[MUSIC] I would now like to present you methods to identify genetic genetic loci affecting diabetes and pre diabetes traits. When you are considering the methods, the most important thing probably is planning the study. Here you should think about the study design, will you focus on association studies, should it be linkage studies in families where you look for co-segregation of genetic low side with a trait of the disease. Or should it for example, be studies of the effect of treatment of the patients? You should think about how much DNA is needed for the genetic studies. You should think about the phenotypes you would like to examine. Should it be more that eight and six, probably, it should. You would like to have information of body mass, you would like to have information for example on beta cell function on insulin sensitivity, on lipids, on blood pressure et cetera. You should make sure that you will get informed consent from the patients, and you should make sure that you have approval for your studies by the local ethical committee. Then, when you have all the approval in the place you can start the actual study, and for the genetics studies you need to obtain the material which you can actually use for studying the genes. And normally we use EDTA full blood, which you then can store and later extract DNA. Alternatives could be immortalized cell lines, DNA from clotted blood, cheek swaps, or dried blood. When you have the blood you should process it. If it is an EDTA blood sample, you could leave it at room temperature for several days before you proceed. You can freeze the entire sample at minus 80. Or you can, as many times, you can centrifuge the sample. You can pipette the white blood cells, what we call the buffy coat, and store that at -80 indefinitely. Then there are several methods to extract the DNA, there are called methods, there are salt-out precipitation methods, and others. But when you have extracted the DNA is important that you do the quality control, that could be measurement of the amount of DNA, there are many many different methods. One method is measuring the Optical Density at the 200 wavelength, 260 and 280 and look at the ratio. You could run an agarose gel and you could do a PCR reaction to check that your DNA actually works. Then you can store the, the DNA. The primary stock solution could be stored in a concentration typically between 100 and 200 nanogram per millilitre, at minus 20 or minus 80. And you can have a working solution which often is between five to 20 nanogram per millilitre, and that is in TE buffer and you can keep that and when you're working with it, in a refrigerator at 4 degrees Celsius. So now you are ready to start your genetic studies. And what are we looking for? We are looking normally in the entire human genome, which is comprised by more than 3 billion base pairs. Only 1 to 1.5% of the human genome is protein coding. And then there are outside the protein coding regions, other important elements that could be non-coding, regions which are coding for non-coding RNA, it could regulatory regions. It could be introns, and it could be non-coding RNA. What we often are looking at when we are doing the genetic studies, that is the single nucleotide polymophism. And what do we mean by a single nucleotide polymophism? That is, that's for approximately each 1000 base pairs, there is a common variation in the population. And as you can see at the upper part of this figure, there is a double helix of DNA and there is a C, C nucleotide at the red part of the DNA strand, and at the lower part and part of the population, this has been shifted to a T. So part of the population for each 1,000 base pairs there would be common variants. The variant with the lowest frequency we call the minor allele variant and usually these variations, they are what what we call biallelic, sib pair to achieve but sometimes they could be for example three allelic. Could be C, T, or A at this specific position, for example. When we're searching for the underlying genetics of diabetes, there're two main types of genetics variation we are looking at. There's a single nucleotide polymorphism SNP and structural variation. The SNP they could be outside of the gene, and there they might be regulatory of the expression of genes, and thereby causing disease, but many of them will just be nonfunctional. It could also be inside of the genes, where there could be reasons or non-synonymous, that means the change in amino acids. It could be silent or synonymous, they do not change amino acid. Or they could for example, be on the splice site where they create or destroy a splice site, and thereby the function of the protein. Then there is, a huge number of structural variation that could be a change of the number of nucleotides. There could be a change in translation in the reading frame. And then there might be a disruptions of entire exons of genes. So, when we want to dissect diabetes, we should also consider which statistical genetic methods we would like to use. There's the linkage approach, that is normally, we are starting large families affect the [UNKNOWN] pairs or we will then families looking, are looking for quantitative traits. Then there's the candidate gene approach, where we have selected a gene which we believe if it is mutated, then it will cause disease and we can choose these genes, for example from biochemical or biological knowledge. It could be from expression experiments, it could be from animal models and it could, for example be for Bioinformatics studies. Then there is the Genome-wide association studies which I will tell you more about. This is recent technology where we can look at thousands of variants and compare between cases and controls, and look for quantitative traits in the population. And very recent there is the next generation sequencing technologies where we also can look at a different forms of variation and look for their association to disease. The linkage approach is primarily used, to identify monogenetic forms of disease. Sometimes also complex forms. A candidate gene approach is used both for monogenetic and complex disease. The Genome-wide association disease studies, are mainly used for complex disease and the next generation sequencing methods are used both for monogenetic and complex disease. [MUSIC] [BLANK_AUDIO]
