Introduction to the Molecular Biology of the Cell

Outstanding technological advances over the past decade have allowed the comparison of human individual genomes by analyzing tens of thousands of single-letter variations, termed single-nucleotide polymorphisms (SNPs). In addition to SNPs, another type of structural genetic variations was discovered in 2004: large chromosomal regions ranging from tens to hundreds of kilobases (kb) in length were deleted, duplicated, or inverted. These large-scale structural variations, called copy-number variations (CNVs), are less common than SNPs, but they occupy up to 13% of the human genome. Moreover, variable number tandem repeats (VNTRs), such as microsatellites (also known as short tandem repeats [STRs]), minisatellites, and satellites, have been used to map human disease genes within families using a linkage approach. For the last two decades, great progress has been made in identifying specific gene mutations that result in monogenic disease, caused by defects in a single gene. Examples of monogenic diseases include cystic fibrosis, Huntington’s disease, and various cardiovascular disorders (CVDs), such as atherosclerosis, hypertrophic and dilated cardiomyopathies, familial forms of hypertension, long QT syndrome, and structural anomalies of the heart and great vessels. However, a number of human disorders—called complex or polygenic diseases—are caused by defects in several genes. Multiple genetic loci interact with each other and with a variety of environmental factors to produce highly heterogeneous disease phenotypes.

The field has been further revolutionized with the development of a novel powerful tool—genome-wide association studies (GWAS)—that allows scanning of genomic variants in tens of thousands of individuals to identify associations between specific chromosomal loci and complex human diseases. These technological advancements have uncovered previously unsuspected common genetic variants that underlie the risk of complex diseases such as coronary artery disease, type 2 diabetes, and stroke.

Another breakthrough technology, next generation sequencing (NGS), enables whole-genome sequencing. This technique relies on massively parallelized sequencing of millions of short DNA fragments from the human genome combined with unique imaging and data analysis. NGS has successfully been applied to identify variants that cause genetically heterogeneous CVDs, such as long QT syndrome and hypertrophic and dilated cardiomyopathy. All of these new advances will be examined in this chapter.

Keywords

cardiovascular disorders; monogenic disease; tandem repeats; polygenic diseases; single-nucleotide polymorphisms; copy-number variations; GWAS; next-generation sequencing

Nucleic Acids, Genes, Chromatin, and Chromosomes

The central dogma of molecular genetics—DNA → RNA → protein—defines a principal flow of genetic information in all living organisms (Figure 1.1). It also introduces the key macromolecules of the cell—nucleic acids (deoxyribonucleic and ribonucleic acids; DNA and RNA, respectively) and proteins—which define all unique features of any living cell.

The central hereditary molecule, DNA, is a long, unbranched polymer chain composed of four different building blocks, deoxyribonucleotides. The deoxyribonucleotides contain the purine bases, adenine (A) and guanine (G), and the pyrimidine bases, cytosine (C) and thymine (T). The bases are attached to the sugar (deoxyribose)-phosphate chain, in which the 5’ carbon of one deoxyribose group is linked by a phosphodiester bond to the 3’ carbon of the next (Figure 1.2). DNA is a very long molecule; the length of a typical mammalian DNA is approximately 3×109 base pairs (bp). The number of different possible sequences in such molecule is very large: 43×109!

DNA is composed of two such antiparallel strands that entwine, forming a right-handed helical structure with the sugar-phosphate backbone on the outside and the bases on the inside of the double helix. A vital characteristic of the DNA molecule is complementary base pairing between two strands: a larger purine base A or G on one strand pairs via hydrogen bonds with a smaller pyrimidine base T or C, respectively, on the other strand (Figure 1.2). Pairing between A and T involves two hydrogen bonds, whereas pairing between G and C is slightly stronger and involves three hydrogen bonds.

RNA is also a polymer composed of a linear sequence of four nucleotides; however, unlike DNA, T is replaced by uracil (U), and the sugar–phosphate backbone contains ribose instead of deoxyribose. Moreover, in contrast to DNA, RNA is a single-stranded molecule; but it contains regions that form double-helical structures via complementary base pairing, in which A pairs with U instead of T.

The gene is the fundamental unit of inheritance and represents a region of DNA that carries genetic information for a polypeptide and/or RNA in a form of the linear sequence of nucleotides. The organism’s total DNA content, the sum of all genetic information, represents its genome. The genome size of prokaryote Escherichia coli is 4.6×106 bp and lower eukaryotic unicellular organism Sacharomyces cerevisiae is 12.1×106 bp, whereas the human genome contains 3.2×109 bp.

A typical eukaryotic protein-coding gene is composed of regulatory noncoding regions, which flank the coding regions. The completion of the Human Genome Project revealed that regulatory regions, called cis-regulatory elements, can be located not only near the coding regions but many thousands of bases away from them, sometimes in introns of neighboring genes. In addition, it has also been discovered that the human genome is full of overlapping genes. Cis-regulatory elements provide the binding sites for trans-acting regulators, transcription factors, and regulators. Cis-regulatory regions include the promoters, the DNA sequences that are recognized and bound by the transcription machinery to transcribe the coding region, and terminators, the regions at the 3’ end of the gene to terminate the movement of the transcription machinery. Additional cis-regulatory elements include enhancers and silencers, which can significantly modulate the rate of gene transcription. In humans, most of the coding regions of the protein-coding genes are composed of exons, which encode the fragments of polypeptide chains, interspersed with noncoding introns. Surprisingly, it has recently been demonstrated that protein-coding exons from one genome region combine with exons from another distant region located hundreds of thousands of bases away, with multiple other genes between them.1–3 The Human Genome Project demonstrated that exons account for only approximately 2% of the genome, whereas introns account for 8–10% of the genome.4 Thus the vast majority (up to 90%) of the genome appears not to be essential; however, emerging evidence strongly suggests that a significant fraction of this so-called junk DNA is transcribed generating several types of regulatory RNAs, which control the expression of the coding genes.5,6 The precise roles and the mechanism of action of these regulatory RNAs are largely unknown. In light of this high complexity, which was not anticipated, developing a precise, single definition of a “gene” is a challenging task.7,8

The human cell contains approximately 2 m of genomic DNA, which is packaged in a highly compact configuration inside the cell nucleus. Eukaryotic genomic DNA is organized into a DNA–protein complex called chromatin.9 The long genomic DNA chain is arranged in arrays of nucleosomes, the basic structural units of chromatin. Each nucleosome contains 147 bp of DNA, wrapped in 1.7 superhelical turns around a core histone octamer, consisting of the histones H2A, H2B, H3, and H4, and is connected by 20–50 bp of linker DNA with neighboring nucleosomes.10–12 The nucleosomes form a “beads on a string” structure, also known as the 10 nm fiber. The linker histones H1 and H5 interact with linker DNA and contribute to less characterized, higher-order chromatin compaction, forming the so-called 30 nm chromatin fiber. Multiple nonhistone proteins assist in further...