The historic completion of the Human Genome Project in 2003 and its subsequent refinement undoubtedly marked the beginning of a new era for biomedical research. The Human Genome Project has helped us understand the most precise sequence of the 3 billion base pairs of DNA that make up the human genome. With this development, we have witnessed a paradigm shift in which medicine has become personalized, predictive and preventive, especially in life-threatening diseases like cancer and other chronic diseases. There has been growing interest in extending these successes to “rare diseases” (RDs), which are often progressive, frequently devastating and life-threatening clinical conditions. Although DRs affect a limited fraction of individuals in the general population (1 in 5000 people or less in the Indian context), overall they represent a significant challenge for global health systems.
There are approximately 5,000 to 8,000 different rare diseases in the world, and they come in many forms and include certain cancers, autoimmune diseases, metabolic disorders, blood disorders, neurological disorders, and inherited defects. Available literature suggests that India is home to nearly 70 million people with rare diseases, and some of the common examples are Primary Immunodeficiencies, Hemoglobinopathies, Muscular Dystrophies, Lysosomal Storage Disorders, Niemann-Pick Disease, ethylmalonic encephalopathy, familial hypercholesterolemia, mucopolysaccharidosis type I. and type II, rhizomelic chondroplasia punctata type 1, pseudorheumatoid dysplasia, ichthyosis, dystrophic epidermolysis bullosa, sporadic acrokeratosis, Tay-Sachs syndrome, Von Willebrand disease, Werner’s syndrome, spastic paraplegia 79 and many others. The majority of these diagnosed DRs are so rare that it is extremely difficult to identify them clinically, the patient may need 7 to 8 medical consultations and it sometimes takes several years to arrive at a conclusive diagnosis (Fig-1). Regarding the etiology of DRs, the exact cause of many rare diseases remains unknown. Yet, for a significant part, the problem can be traced to mutations in a single gene (genetic origin). As these conditions are difficult to recognize clinically, genetic and genomic testing has become the backbone of diagnostic modality in recent times. Identifying pathogenic DNA changes remained a great challenge at the time (late 1970s), when basic information about the DNA sequence and genomic location of an abnormal gene had to be worked out through to a tedious, time-consuming and expensive laboratory process called cloning followed by first-generation Sanger sequencing of the cloned product.
Technological advances in DNA sequencing were boosted after 2003, with the introduction of the first high-throughput sequencing technology (Next Generation Sequencing, NGS) by Roche in 2005, capable of detecting genetic variation with high precision. and high accuracy. In no time, NGS became the real game changer and brought about a complete “genome revolution” by providing the ability to read whole genomes, rather than individual genes. This gave researchers the ability to identify potentially pathogenic variants across the genome much faster than before. The past decade and a half has seen rapid advances in NGS technologies in terms of sequencing chemistry, data generation, longer read lengths, and improved bioinformatics tools. This dramatically increased sequencing data outputs into the gigabases range per instrument cycle, resulting in a much more affordable cost compared to Sanger’s traditional first-generation sequencing method, and at a much faster rate. quick.
Today, larger multigene panels (checks a few clinically relevant genes), whole exome sequencing (checks all coding exons in the genome, WES), clinical exome sequencing (checks approximately 6000 known genes for cause human diseases, CES) and whole genome sequencing (verifies all exons and non-coding regions of the genome, WGS), are the most preferred testing methodologies in the diagnosis of DR. Patients are now increasingly freed from the long-standing diagnostic bottleneck. Many can be diagnosed in just 2-3 months using modern genomic tools, compared to 7-8 years traditionally, with an accuracy that remains unmatched in medicine.
Typically, a referring physician will consider high-end genomic testing, if a patient comes to their clinic with overlapping nonspecific symptoms and unexplained illness, or known to have multiple affected family members, or couples who have undergone a consanguineous marriage. Also, many pediatricians prefer to perform genomic testing if they see the early onset of the disease in a child or repeated episodes of seizures in newborns. As shown in (Fig-1), the strategy begins with collecting detailed clinical information about the patient and their family history. Based on clinical suspicion and prior knowledge of the molecular etiology of suspected disorders, two different approaches are considered for genetic screening. In cases of clinically suspected but genetically characterized DR, screening for known candidate genes associated with the disease through a targeted multigene panel or a clinical exome-based NGS approach is recommended.
On the other hand, in case of clinically suspected but genetically uncharacterized DR, the WES or WGS method is adopted. Some examples of the use of the above strategy include the diagnosis of Duchenne muscular dystrophy (DMD) in a negative deletion/duplication case, in which pathogenic variants of the dystrophin gene have been identified using a targeted NGS panel with a success rate of up to 100%. Similarly, the diagnosis of lysosomal storage disorders (LSD), characterized by an overlapping disease phenotype and the involvement of multiple genes, requires clinical or whole-exome sequencing for faster diagnosis. Similarly, disease associated with mitochondrial disorders is extremely difficult to diagnose due to overlapping phenotypes and the involvement of multiple systems. In such cases, mitochondrial whole genome sequencing together with nuclear gene sequencing using exome sequencing is the best practical approach to establish the definitive diagnosis. Whole exome sequencing has also aided in the identification of pathogenic variants for the rapid diagnosis of skeletal dysplasias, spastic ataxia and progressive pseudorheumatoid dysplasia. It is now very well established that in the clinical setting, depending on the type of disease and the selection of patients, it is estimated that exome sequencing leads to a diagnosis in 30 to 50% of rare Mendelian diseases.
Finally, whole genome sequencing (WGS) is the most comprehensive test to detect multiple types of variants at once across the entire human genome. Of all genomic testing methods, WGS offers the highest probability of finding a diagnosis, and as a result, a change in patient management has been reported in 49-75% of pediatric outpatients who received a diagnosis. by WGS. A rare disease like beta-propeller protein-associated neurodegeneration (BPAN) and Sawyer’s disease has been diagnosed due to the identification of pathogenic variants in WDR45 and TRIP12 genes respectively using the WGS approach.
Thanks to this recent technological advance in genomics, the number of Mendelian diseases with a known genetic cause has increased from 1,257 in 2001 to 4,589 in 2022. The genome revolution has opened the door to true personalization in disease management, and it is now truly improving people’s health. With several success stories emerging around the world, including in India, genomics will become a mainstay in the diagnosis of rare genetic diseases in the near future.
by Dr. Firoz Ahmad, Section Head, Molecular Pathology, SRL Diagnostics, Mumbai
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