From peapods to laboratory medicine: molecular diagnostics of inheritable diseases

Gregor Johann Mendel, a botanist and plant experimenter, was born in 1822 in Moravia (todays Czech Republic) and died in 1884. He became an Augustinian monk in 1843 and later studied at the University of Vienna. In 1856, working in his monasterys garden, he began the experiments that led to his formulation of the basic principles of heredity. Unfortunately for Mendel, he achieved fame only after his death, through the works of Correns, Tshermak, von Seysenegg and de Vries, who independently obtained similar results and rediscovered the experimental data and general theory that Mendel had published 34 years previously.

The study of genetics, born from the curiosity of a monk (now referred to as the genius of genetics) 147 years ago, exploded into the fastest area of growth in laboratory medicine. Today, discoveries of new genetic markers of disease frequently appear in newspaper and magazine headlines. The time from discovery to clinical application is measured in months, not decades. The modern laboratorian must become conversant with the terminology of molecular diagnostics and proficient in understanding and meeting the growing demands of its clinical application and performance.

In 1999, a task force defined genetic tests as the analysis of human DNA, RNA, chromosomes, proteins and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes or karyotypes for clinical purposes.1 There are a great many potential targets and laboratory methods that can be employed in genetic testing. This article is limited to molecular genetics, the clinical application of molecular diagnostics in the analysis of DNA by clinical laboratories. (Although the term diagnostics should be strictly applied to tests that are diagnostic, the more widely accepted definition includes the full spectrum of laboratory tests that may be applied for screening, diagnosis, treatment selection, monitoring and prognosis.)

In the past decade, with the availability of technology technology and knowledge fueled by the investment and interest in the Human Genome Project, molecular diagnostics has recently enabled laboratories to offer diagnostic and predictive tests for inherited disorders.2 The technology will continue to become easier to apply and more affordable. Large amounts of genetic information can be determined at increasing efficiencies. Many of the challenges of more widespread use of molecular diagnostics are listed in
Table 1 (see below).

Table 1. Challenges for widespread application of molecular diagnostics

Defining the appropriate circumstances for ordering
  • Developing therapies that correct or address specific genetic defects or genetic risk factors
  • Developing consensus on standards of care

Educating physicians and patients concerning the potential information from testing

  • Providing access to affordable testing and services
  • Providing adequate controls to prevent discrimination at work, within communities and for insurance coverage

Performing tests on platform technologies that are easy to control

  • Developing adequate proficiency testing and consistency among laboratories
  • Training a sufficient number of medical technologists to perform highly complex testing

Interpreting the results in the context of the clinical history and other results

  • Integrating results with other family members and ethnicity, while complying with HIPAA
  • Providing adequate genetic counseling support for physicians and patients

Obtaining adequate reimbursement for performing the tests and the potential liability

  • Obtaining necessary regulatory approval and coding for reimbursement
  • Convincing carriers and payers of the merits of providing payment coverage


Historically, genetic testing focused on single-gene disorders, where a disease is caused by a mutation in one gene. The classic example is sickle cell anemia. A single nucleic acid base change is responsible for the sickle cell trait, when inherited from one parent, or sickle cell disease when the nucleic acid base change is inherited from both parents. Although molecular diagnostics is available, sickle cell disease is most often diagnosed with hemoglobin electrophoresis.
Table 2 (see below) lists other genetic diseases for which a single-gene mutation is associated with disease. Excluded from this table are genetic mutations associated with increased risk of morbidities, such as factor V (Leiden) mutation.

Table 2.
Examples of single-gene disorders and their associated genes and

HD 1
in 1,000 (carriers); 30,000
the disease
oncogene (multiple endocrine neoplasia type-2, MEN-2)
RET Approximately
200 new cases of hereditary MTC annually
(C282Y, H63D)
to 0.5% are homozygous; 10% to 15% of European-Americans are
adenomatous polyposis of the colon
APC 2.3
to 3.2 per 100,000 (prevalence)
in 25 Caucasians (carriers); 1 in 2,500 Caucasians (disease)
in 40 Ashkenazi Jews are carriers (heterozygous)
X syndrome
FMR1 The
full mutation appears in approximately 1 in 3,600 males; 1
in 4,000 to 6,000 females
PI (ATT) The
Z mutation is carried in 1 in 50 European-Americans; the S
mutation is carried in 1 in 25 people 
Neurofibromatosis NF 1 , NF
in 10,000 (prevalence)
FBN1 1
in 3,000 to 10,000 (prevalence)
in 5,600 to 14,000 (prevalence)
DM 1 1
in 8,000
polycystic kidney disease
in 500 to 1,000 (prevalence)


For single-gene disorders, mutation analysis is useful in diagnosis, confirmation of diagnosis, predisposition, prognosis and family planning. As with Huntingtons disease, the identification of the gene and availability of testing opened another door to additional social and medical issues. In contrast, testing an individual with multiple endocrine neoplasia type-2 (MEN-2) for the RET proto-oncogene can avoid medullary carcinoma if the individual is positive and undergoes a prophylactic thyroidectomy.

Huntingtons disease gene

Dr. George Huntington, along with his father and grandfather, published their observations in 1872 on familial cases of chorea near their home on Long Island, NY. The genetic disorder they described is now known as Huntingtons disease (HD). Nearly 1% of Americans has HD or is at risk of passing along the disease to a child. HD affects as many people as hemophilia, cystic fibrosis or muscular dystrophy combined. In 1993, the HD gene was isolated and, eventually, a direct genetic test was developed that can accurately determine carrier status for the HD gene. The HD gene was found to contain a specific section with a pattern of so-called trinucleotide repeats which is expanded in people with HD. In most cases, the repeated pattern occurs 30 times or less. In HD, it occurs more than 40 times.

The test cannot predict when symptoms will begin, and therapy is palliative. In the absence of effective treatment and a cure, most individuals at risk elect not to take the test. HD is one of many trinucleotide expansion diseases characterized by genetic anticipation. This phenomenon manifests when a genetic disease either presents earlier, or presents with more severe symptoms in subsequent generations. For example, myotonic dystrophy has a broad spectrum of presentation varying from congenital myotonia to the seventh decade of life. Because of genetic anticipation, early and accurate diagnosis is vital for parents who may have minimal or late onset symptoms but can have children who are more severely affected.

The advent of pre-implantation genetics allows for the detection of embryos that are homozygous for inheritable recessive disorders. Through detection of a mutation of the dystrophin gene in a fertilized cell, a couple can avoid bringing to term a child with Duchennes muscular dystrophy. Approximately 30 other diseases like Huntingtons disease, Tay-Sachs, cystic fibrosis and familial dysautonomia can be diagnosed on an embryo prior to implantation in the womb.

2 RET proto-oncogene

Multiple endocrine neoplasia, type 2 (MEN 2) presents as two syndromes, 2A and 2B. In MEN 2A, medullary thyroid carcinoma (MTC) involving the thyroid interstitial C-cells is ultimately found in some 90% to 95% of affected individuals, pheochromocytoma in 25% to 50% and hyperparathyroidism in 15% to 20%. MEN 2B is characterized by MTC, pheochromocytoma, mucosal neuromata and marfanoid habitus.

Classical linkage studies initiated in the early 1980s led to mapping of the MEN 2 gene to a centromeric chromosome 10 locus in 1987. Mutations of the RET proto-oncogene causative for MEN 2 were identified in 1993. Clinical laboratory testing became available a few years later.3

The discovery of molecular abnormalities of the
RET proto-oncogene in MEN 2 and FMTC has significantly improved clinical management of these disorders. The identified mutations are responsible for 90% to 95% of all hereditary MTC. Six to seven percent of patients with apparent sporadic MTC have been found to have germline mutations of RET indicative of hereditary MTC. Application of these tests in the management of MTC has improved diagnostic accuracy, lessened the likelihood that hereditary disease will be missed in the context of apparent sporadic MTC and improved the clinical management of this disease.

Many of the common disorders that plague humans are multifactorial gene disorders, including cardiovascular disease, diabetes and most types of cancer. Some genes associated with these disorders may indicate increased predisposition, just as total cholesterol and LDL cholesterol serve as important but not exclusive predictors of cardiovascular disease. With additional studies, more genes are being identified with behaviors. These genes may become more important in the future as we develop a fuller understanding of the impact of genetic factors on behavioral disorders and characteristics.
Table 3 (see below) lists examples of common disorders with multifactorial genes.

3. Examples of common disorders with multifactorial genes

onset diabetes mellitus
Breast and
ovarian cancer
ras, TP53, HNPCC, others (see
gene, HFE)


Common disorders for which intensive research is underway include cardiovascular disease, diabetes, cancer, neurological diseases (including Alzheimers), bipolar disease, osteoporosis and behavioral disorders. The diagnostic tests that will be developed will complement current and future tests, including those involving proteomics and metabolic analysis. Many of these diagnostic tests may not live up to initial expectations and others may exceed them. Academic centers and large reference laboratories will be on the forefront of this frontier. The prospectors and pioneers will be followed by a growing group of laboratories as molecular diagnostic tests are more widely accepted and adopted. This is the same pattern that was observed with testing for HIV.

Factor V (Leiden) mutation

The factor V (Leiden) mutation (1691G>A) occurs primarily in the Caucasian population and is a major risk factor for venous thrombosis (a lifetime risk of 12% to 30% in affected individuals) and a lesser risk factor for arterial thrombosis (cardiovascular disease). Additionally, factor V (Leiden) mutation is associated with arterial thrombosis (especially in smokers), complications of pregnancy (including fetal loss) and increased levels of factor VIII.

The factor V (Leiden) mutation leads to the laboratory finding of activated protein C resistance (APCR) and a sevenfold increase in venous thromboembolic events in heterozygous individuals and an eightyfold increase in homozygous subjects. Due to a synergistic increase in venous thrombosis risk, individuals heterozygous for the factor V mutation are at greater risk when taking oral contraceptives. When a heterozygous mutation is coupled with oral contraceptive use, risk increases synergistically to thirtyfold.

Since laboratory tests for APCR are highly sensitive, specific and simpler to perform, APCR is usually the test of choice; however, factor V mutation analysis is recommended to confirm positive APCR tests. It is also recommended in place of APCR for patients with lupus anticoagulant, since such patients often have a false-positive APCR test. Although this test is highly specific, identification of a mutation may occur in the absence of APCR in rare cases. Sensitivity of this test for APCR is 94%; thus, a negative result does not rule out APCR or an increased risk of venous thrombosis.

More than half of thromboembolic events associated with factor V (Leiden) mutation occur in the presence of additional risk factors, such as surgery and use of oral contraceptives. Thus, factor V (Leiden) mutation is a risk factor and not an indication of thromboembolic disease.5

Cystic fibrosis gene, CFTR

Cystic fibrosis (CF) is one of the most commonly inherited diseases in the United States, affecting one infant out of every 3,300 live births. Those affected have high levels of sodium and chloride (salt) in their sweat. More importantly, a thick, sticky mucous in the lungs causes persistent coughing, wheezing and frequent lung infections, including pneumonia.

In 1989, a research team led by Dr. Francis Collins who was at the University of Michigan and Lap-Chee Tsui and John Riodan at Torontos Hospital for Sick Children discovered the gene responsible for cystic fibrosis. The protein is the cystic fibrosis transmembrance conductance regulator (CFTR). (Dr. Francis Collins was instrumental in the discovery of the genes for neurofibromatosis and Huntingtons disease. Dr. Collins is now widely known as the second director of the National Center for Human Genome Research, following Dr. James Watson, who first served in that role. The Center led the federal effort in the Human Genome Project.)

Since the discovery of the most common mutation that causes cystic fibrosis, approximately 1,000 additional mutations have been identified. The National Institutes of Health convened a Consensus Development Conference on cystic fibrosis in 1997. Following the conference, the American College of Medical Genetics and the American College of Obstetricians and Gynecologists, in conjunction with the National Human Genome Research Institute, formed a steering committee to implement strategies to make cystic fibrosis testing standard medical practice.6 Given that approximately 10 million Americans are carriers for cystic fibrosis and 30,000 have the disorder, testing for cystic fibrosis carrier status prior to conception (or if necessary, early after conception) is currently recommended. While cystic fibrosis is not curable, there are some treatments that greatly increase the life span and quality of life for patients with CF. Today, cystic fibrosis mutational analysis is the most commonly performed molecular diagnostic test for inheritable disorders.

Hemochomatosis gene,

Hemochomatosis is an excess accumulation of iron that causes damage to organs, leading to such diseases as cirrhosis, cardiomyopathy, diabetes and arthritis. Two mutations in the HFE gene C282Y and H63D are associated with hemochromatosis.7 Disease develops in less than 1% of individuals with these genotypes. These mutations have low penetrance. Other factors, such as diet, hepatotoxins and likely other genes, are important factors that lead to hemochromatosis. The role of testing may be limited to family members of individuals with hemochromatosis. Even for these individuals, biochemical testing remains the cornerstone for diagnosis.

Many molecular diagnostic tests may be similar to the HFE gene tests that have a limited role and must be interpreted in the context of family, clinical history, and other risk factors and laboratory tests.

Colon cancer gene,

Dr. Bert Vogelstein generated enormous excitement 15 years ago when he and his colleagues at Johns Hopkins described a series of genetic alterations leading to colorectal cancer,8 that occurs in the different phases of cancer development, starting from normal epithelium and moving through adenomatous polyps to cancer. This suggested a genetic pathway in tumor development. Unfortunately, the number and nature of the genetic alterations may vary in a population. There may be a spectrum of routes that describes the genetic alterations leading to cancer. The interaction with environmental factors, including diet and other genetic factors, is open for exploration. Today, genetic testing for colorectal cancer is quite limited.

Beyond the complexity of genetic sequencing, with rigorous quality control and assurance, is the difficulty in interpreting the results. As with HIV genotyping, some mutations have no known clinical significance and others impart antiretroviral drug resistance. Sometimes, two mutations act to cancel out the impact of one another. Likewise, some mutations in the gene associated with Gauchers disease cause neuronopathic disease and other mutations do not. As a final example, some mutations in the CFTR cause cystic fibrosis. Other mutations have low penetrance. That is, these genes seem to have no clinical significance in the majority of individuals, but when combined with other gene expressions, impart classical or nonclassical cystic fibrosis disease.

There are a myriad of technologies capable of detecting single mutations or SNPs. These platforms are capable of performing medium throughput testing with standard laboratory robotic liquid handlers and at a reasonable hardware cost. These methods include allele-specific oligonucleotide hybridization, PCR RFLP, allele-specific PCR, Line Probe Assays (reverse dot blots), Invader (Third Wave Technologies), ReadIT (Promega), Nanochips (Nanogen), Homogenous PCR (includes TaqMan and Molecular Beacon) and many others. Gene quantitation can be done by several techniques, including fluorescent in situ hybridization (FISH), usually performed in the cytogenetics laboratory with the use of a computerized fluorescent microscope, real-time PCR analysis and comparative genome hybridization.

Whats coming

The future will certainly include more demand for information that will improve the lives of individuals, including testing of embryos and fetuses. Technology will make it easier for laboratories to perform molecular diagnostics of common disorders. The application of microarrays will aid in the discovery of complex patterns of genes, whose functions may not be understood, that define prognostic patterns and lead to therapeutic recommendations. Microarrays will likely be used in one of four ways: expression arrays, resequencing microarrays, multiple genotyping arrays and comparative genome hybridization arrays.

Expression arrays consist of DNA probes immobilized on chips that are used to detect mRNA, primarily in solid tumors. Promising data suggests these expression arrays can be used to predict malignant potential in stage 1 breast cancer and other malignancies.

Resequencing microarrays are being developed to replace expensive DNA sequencing assays for large genes, such as BrCa1 and BrCa2. These chips may reduce the cost of BrCa1 sequencing. If this can be accomplished, the indications for BrCa1 and BrCa2 testing may be broadened.

Multiple SNP analysis is currently necessary for cystic fibrosis carrier detection, which requires 25 mutations and six polymorphisms to be analyzed simultaneously. A low-density microarray has been developed for this purpose. Suggestions have been made to screen patients for multiple drug sensitivity pharmacogenetic SNPs using a single chip. This approach suffers from HIPAA and compliance issues, as genetic testing would be performed without a definite indication.

Comparative genome hybridization microarrays hold enormous hope in the field of oncology. Many solid and hematologic malignancies have gene duplications and/or gene deletions associated with them. Amplification of the proto-oncogene Her2/neu has been demonstrated to correlate with a higher grade of malignancy and with tumor response to the chemotherapeutic agent Herceptin. Comparative genome hybridization microarrays have the ability to scan the entire genome for such insertions and deletions and may revolutionize drug development and prognostic testing.

These complex tests may need to be interpreted with additional clinical and routine test results. Regulatory oversight and ethical debate will strive to keep pace with the rapid advances and applications.

Genetic testing, as all clinical laboratory testing, must always be interpreted in the light of clinical findings. Molecular testing for hemochromatosis, alone, is not capable of making the diagnosis of hemochromatosis. Less than 5% of patients homozygous for C282Y in the HFe gene will ever develop symptoms of hemochromatosis. Molecular genotyping tests are extremely sensitive and specific for the mutations they are designed to detect, but may be less so for disorders associated with those diseases.

Molecular diagnosis of inherited diseases will expand rapidly into clinical labs in the coming years. Laboratorians must develop internal capabilities to perform and interpret selective tests that are appropriate for their setting, understand the clinical application of these tests, examine the technical issues with performing these tests and maintain the expertise to interpret the test results in the full context of the patient and the family being tested.

From Mendels rudimentary study of peapods a century and a half ago, to the sophisticated clinical laboratories of today, the rapid growth of molecular genetics provides unsurpassed diagnostic insights into complicated hereditary diseases that affect the lives of human beings worldwide. From these insights, medical science can contribute solutions to the families who struggle to resolve hereditary issues. After all, life should be as simple as she has your smile and my eyes.

Dr. Harvey W. Kaufman is medical director, hospital services, and Dr. Charles M. Strom is medical director, genetics, at Quest Diagnostics Nichols Institute, Teterboro, NJ.


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July 2003: Vol. 35, No. 7

© 2003 Nelson Publishing, Inc. All rights reserved.

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