor β (TGF-β) receptor complex. There is a genetic connection between PPH and hereditary hemorrhagic telangiectasia (HHT), a bleeding disorder characterized by local telangiectasias and sometimes with pulmonary hypertension. In HHT, mutations in ALK1 (activin like kinase type 1) and Endoglin, another members of the TGF-β signaling pathway are found. In this study we identified all of the Finnish PPH patients for the years 1986-1999 using the hospital discharge registries of Finnish university hospitals. During this period we found a total of 59 confirmed PPH patients: 55 sporadic and 4 familial representing 3 different families. In 1999 the prevalence of PPH was 5.8 per million and the annual incidence varied between 0.2-1.3 per million. Among 28 PPH patients studied, heterozygous BMPR2 mutations were found in 12% (3/26) of sporadic patients and in 33% of the PPH families (1/3). All the mutations found were different. Large deletions of BMPR2 were excluded by single-stranded chain polymomorphism analysis. As a candidate gene approach we also studied ALK1, Endoglin, Bone Morphogenetic Receptor Type IA (BMPR1A or ALK3), Mothers Against Decapentaplegic Homolog 4 (SMAD4) and Serotonine Transporter Gene (SLC6A4) using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. Among patients and family members studied, we found two mutations in ALK1 in two unrelated samples. We also identified all the HHT patients treated at the Department of Otorhinolaryngology at Helsinki University Central Hospital between the years of 1990-2005 and 8 of the patients were studied for Endoglin and ALK1 mutations using direct sequencing. A total of seven mutations were found and all the mutations were different. The absence of a founder mutation in the Finnish population in both PPH and HHT was somewhat surprising. This suggests that the mutations of BMPR2, ALK1 and Endoglin are quite young and the older mutations have been lost due to repetitive genetic bottlenecks and/or negative selection. Also, other genes than BMPR2 may be involved in the pathogenesis of PPH. No founder mutations were found in PPH or HHT and thus no simple genetic test is available for diagnostics.

Search for genetic variants influencing human height

Human growth and attained height are determined by a combination of genetic and environmental effects and in modern Western societies > 80% of the observed variation in height is determined by genetic factors. Height is a fundamental human trait that is associated with many socioeconomic and psychosocial factors and health measures, however little is known of the identity of the specific genes that influence height variation in the general population. This thesis work aimed to identify the genetic variants that influence height in the general population by genome-wide linkage analysis utilizing large family samples. The study focused on analysis of three separate sets of families consisting of: 1) 1,417 individuals from 277 Finnish families (FinnHeight), 2) 8,450 individuals from 3,817 families from Australia and Europe (EUHeight) and 3) 9,306 individuals from 3,302 families from the United States (USHeight). The most significant finding in this study was found in the Finnish family sample where we a locus in the chromosomal region 1p21 was linked to adult height. Several regions showed evidence for linkage in the Australian, European and US families with 8q21 and 15q25 being the most significant. The region on 1p21 was followed up with further studies and we were able to show that the collagen 11-alpha-1 gene (COL11A1) residing at this location was associated with adult height. This association was also confirmed in an independent Finnish population cohort (Health 2000) consisting of 6,542 individuals. From this population sample, we estimated that homozygous males and females for this gene variant were 1.1 and 0.6 cm taller than the respective controls. In this thesis work we identified a gene variant in the COL11A1 gene that influences human height, although this variant alone explains only 0.1% of height variation in the Finnish population. We also demonstrated in this study that special stratification strategies such as performing sex-limited analyses, focusing on dizygous twin pairs, analyzing ethnic groups within a population separately and utilizing homogenous populations such as the Finns can improve the statistical power of finding QTL significantly. Also, we concluded from the results of this study that even though genetic effects explain a great proportion of height variance, it is likely that there are tens or even hundreds of genes with small individual effects underlying the genetic architecture of height.

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.

Novel Multiple Sclerosis Predisposing Genetic Variants Outside the HLA Region

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Both environmental factors and several predisposing genes are required to generate MS. Despite intensive research these risk factors are still largely unknown, the pathogenesis of MS demyelination is poorly understood, and no curative treatment exists. Both prevalence and familial occurrence of MS are exceptionally high in a Finnish population subisolate, Southern Ostrobothnia, presumably due to enrichment of predisposing genetic variants within this region. Previous linkage scan on MS pedigrees from Southern Ostrobothnia detected three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in other populations have also provided independent evidence for the location of MS susceptibility genes in these regions. Further, these loci are syntenic to the experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents. In this thesis work an effort was made to localize MS predisposing alleles of the linked loci outside the HLA region by studying familial MS cases from the Southern Ostrobothnia isolate. Analysis of the 5p locus revealed one region, flanking the complement component 7 (C7) gene. The identified relatively rare haplotype seems to have a fairly large effect on genetic susceptibility of MS (frequency MS 12%, controls 4%; p=0.000003, OR=2.73). Evidence for association with alleles of the region and MS was seen also in more heterogeneous populations. Convincingly, plasma C7 protein levels and complement activity correlated with the risk haplotype identified. The finding stimulated us to study other complement cascade genes in MS. No evidence for association could be observed with the complement component coding genes outside 5p. A scan of the 17q locus provided evidence for association with variants of the protein kinase C alpha (PRKCA) gene (p=0.0001). Modest evidence for association with PRKCA was observed also in Canadian MS families. Finally we used a candidate gene based approach to identify potential MS loci. Mutations of DAP12 and TREM2 cause a recessively inherited CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located in MS regions on 6p and 19q, and we tested them as potential candidate genes in the Finnish MS sample. No evidence for association with MS was observed. This thesis provides an example of how extended families from special populations can be utilized in fine-mapping of the linked loci. A first relatively rare MS variant was identified utilizing the strength of a Finnish population subisolate. This variant seems to have an effect on activity of the complement system, which has previously been suggested to have an important role in the pathogenesis of MS.

Molecular Genetic Background of Tumours in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.

Genetic studies on recurrent miscarriage

Recurrent miscarriage (RM) is defined as three consecutive pregnancy failures and is estimated to affect ~1% of couples trying to conceive. The cause of RM remains unknown in approximately 50% of cases. In this study, it was hypothesized that some of the underlying factors yet to be discovered are genetic. The aim was to search for mutations in genes AMN, EPCR, TM, and p53 known to cause miscarriage in mouse models and thereby find new genetic causes for unexplained miscarriages in humans. In addition, the mitochondrial genome was studied because mitochondria are involved in processes important in early development. Furthermore, sex chromosome characteristics suggested to underlie miscarriage were also studied. A total of 40 couples and 8 women with unexplained RM were collected for this study and screened for mutations in the candidate genes. Six interesting exonic or potential splice site disrupting variations were detected. However, their phenotypic effects cannot be determined without further investigations. Additionally, an association between the C11992A polymorphism of the p53 gene and RM was detected. The results indicate that women carrying the C/A or A/A genotype have a two-fold higher risk for RM than women with a C/C genotype. This strengthens the results of previous studies reporting that p53 sequence variations may cause miscarriage. The role of variation C11992A in embryonic development is, however, difficult to predict without further studies When screening the mitochondrial genome a heteroplasmic mtDNA variation was found in an unexpected high number of women, as heteroplasmic variations are reported to be rare. One novel variation and 18 previously reported polymorphisms were detected in the mitochondrial genome. Although the detected variations are likely to be neutral polymorphisms, a role in the aetiology of miscarriage cannot be excluded as some mtDNA variations may be pathogenic only when a threshold is reached. Recent publications have reported skewed X chromosome inactivation and Y chromosome microdeletions to be associated with RM. Therefore, these sex chromosome abnormalities in the context of RM were investigated. No associations between skewed X chromosome inactivation or Y chromosome microdeletions and RM in the Finnish patients were detected. Data on ancestral birthplaces of the patients were collected to study any possible geographic clustering, which would indicate a common predisposing factor. The results showed clustering of the birthplaces in eastern Finland in a subset of patients. This suggests a possibility of an enriched susceptibility gene which may contribute to RM.