The New Biology Enters the Generalist Pediatrician’s Office:Lessons from the Human Genome Project (1999)

Birth defects are the leading cause of infant mortality in the United States, representing more than 20%of all infant deaths. This infant mortality rate from birth defects exceeds that from sudden infant death syndrome, low-birthweight/short gestation, respiratory distress syndrome,and maternal complications. In addition, birth defects and genetic diseases represent major sources of morbidity for those who survive. As our ability increases to care effectively for those who have infectious diseases and other acute illnesses,individuals who have chronic illnesses due to genetic etiologies represent an increasing proportion of patients seen in the general pediatrician”s office.The Human Genome Project was initiated on October 1, 1990, and has a projected funding period of 15 years. The goal is to sequence the entire human genome,representing three billion base pairs that contain the coding sequences for approximately 75,000 genes.During the latter half of this century, investigations into the genetics of disease gathered increasing momentum. In addition to fundamental investigations into human genetics, technologic tools were developed that permitted large-scale genomic sequencing. These tools included the polymerase chain reaction (PCR), which permits amplification of hundreds of thousands or even millions of copies of DNA and requires only limited sequence data for its success; automated DNA sequencing, which allows increased sequence processing and decreased cost compared with manual methods; and improved information systems, which permit sophisticated analysis and assembly of the three billion base pairs of DNA in the human genome. Thus, the Human Genome Project represents the current chapter in our understanding,but it is neither the first nor the final chapter in this story. Once we know the sequences of all of the human genes, we must learn their functional roles in human development and disease pathogenesis.The Human Genome Project has been referred to as the “moon shot of biology.” Not only does this biomedical initiative represent a significant funding source, but like the space program, it will lead to major technologic advances. The project will have a far-reaching influence on the daily practice of medicine that will extend into all physicians”offices. Through home care products and services, the effects of this initiative will be evident in each of our residences. The ability to manage huge genome sequence databases in ways that will make them accessible to professionals and the public will be another area of impact. Individual patients and their families already can search the Internet and quickly become knowledgeable about specific disorders. In addition, the existence of massive amounts of basic science information about human genetic diseases requires that information gained from the project be mined assiduously, so that new knowledge and existing technology can be combined to improve the prevention, diagnosis, and management of disease.Identification of genes permits not only improved diagnosis, but a better understanding of the pathogenesis of disease. Strategies for gene identification may be developed with traditional approaches, such as targeting specific steps in the pathogenesis of a disease. Alternatively, a genetic approach may be used that does not proceed from a preconceived view of the function of the gene product.Cloning genes by the candidate gene approach is the traditional method for gene identification. This strategy requires knowledge of the pathogenesis of the disease and an understanding of the function of one or more proteins that may be involved in the disease. These gene candidates are targeted for mutation identification, and once a mutation in one of the genes is identified in a patient who has the disease, the gene is recognized as responsible for the disease.Osteogenesis imperfecta, which is associated with frequent and often severe pathologic fractures that may be misdiagnosed as child abuse,represents a group of clinical disorders for which the genes were identified by the candidate gene approach. The pathology and pathogenesis of osteogenesis imperfecta indicated that the altered genes for these disorders would result in abnormalities in the integrity of connective tissue. Investigations in the 1970s involving cultured cells and tissues from patients who had osteogenesis imperfecta documented abnormalities of collagen biosynthesis in affected patients. Decreased procollagen mRNA and collagen structural mutations subsequently were identified. Normal collagen molecules twist together to form a much stronger fibrous element, like the strands of a rope, that provides structural stability to bones and connective tissues. A mutation in a collagen gene alters one or more strands in the fiber, weakening these structural elements and predisposing the bones to pathologic fractures. Because of this mutation of the collagen gene, the bones may break with only minimal trauma, such as that which occurs during a normal vaginal delivery, or with no apparent trauma other than the simple acts of normal living, such as walking,standing, or coughing.Positional cloning does not require knowledge of the pathogenesis of a particular disease. The investigator determines the chromosomal region likely to contain the gene for the disease by linkage analysis or chromosomal rearrangements, then develops a physical map of this genomic region made up of a series of overlapping contiguous genomic sequences referred to as a “contig”. Coding sequences for expressed genes in this region of the genomic DNA are identified and characterized. Mutation analysis among the expressed sequences in this region will determine which of the genes is responsible for the disease under investigation.Cystic fibrosis is a disease for which the responsible gene was identified by positional cloning without any chromosomal rearrangement to indicate the specific region of the genome that contained the responsible gene. The genomic region of interest was localized to the long arm of chromosome seven(7q) by linkage analysis that involved evaluation of inheritance patterns of genetic markers within families that contained individuals who had cystic fibrosis. Subsequently, a contig was developed within this chromosomal region, and open reading frames (ORFS),representing the coding sequences for genes, were identified. Mutations were found in a gene for a protein that had the features of a membrane channel, indicating that this channel,referred to as the cystic fibrosis transmembrane regulator (CFTR),was involved in controlling the movement of chloride across the cell membrane.The positional candidacy approach involves determining the chromosomal region most likely to contain the genes for the disease of interest,just as for positional cloning. Once the region is identified, the expressed genes in that region can be searched for ones that appear to be likely candidates. Mutation analysis among these candidate genes leads to identification of the specific gene causing the disease.Marfan syndrome, which is associated with connective tissue disorders that involve bones, ligaments,and arterial walls, is one example of the positional candidacy approach that demonstrates the speed of this process. In 1986, fibrillin, a 350 kDa glycoprotein, was observed by immunohistochemistry to be decreased in patients who had Marfan syndrome. In 1990, the Marfan syndrome locus was linked to a specific region on the long arm of chromosome 15 (15q15-q23). In 1991, the fibrillin gene (FBN-1) and the Marfan syndrome locus were colocalized; during that same year,fibrillin gene mutations were identified in patients who had Marfan syndrome.With the increasing sequence information available from the Human Genome Project, laboratories now routinely turn to the computer before they approach the laboratory bench. Some refer to this process as“cyber-cloning.” The investigator initially searches a variety of databases for genomic DNA and expressed gene sequences in the chromosomal region of interest. For example, an investigator who is trying to identify a gene associated with mental retardation may search a genomic database for sequences characteristic of genes that may be expressed in the brain. Alternatively,the investigator might examine a database of expressed sequence tags(ESTs) for the brain for ones that are fragments of transcribed genes identified from individual tissues and specific developmental stages. Once a potential gene or an EST is identified that maps to the genomic region of interest, it can be used as a probe in the laboratory to identify the full-length cDNA for that gene from a library in the tissue of interest, such as the brain for mental retardation. Mutation analysis of DNA samples from family members affected with the disease is used to determine which of the genes in the region is responsible for the disease of interest. The sequence databases that already have been generated by the Human Genome Project have accelerated the process of gene identification dramatically, from periods of years to only weeks or months in many cases.The Human Genome Project is nearly two thirds through its originally planned 15-year life span and is considered to be ahead of schedule at this time. A consortium of private organizations, building on the information and technology developed by this government-sponsored project, recently announced a proposed strategy for completion of this initiative in 2001,4 years earlier than originally planned by federally funded research. In response, the National Institutes of Health-funded Human Genome Project plans a complete draft of the human genome in 2000,with the total sequence available shortly thereafter. Investigators already are considering how the information that derives from the completed Project can be used in the postgenomic era.The area of functional genomics will include analyses of the activities of gene products for which only sequence information may be known. The temporal expression of these genes and their gene products will provide a better understanding of their role in developmental biology, an area of critical importance to pediatrics in improving our insight into the origins of and potential treatments for birth defects. We also will begin to explore the basis of individual variation and the subtle sequence differences involving one or several genes that are responsible for complex conditions such as heart disease, cancer, and diabetes. Already we have begun to recognize that not all changes in an individual”s DNA will lead inevitably to a specific disease. Rather, it may place that individual at increased risk for one of these complex diseases.Technologic advances from the Human Genome Project, such as rapid amplification of small quantities of DNA by PCR over time periods measured in minutes rather than hours, will permit “real-time”genetic diagnosis. DNA chips containing tens or hundreds of thousands of sequence probes developed for rapid DNA sequencing and gene expression analysis will permit rapid diagnosis of genes for common,complex genetic diseases.Among the consequences of the intense investigational effort that is part of the Human Genome Project has been recognition that not all genetic diseases are caused by simple point mutations. Novel mechanisms can involve deletion of a section of DNA that contains several genes, expansion of triplet repeat sequences in any part of a gene, and alterations in certain genes that are differentially expressed from the maternal or paternal allele.Contiguous gene syndromes involve the loss of a section of chromosomal DNA that contains two or more contiguous genes. Frequently, the clinical recognition of the syndrome precedes the discovery of its chromosomal etiology. Cytogenetic detection of the DNA deletion may require high-resolution chromosome analysis. However, not all deletions may be detected cytogenetically,suggesting that some affected individuals have submicroscopic abnormalities. Eventually, as the contiguous gene syndrome is dissected into the individual genes responsible for the phenotype, specific features of the syndrome will be recognized to occur as simple mendelian conditions.One of the earliest recognized contiguous gene syndromes is complex glycerol kinase deficiency, in which a deletion of a region of the short arm of the X-chromosome(Xp21.3) may range from one megabase (one million base pairs) or fewer to more than 10 megabases. Affected boys have involvement of the gene for the enzyme glycerol kinase that leads to glycerol kinase deficiency, which is associated with episodes of acidemia and central nervous system deterioration with or without hypoglycemia. They also have involvement of the contiguous loci for the Duchenne muscular dystrophy gene, which causes muscular pseudohypertrophy and progressive muscle weakness, and/or the gene for X-linked adrenal hypoplasia congenita, which results in adrenal cortical insufficiency and hypogonadotropic hypogonadism. In addition,these patients may have bony abnormalities, mental retardation, or both,suggesting involvement of additional genes that have yet to be identified in this region of the short arm of the X-chromosome.Anticipation is a clinical characteristic of a genetic disorder in which the disease severity increases in succeeding generations. It is caused by the expansion of triplet nucleotide repeats in the DNA sequence for that gene. This triplet repeat expansion may alter the expression of the gene or if the triplet represents the codon for an amino acid within the coding sequence of the gene,expanded stretches of that amino acid may alter the function of the protein product.Fragile X syndrome is the prototypic disorder associated with anticipation in which the severity of the phenotype increases over subsequent generations. Classic fragile X syndrome is associated with large head size, rapid growth rate, prominent jaw, large ears, macro-orchidism,and significant behavioral abnormalities and mental retardation. Less characteristic physical features,behavioral disturbances, and intellectual impairment may be seen in adults in the family. What was recognized under the microscope as a fragile site, or an apparent disruption of the normal chromosomal architecture at the tip of the long arm of the X chromosome, now is known to be due to expansion of a triplet repeat. As the triplet repeat expansion increases in length, the expression of the FMR-1 gene containing the triplet repeat is decreased and the severity of the mental retardation and somatic phenotype is increased.The central dogma of human mendelian genetics includes the following statements: The normal individual has 23 pairs of chromosomes;we received one copy of each of the chromosomes and, therefore, one copy of each paired gene from our father and one copy from our mother; and the maternal and paternal copies of the genes are expressed equally. However, this is not true for all genes; both copies of some genes may not be expressed in every cell at all times during development. An imprinted gene is one for which there is differential expression of the maternal or paternal copy of the gene. Imprinted genes may be paternally expressed and maternally silenced or maternally expressed and paternally silenced.Two syndromes are due to alterations in imprinted genes in the same chromosomal region, the long arm of chromosome 15 (15q11-q13):Prader-Willi syndrome, associated with severe hypotonia in infancy as well as uncontrolled eating, obesity,and mental retardation among older children and adults; and Angelman syndrome, associated with uncontrolled and inappropriate laughing and mental retardation. The Prader-Willi syndrome is caused by either paternal deletion of this portion of the long arm of chromosome 15 or maternal uniparental disomy (ie, the paternal DNA in this region or perhaps the entire chromosome has been lost and there are two copies of the maternal chromosomal DNA). The Angelman syndrome is caused by either maternal deletion of this portion of chromosome 15 or paternal uniparental disomy. Uniparental disomy may be caused by abnormalities in germ cell such as with of the by loss of one of the chromosomes the paternal chromosome is the individual will have Prader-Willi if the maternal chromosome 15 is individual will have Angelman syndrome. Because both Prader-Willi and Angelman syndromes result from of genes on chromosome which is an there is no the of the and these both boys and can have Prader-Willi or Angelman syndrome. 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