So, correlating genotype and phenotype. So there's a way to describe a particular genotype responsible for a particular phenotype. So allelic heterogeneity, different variants in a gene resulting in the same phenotype. This is really common, right? So different mutations resulting in a particular gene disorder. This is all, you know, like the familial hypercholesterolemia, different mutations can result in very similar phenotype of high cholesterol, and so forth. Locus heterogeneity describes variants in different genes causing the same phenotype. This is also commonly seen, the familial hypercholesterolemia, the LDL receptor gene causes familial hypercholesterolemia, which is the most well-known, but PCSK9 and APOB gene, certain regions of it can cause hypercholesterolemia, you know, familial hypercholesterolemia. So that's the locus heterogeneity, different locus causing the same disease. So clinical or phenotypic heterogeneity, different mutation in a gene causing different phenotype. So these are called allelic disorders. So Duchenne muscular dystrophy is one type, but Duchenne muscular dystrophy and Becker muscular dystrophy, they are caused by mutations in DMD gene, Duchenne muscular dystrophy gene, and also like laminate mutations. Some causes cardiomyopathy, some causes muscular dystrophy, some causes progeria, lipodystrophy, neurological disorders as well. So, you know, the same gene, but depending on the mutation and what's surrounding there, or, you know, like the modifier effect or environment that phenotype of one gene changes. So that's clinical phenotypic heterogeneity, allelic disorders, and that's very interesting. So how do we identify DNA sequence? So Sanger sequence is the gold standard of sequencing. It's a radio-labeled dideoxynucleotide sequencing on the gel electrophoresis. So this was a very tedious method, and I've done it myself with a radio-labeled dideoxynucleotide gel, and you have to have a straight gel. Sometimes it gets crooked, so the lines get... This is a really pretty gel that's printed out on a film, but mine was not this pretty. Sometimes like crooked, it's difficult to read. We have to read it from the bottom up. But then fluorescent-labeled dideoxynucleotide on capillary electrophoresis was developed and detected by laser-induced fluorescent readouts. It's so pretty, different, like ribbon-like readouts that come out with different colors and easy to read the sequence, and this was a tremendous advancement at the time, I thought. I've done the fluorescent-labeled dideoxy electrophoresis as well for sequencing. So I figured this is so amazing, because everything was already in the kit. You just have to add the DNA, and that was it. So turn of the century, next-generation sequencing was developed. This changed the outlook of molecular genetics tremendously. I couldn't believe this could have happened. When I was learning about genetics, I could not have imagined. So they fragment DNA into multiple pieces, and they add the unique sequences and adapters, and then you kind of gather known adapters together. They reassemble this sequence, and genomic sequence would be reassembled together to read the sequence. And you are able to read multiple genes at the same time, and all automated and easy to do, and it takes much shorter time, and cost has been declining. So sequencing has gotten so much easier to order for clinical testing as well. So that whole-exome sequencing and whole-genome sequencing are available. So whole-exome sequencing basically focuses on protein-coding regions of the genome, and 85% of known disease variants are included in there. And then comprehensive coverage of coding variants, such as single nucleotide variants, or some insurgent deletions. There might be a limit, but some insurgent deletions are detectable with the whole-exome sequencing. So whole-genome sequencing is amazing. It can analyze the whole entire genome, covering coding, non-coding, mitochondrial DNA, information, single nucleotide, structural, and copy number variant, including translocation. So it can do so much. However, it requires sophisticated bioinformatic expertise, and more cost and time required for analysis. But I've heard recently that the whole genome can be done within a month or two. So that's incredible. It would have taken us like over a year to finish the genome sequence the way we were doing before. So this next-generation sequencing, the technology development has changed the outlook of genetic field for sure. So when you examine such a big region of the genome, there's always going to be unexpected identification of the variant, which is not expected, and which wasn't the intention of performing whole-genome or whole-exome sequencing. American College of Medical Genetics provides on their website 73 genes that should be identified as secondary finding, because these genes or variant have, or diseases have, some type of preventative measures or medications that can be implemented to prevent a disease or ameliorate the severity of the disease. So these 73 genes, if the patient would like to be disclosed, they can be disclosed to the patient when they order whole-exome or whole-genome sequencing. So the advantage of whole-exome or whole-genome allows for identification of novel genetic variants. So it's great for research as well, because it would not have been possible without next-generation sequencing to identify novel variants so easily.

genotype

phenotype

allelic heterogeneity

locus heterogeneity

DNA sequencing