Zusammenfassung Viele Untersuchungen deuten darauf hin, dass das Surfaktantprotein A (SP-A) sowohl an der Regulation des Surfaktanthaushalts als auch als unspezifisches Opsonin an der Abwehr von Pathogenen in der Lunge beteiligt ist. Zahlreiche Polymorphismen kennzeichnen die Gene der Proteinuntereinheiten SP-A1 und 2. Die häufigste Aminosäuresubstitution Val50Leu befindet sich in der kollagenartigen Domäne, die an den Kollektinrezeptor der Phagozyten bindet. Weitere existieren in der an der Bindung an Lipopolysaccharide, Surfaktantbestandteile und Rezeptoren auf Pneumozyten beteiligten Kohlehydraterkennungsregion (CRD) der globulären Domäne. Träger des schwach exprimierten Wildtypallels 1a0 des SP-A2-Gens haben ein erhöhtes Risiko, am Atemnotsyndrom des Neugeborenen (RDS) zu erkranken. Bei der Alveolarproteinose akkumulieren die hydrophilen Surfaktantproteine A und D in den Alveolen. In der vorliegenden Arbeit wurde eine nested PCR zur isolierten Amplifikation beider SP-A-Gene etabliert. 31 Patienten mit Verdacht auf einen Surfaktantproteindefekt wurden auf neue Restriktionsfragmentlängenpolymorphismen (RFLP) im SP-A1-Gen untersucht. Der in einer Familie konstante NcoI-Polymorphismus 1162C>T in Codon 39 und der NdeI-Polymorphismus 3138T>C in Codon 184 wurden mit einer Allelfrequenz von etwa 11 % detektiert. Die Sequenzen der entsprechenden Allele wurden kloniert. Bei 14 Patienten mit idiopathischer Alveolarproteinose, therapierefraktärem Surfaktantmangel oder rezidivierender Pneumonie wurden die SP-A-Gene sequenziert. Der bisher nur SP-A1 zugeschriebene Aminosäureaustausch Val50Leu wurde als Substitution 1220G>C bei zwei Patienten im SP-A2-Gen nachgewiesen. Drei Patienten mit Alveolarproteinose waren homozygot für die Substitution Gln223Lys in der CRD des SP-A2. Bei einem Patienten handelte es sich möglicherweise um eine somatische Mutation der Leukozyten-DNA im Rahmen einer Leukämie mit sekundärer Alveolarproteinose. Ein anderer war heterozygoter Träger des seltenen Allels 6a4 mit der Aminosäuresubstitution Arg219Trp in der CRD des SP-A1 und hatte die Alveolarproteinose erst im Erwachsenenalter entwickelt. Der dritte war homozygoter Träger des sehr seltenen Allels 1a3 des SP-A2 und verstarb im Alter von 6 Wochen an konnataler Alveolarproteinose (CAP), ohne dass ein bekannter Defekt des SP-B- oder des GM-CSF-Rezeptorgens vorlag. Die SSCP-Analyse konnte allelische Varianten als Einzelstrangkonformationspolymorphismen unterscheiden, war jedoch als Suchtest in heterozygoten Proben zu unspezifisch. Der hohe Gehalt an Polymorphismusinformation (PIC) macht den SP-A-Genort sftp1 zu einem nützlichen Marker bei der Untersuchung der Surfaktantproteine und anderer auf Chromosom 10 lokalisierter Gene. / Abstract Many studies give evidence of the role of surfactant protein A (SP-A) in the regulation of surfactant homeostasis and the defence from pathogens in the lung by opsonisation. The genes for the two protein subunits SP-A1 and SP-A2 are characterised by numerous polymorphisms. The most frequently substituted amino acid Val50Leu is located within the collagen-like region, which is recognised by the collectin-receptor on phagocytes. Further amino acids are substituted in the globular region, which is involved into the binding to lipopolysaccharides, surfactant particles, and receptors on pneumocytes by its carbohydrate recognition domain (CRD). Individuals carrying the weakly expressed wild-type allele 1a0 of SP-A2 have an increased risk of developing the respiratory distress syndrome (RDS) of the new-born. Alveolar proteinosis is a disease with accumulation of the hydrophilic surfactant proteins SP-A and SP-D in the alveoli. In this study a nested PCR for separate amplification of the two SP-A genes has been established. 31 patients with suspected deficiency of a surfactant protein has been investigated for new restriction fragment length polymorphisms (RFLP) in the SP-A1 gene. The NcoI-polymorphism 1162C>T in codon 39, which was constantly inherited in one family, and the NdeI-polymorphism 3138T>C in codon 184 have been detected with an allele frequency of around 11 %. The DNA sequences of these alleles have been cloned. In 14 patients suffering from idiopathic alveolar proteinosis, therapy-refractory surfactant deficiency, or recurrent pneumonia the SP-A genes have been sequenced. The substituted amino acid Val50Leu, which was previously considered exclusively in SP-A1, has been detected in SP-A2 in two patients. Three patients with alveolar proteinosis proved to be homozygous for the substitution Gln223Lys within the CRD of SP-A2. One of these patients might have a somatic mutation in the DNA of his leucocytes, with alveolar proteinosis developing secondary to his leukaemia. Another one developed alveolar proteinosis as an adult and was heterozygous for the rare allele 6a4 which includes the substituted amino acid Arg219Trp in the CRD of SP-A1. The third one proved to be homozygous for the very rare allele 1a3 of SP-A2 and died at 6 weeks of age from congenital alveolar proteinosis (CAP) without having one of the known mutations responsible for this condition within the genes for surfactant protein B (SP-B) or the GM-CSF receptor protein. The allelic variants could be differentiated by single strand conformation polymorphism but the SSCP-analysis was not enough specific for the screening of heterozygous DNA. Due to its high polymorphism information content (PIC), the SP-A gene locus sftp1 is a useful genetic marker for the analysis of the surfactant proteins and other genes located on chromosome 10.

Background: Genome-wide association studies (GWAs) have identified almost one hundred genetic loci associated with variances in human blood lipid phenotypes including very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol and triglycerides. Nevertheless the revealed loci only explain a small fraction of heritability and therefore a subtile phenotype of cholesterol homoestasis was examined in our study for the very first time. Methods and Results: Using a multi-stage approach of a GWA, firstly, a genome-wide analysis (Affymetrix 500K GeneChip) for serum lanosterol and serum total cholesterol using LC-MS/MS was conducted in 1495 participants of the KORA-S3/F3 cohort with subsequent replication in two additional independent samples of the the KORA-S3/F3 cohort (n = 1157) and CARLA cohort (n = 1760). Two genetic variants, SNP rs7703051 and rs17562686, in the HMGCR locus were significantly associated with serum lanosterol and showed similar effects of elevated serum lanosterol for each minor allele (combined n = 4412: p = 1,4 x 10-10, +7,1% and p = 4,3 x 10-6, +7,8%). Furthermore, rs7703051 showed a nominal statistical significance to serum cholesterol (p = 0,04). A combined analysis of both SNPs demonstrated that observed associations of rs17562686 can be partly explained by LD with rs7703051 being the primary polymorphism in that study. Nevertheless, rs17562686 shows consistent independent effects on serum lanosterol, thus being associated to a lipid phenotype for the very first time. The following SNP-fine mapping of the HMGCR locus was carried out in the CARLA cohort with subsequent validation in the LE-Heart cohort (n = 1895). The recently published SNP rs3846662 being in tight LD with rs7703051 could be associated with variances of serum lanosterol in both cohorts and functional in vivo studies of gen expression using qRT-PCR assays demonstrated a highly significant association of higher expression of alternatively spliced HMGCR mRNA lacking exon 13 with homozygosity for the rs3846662 major allele in 51 human liver samples (p < 0,01) and 958 human PBMCs (p = 2,1 x 10-7). The overall HMGCR gen expression was not affected. Further investigation of in vivo HMG-CoA reductase enzyme activity in both human samples (n = 48 and n = 55) using anionic exchange column chromatography and scintillation counting of [3-14C]-HMG-CoA and [5-3H]-mevalonolacton did not show any significant results. In addition there was not any association in the LE-Heart cohort between these SNPs and the development of CAD. Finally, rs7703051 could be replicated for already published total cholesterol (combined n = 4412) and rs3846662 for LDL-cholesterol (LE-Heart n = 1895). Since fine mapping in CARLA showed several SNPs throughout the HMGCR locus being in LD with rs17562686 we performed a DNA sequencing of the extended 5´-HMGCR promotor region in six human liver samples. A unknown SNP was discovered in the promotor but could not be associated with any of the examined phenotypes mentioned above. The minor allele of SNP rs5909 situated next to the stop codon and being in high LD with rs17562686 was associated with elevated serum lanosterol and slightly reduced HMGCR gen expression, but further studies including the above mentioned as well as measurement of 3’-UTR transcript lengths using qRT-PCR assays did not produce significant results. Conclusion: The phenotype serum lanosterol could be associated with genetic polymorphisms (e.g. rs7703051) in the HMGCR locus. Therefore already published associations of HMGCR with total cholesterol and LDL-cholesterol can be explained by variances of cholesterol homeostasis. The SNP rs17562686 could be associated with a phenotype of human blood lipids for the very first time. Subsequent gen expression analyses demonstrated a highly significant association of rs3846662 with variant patterns of HMGCR alternative splicing. A significant effect of alternatively spliced protein on enzyme activity and a association of these SNPs with CAD could not be shown.

Background: Genome-wide association studies (GWAs) have identified almost one hundred genetic loci associated with variances in human blood lipid phenotypes including very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol and triglycerides. Nevertheless the revealed loci only explain a small fraction of heritability and therefore a subtile phenotype of cholesterol homoestasis was examined in our study for the very first time. Methods and Results: Using a multi-stage approach of a GWA, firstly, a genome-wide analysis (Affymetrix 500K GeneChip) for serum lanosterol and serum total cholesterol using LC-MS/MS was conducted in 1495 participants of the KORA-S3/F3 cohort with subsequent replication in two additional independent samples of the the KORA-S3/F3 cohort (n = 1157) and CARLA cohort (n = 1760). Two genetic variants, SNP rs7703051 and rs17562686, in the HMGCR locus were significantly associated with serum lanosterol and showed similar effects of elevated serum lanosterol for each minor allele (combined n = 4412: p = 1,4 x 10-10, +7,1% and p = 4,3 x 10-6, +7,8%). Furthermore, rs7703051 showed a nominal statistical significance to serum cholesterol (p = 0,04). A combined analysis of both SNPs demonstrated that observed associations of rs17562686 can be partly explained by LD with rs7703051 being the primary polymorphism in that study. Nevertheless, rs17562686 shows consistent independent effects on serum lanosterol, thus being associated to a lipid phenotype for the very first time. The following SNP-fine mapping of the HMGCR locus was carried out in the CARLA cohort with subsequent validation in the LE-Heart cohort (n = 1895). The recently published SNP rs3846662 being in tight LD with rs7703051 could be associated with variances of serum lanosterol in both cohorts and functional in vivo studies of gen expression using qRT-PCR assays demonstrated a highly significant association of higher expression of alternatively spliced HMGCR mRNA lacking exon 13 with homozygosity for the rs3846662 major allele in 51 human liver samples (p < 0,01) and 958 human PBMCs (p = 2,1 x 10-7). The overall HMGCR gen expression was not affected. Further investigation of in vivo HMG-CoA reductase enzyme activity in both human samples (n = 48 and n = 55) using anionic exchange column chromatography and scintillation counting of [3-14C]-HMG-CoA and [5-3H]-mevalonolacton did not show any significant results. In addition there was not any association in the LE-Heart cohort between these SNPs and the development of CAD. Finally, rs7703051 could be replicated for already published total cholesterol (combined n = 4412) and rs3846662 for LDL-cholesterol (LE-Heart n = 1895). Since fine mapping in CARLA showed several SNPs throughout the HMGCR locus being in LD with rs17562686 we performed a DNA sequencing of the extended 5´-HMGCR promotor region in six human liver samples. A unknown SNP was discovered in the promotor but could not be associated with any of the examined phenotypes mentioned above. The minor allele of SNP rs5909 situated next to the stop codon and being in high LD with rs17562686 was associated with elevated serum lanosterol and slightly reduced HMGCR gen expression, but further studies including the above mentioned as well as measurement of 3’-UTR transcript lengths using qRT-PCR assays did not produce significant results. Conclusion: The phenotype serum lanosterol could be associated with genetic polymorphisms (e.g. rs7703051) in the HMGCR locus. Therefore already published associations of HMGCR with total cholesterol and LDL-cholesterol can be explained by variances of cholesterol homeostasis. The SNP rs17562686 could be associated with a phenotype of human blood lipids for the very first time. Subsequent gen expression analyses demonstrated a highly significant association of rs3846662 with variant patterns of HMGCR alternative splicing. A significant effect of alternatively spliced protein on enzyme activity and a association of these SNPs with CAD could not be shown.