CHAPTER 20
Pediatric Genomic Medicine
Moin Vera, MD, PhD, and Henry J. Lin, MD
CASE STUDY
A 4-year-old boy with moderate global developmental delay is brought to his pediatrician’s office for evaluation. The patient has an unremarkable family history and normal physical examination findings. Previous evaluation included normal karyotype and fragile X syndrome DNA test results. The patient’s parents would like to know whether there is anything else that can be done to determine the etiology of the delay. In addition, his mother has recently read about companies that offer multiple genetic tests to consumers and wonders whether these tests will be useful as well.
Questions
1. What is microarray technology, and how is it useful in pediatric practice?
2. How is next-generation sequencing technology affecting current practice?
3. What are the limitations of these new technologies?
4. What is direct-to-consumer genetic testing?
The National Human Genome Research Institute at the National Institutes of Health describes genomic medicine as the incorporation of an individual’s genomic information into clinical care. In this way, care involves diagnosis, therapeutic decision-making, health outcomes, and policy implications. The Human Genome Project (HGP) was an extensive, broad-based, and multidisciplinary research effort to develop knowledge of biology and disease—leading to the potential for so-called precision medicine, based on genome sequence information. The first essentially complete human sequence was published in 2003. The cost of sequencing a human genome has decreased from approximately $100 million in 2003 to about $1,000 in 2018, according to National Human Genome Research Institute data.
Whole exome sequencing (WES) uses next-generation sequencing to focus on exons, the regions of the genome that contain the actual DNA code for making proteins. Overall, the exons amount to about 1.5% of the human genome. Whole exome sequencing costs less than whole genome sequencing, and the functional significance of variants within exons is easier to interpret. Whole exome sequencing or whole genome sequencing is an appropriate study when a single-gene (or mendelian) disorder is suspected.
Chromosomal microarray analysis—compared with standard chromosome analysis—dramatically increased diagnostic yields for patients with intellectual disability, autism spectrum disorder (ASD), and multiple congenital anomalies. Genetic testing via gene sequencing panels (eg, 20–1,000 genes) and WES have driven diagnostic yields still further. Direct-to-consumer genetic testing is also available, designed to provide information without physician input. Pharmacogenomics, which promises individualized drug therapy based on genomic data, is moving toward applications to common diseases.
Epidemiology: Human Genome Anatomy
Human cells have 2 haploid genomes (ie, 23 pairs of chromosomes, 1 pair of most genes), each containing 3 billion base pairs with an estimated 20,000 to 25,000 protein-encoding genes, plus a variable number of copies of the mitochondrial genome. On average, 2 humans share 99.9% of their DNA. There are at least 10 million single nucleotide polymorphisms (SNPs), which are single base changes that are present in a substantial percentage of the population (>1%). A small percentage of SNPs fall within known coding or regulatory regions of genes and directly influence gene function. The remaining SNPs may have unclear effects on gene function, but they may be inherited in recognizable patterns (haplotypes) with other SNPs.
Recently, it has become evident that most human DNA variation is not represented by SNPs. Instead, copy number variants, in which DNA segments containing up to several million base pairs are duplicated or deleted, account for a substantial portion of variation among individuals. Approximately 12% of the genome can exhibit copy number variation, but the effect of such variation on individual phenotypes is unknown.
Autosomal recessive disorders are caused by pathogenic variants in each of the 2 copies of a disease-associated gene. Carriers of such disorders, with only 1 variant or disease allele, are typically asymptomatic. Although most autosomal recessive disorders are rare, most humans are carriers of several different recessive disease alleles. In a 2011 study, an average of 2.8 recessive variants was observed per person, among 448 genes involved in severe autosomal recessive disorders.
Pathophysiology: Genotype and Phenotype Correlations and Environment
Genomic data provide information about the genes on which pathogenic mutations are found. But many diseases are caused by a combination of genetic susceptibility and environmental factors. The first examples of such interactions were monogenic conditions, such as complement deficiency (which predisposes patients to bacterial meningitis) or mendelian cancer syndromes (which cause extreme radiation sensitivity). However, the gene-environment connection is now recognized to be more complex. For example, certain environmental conditions have been found to cause DNA methylation, a mechanism for gene silencing without changing the DNA sequence. These so-called epigenetic changes may persist across generations. As an example, mothers exposed to wartime famine may birth children who are predisposed to conservation of energy. When these children are fed a typical American diet, they are prone to development of obesity and diabetes. Also, fetal cells that persist in maternal circulation (fetal microchimerism) may play a role in tumor prevention and susceptibility to autoimmune disease. Finally, our bodies contain more bacteria than human cells, and the interaction of the bacterial and human genomes is thought to play an important role in the development of some diseases.
Clinical Presentation, History, and Physical Examination
A thorough history and physical examination will continue to be critical components of patient assessment in the genomic age of medicine. Large databases will be needed to correlate human genotypes with corresponding phenotypes defined by the patient’s clinical presentation. Even when genetic and epigenetic sequencing is commonplace, the only way to measure the effect of the disease on the individual is by clinical assessment.
Laboratory Tests
Microarray testing, which includes a wide range of different technologies, has had a dramatic effect on the evaluation of common pediatric conditions, such as intellectual disability and ASD. Comparative genomic hybridization (CGH) microarray testing uses closely spaced DNA probes to detect chromosomal deletions or duplications at 100 to 10,000 times the resolution of standard karyotyping. The diagnostic yields for patients with intellectual disability and multiple congenital anomalies has increased from 3% to 4% to 15% to 20%, using this technology. For example, patients with ASD with normal karyotypes may have microdeletions or microduplications of 16p11.2. Several relatively common genetic conditions, such as 1p36 deletion syndrome, a form of severe intellectual disability that affects 1 in every 5,000 to 10,000 newborns, have been delineated through the use of microarray testing.
Single nucleotide polymorphism microarray testing has largely replaced CGH. It detects the same chromosomal imbalances as CGH, but SNP microarray testing can pinpoint regions of homozygosity, which represent identical sequences on both copies of a chromosome. The information may be useful to consanguineous couples, because it suggests areas where abnormal autosomal recessive disorder genes may be found. In addition, SNP microarray testing can detect some cases of uniparental isodisomy (inheritance of 2 identical copies of a chromosome from 1 parent) and may suggest the presence of an imprinting disorder, such as Prader-Willi syndrome.
Although microarray testing has largely replaced standard karyotyping, microarray testing cannot detect carriers of balanced translocation or patients with mosaicism (when the proportion of abnormal cells is <25%–30%). Some deletions or duplications detected by microarray testing are benign variants. Therefore, lack of availability of parental samples may hinder interpretation of an abnormal finding in a child.
Next-generation sequencing is a term used to describe methods for parallel sequencing of billions of base pairs at relatively low costs. Whole exome sequencing focuses on the 1% of the genome that encodes proteins and has been used for clinical testing in the past several years. A 2013 report shows a diagnostic yield of 25% for this technology in 250 samples studied. Whole genome sequencing has also become clinically available, although perhaps large-scale discovery of variation in regulatory elements located outside the coding regions has yet to be fully realized. These technologies have created a major paradigm shift, by decreasing the time to diagnosis and averting many costly and invasive procedures, such as muscle biopsy. Improvements in the technology and the bioinformatics used to interpret results are expected to increase the use of these tools.
Next-generation sequencing methods have certain limitations that should be addressed as technology progresses. Some genes, particularly those with high guanine-cytosine content, are not well captured or sequenced with current high-volume technologies. Exome sequencing cannot capture triplet repeat conditions, such as fragile X syndrome. Microduplications and microdeletions (of exons) are not normally detected with current technologies and must be assessed separately, using a microarray.
A patient’s next-generation sequencing results will have thousands of differences from reference sequences. Bioinformatic algorithms must be used to sift through the data and determine the changes that are potentially relevant to the patient’s condition. Parental testing often provides an essential reference but may not always be available. The testing laboratory classifies variants found into 5 categories: pathogenic, likely pathogenic, variants of unknown significance, likely benign, and benign. Family counseling may be limited for variants of unknown significance. Technology will drive down the price of sequencing over time, but the cost of bioinformatics will dominate the price of these new technologies as the amount of data increases.
Direct-to-Consumer Genetic Testing
Several commercial testing companies now market genetic tests directly to consumers. These tests purport to provide disease risk information by analyzing multiple SNPs along with common disease mutations. However, the tests may lack sensitivity and specificity, because analyses are based on limited genetic markers without family history or phenotype information. Many consumers (and their physicians) are ill-equipped to understand the results, and patients of color may have indeterminate results. These companies argue that consumers have the right to know their genetic information, and some offer genetic counseling services. In 2010, the US Food and Drug Administration decided to develop regulations for the sale of direct-to-consumer genetic tests.
Management: Pharmacogenomics
Individualized pharmacological treatment has always been 1 of the goals of the HGP. Although pharmacogenomics is a fairly young field, several tests are available that can reduce the risk of an adverse drug reaction. Patients with variant thiopurine methyltransferase alleles may experience severe toxicity to azathioprine and 6-mercaptopurine. Children with a 1555A>G mutation in the mitochondrial genome are susceptible to aminoglycoside-induced hearing loss, even after a single dose of an aminoglycoside antibiotic. Pharmacogenomic treatment of common diseases (eg, asthma) is an active area of investigation and may allow for a more rational choice of drug regimens.
Future Developments
The huge potential of the HGP now influences several areas of medicine, including pediatrics. During the next decade, genomics will likely continue to revolutionize the diagnosis of rare or previously uncharacterized mendelian disorders. The impact of genes on common diseases, such as atherosclerosis, is being investigated through ever larger genomewide association studies and polygenic risk scores. Although molecular diagnoses of previously unidentified diseases have increased over the past several years, our understanding of disease pathogenesis and treatment has not kept pace with the explosion in information. It is unclear how individuals will use genomic information to improve health— whether by altering lifestyles or by use of precision drugs, or both.
In addition, whole exome and whole genome sequencing create various ethical issues. If a patient presents with heart disease and testing shows an increased risk for Alzheimer disease, should this information be returned to the patient? It is certain that pediatricians will need to familiarize themselves with genomic medicine, because the number of tests—and patients seeking testing—will exceed the availability of medical genetics specialists.
| Glossary | |
Autism spectrum disorder | A medical condition resulting in deficits in social communication and social interaction and characterized by restricted, repeated interests and behaviors starting in early childhood. Other formal criteria are also used to establish a diagnosis. |
Comparative genomic hybridization | A chromosomal microarray method in which tens of thousands of DNA probes for regions along the genome can be used to detect chromosome deletions or duplications at 100 to 10,000 times the resolution of standard karyotyping. Comparative genomic hybridization has largely been replaced by single nucleotide polymorphism microarray methods. The DNA probes (for binding to patient DNA) are immobilized on glass slides—called microarrays. |
Copy number variant | Variants in the structure of the genome having different numbers of copies of DNA segments (usually between 1 kilobase and 5 megabases long). Copy number variants form a large part of human DNA diversity, including causes of some genetic conditions. |
Human Genome Project | The worldwide effort to determine the DNA sequence of the Homo sapiens genome (3 billion bases). The project was launched in the United States by the US Department of Energy and the National Institutes of Health. Efforts were also started in France, the United Kingdom, and Japan. Other countries joined later (eg, Germany, China). The project ran from approximately 1988 to 2003. A review of the effort stated: “For everyone, this achievement represents a major turning point in our quest to learn how all the components of the human genome interact and contribute to biological processes and physiological complexity.” |
National Human Genome Research Institute | One of the 27 institutes and centers of the National Institutes of Health. It was established in 1989 and is “devoted to advancing health through genome research.” |
Single nucleotide polymorphism | The most common and simplest type of DNA polymorphism, in which 1 base is changed to another. These polymorphisms occur roughly every 1,000 base pairs in the genome. Those that occur in or around genes may change the amino acid sequence of the encoded protein, may produce or remove a stop codon, may impair the usual processing (splicing) of the messenger RNA, may change how the gene is controlled, or may have no effect at all. |
Single nucleotide polymorphism (SNP) microarray testing | A chromosomal microarray method that has largely replaced comparative genomic hybridization. It may contain a few million oligonucleotide probes (approximately 25 nucleotides long) for regions along the genome. In addition to detecting chromosome deletions and duplications, SNP microarray testing can also detect long regions of homozygosity, indicating uniparental disomy or consanguinity. The diagnostic utility of chromosomal microarray testing among children with intellectual disability, autism spectrum disorder, and congenital anomalies has been estimated to be 10% to 20%. |
Whole exome sequencing (WES) | Sequencing all the known coding regions in the genome. The diagnostic utility of WES among children with intellectual disability, autism spectrum disorder, and congenital anomalies has been estimated to be at least 30% to 40%. |
CASE RESOLUTION
The patient’s microarray testing results show a small microdeletion in chromosome 6. Parental testing indicates that the microdeletion is present in the patient’s father, who has had normal development. Further testing includes WES, which shows a potential missense mutation in CASK, a gene on the X chromosome that may cause developmental delay. This alteration is not found in the patient’s mother, implying that it is most likely deleterious. The parents receive genetic counseling about future pregnancies.
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