Chapter IV.2. Genetic Testing Techniques
Thomas P. Slavin, MD
June 2013

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The editors and current author would like to thank and acknowledge the significant contribution of the previous author of this chapter from the 2004 first edition, Dr. Steven C. Crook. This current second edition chapter is a revision and update of the original authorís work.

A term newborn female was born to a 24 year-old G1P1 mother by vaginal delivery. Apgar scores were 8 and 9 at one and five minutes, respectively. The pregnancy was complicated by abnormal fetal ultrasound findings including nuchal edema, absent left kidney, and intrauterine growth restriction. Her obstetrician was concerned about possible trisomy 21 (Down syndrome). Amniocentesis for genetic testing was declined and the couple deferred maternal serum screening because they said they would not change any decision about continuing the pregnancy based on any abnormal results. She had consistent prenatal care and no other prenatal exposures or complications were identified. There is no family history of congenital anomalies.

Physical exam: T37.1, P130, R26, BP 100/60, oxygen saturation 95%, length 47.5 cm (3%), weight 2.79 kg (5%), head circumference 32 cm (3%). She is alert, active, no distress. Neck exam shows bilateral neck webbing with posterior redundant skin folds. Her breasts show widely spaced nipples. Her chest has a normal anterior-posterior diameter. A II/VI systolic ejection murmur was heard at the right upper sternal border. Lungs are clear. Abdomen shows normal bowel sounds and no organomegaly. Her genitalia are normal. There is swelling of the dorsal surfaces of both hands and feet.

Clinical course: The infant was initially stable in the newborn nursery. A cardiology consultation was completed to evaluate the heart murmur. An echocardiogram showed a coarctation of the aorta with a bicuspid aortic valve. The baby was transferred to the neonatal intensive care unit for management of the coarctation and hypertension. A genetics consultation was requested and a chromosomal microarray was ordered. The results returned in 4 weeks showing a variant of Turner syndrome. A renal ultrasound was completed confirming the prenatal ultrasound finding of a single right kidney with grade II hydronephrosis. The parents were counseled about Turner syndrome and given written resources. The child will continue to follow-up with their pediatrician, geneticist, endocrinologist, nephrologist, and cardiologist.

Genetic testing is a rapidly evolving field. Among our 46 chromosomes reside 20,000-25,000 genes. A haploid genome (23 of the 46 chromosomes) contains around 3.2 billion base pairs or nucleotides; adenine, thymine, guanine, and cytosine (i.e., A, T, G, C). Our genetic code is a blueprint for making proteins and other elements. The genetic code is read in groups of 3 bases called a codon. Codons accept a specific transfer RNA with an attached amino acid. The amino acid can then join the developing amino acid chain to form the coded protein.

Since DNA is a code, it is prone to developing errors. The errors can be large-scale errors such as an extra chromosome, or major deletions or duplications of the genetic material visible under light microscopy. In contrast, some errors may be subtle and impossible to see using light microscopy, such as a single base change (an "A" was substituted for a "T"). Genetic testing allows for confirmation or the diagnosis of many conditions, ranging from chromosome disorders to determining an individualís risk for hereditary breast cancer by sequencing that individualís BRCA1 and/or BRCA2 genes (tumor supression genes that help repair damaged DNA and when mutated or altered, DNA damage may not be repaired properly resulting in a higher risk of female breast and ovarian cancer). Note that based on common nomenclature guidelines, specific genes are written in italics, while the respective proteins are not. For example, the BRCA1 gene codes for the BRCA1 protein.

There are two main types of genetic testing, cytogenetic analysis and molecular analysis. We will discuss cytogenetic testing first. Chromosome analysis using light microscopy, also known as karyotyping, was first developed in the 1920ís (1). As the years went on, techniques to lengthen the chromosomes chemically to improve karyotype resolution were developed and the ability to see smaller deletions and duplications increased. A karyogram is constructed by rearranging the karyotyped chromosomes to make a conventional, systematic image of all of the chromosomes. An example of the use of a karyogram may be to confirm the diagnosis of trisomy 21 (47, XX or XY, +21). The karyogram would show three (instead of two) chromosome number 21ís next to each other on the image.

Fluorescence in situ hybridization (FISH) testing began to be used clinically in the 1990ís. This is a technique that utilizes small, 10 to 10,000 base pair, single stranded DNA probes that can be hybridized to very specific sequences of interest within the genome. The term "hybridization" refers to the process of mixing DNA from two different samples, then unzipping them into single strands to see if they form new double stranded DNA (hybrids), which they should do if the DNA sequences in the two samples are identical or highly similar. A hybridization probe is a single strand of DNA (or RNA) that is bound to a radioactive tracer or a fluorescent molecule (can be seen under fluorescent microscopy) that is complementary to a specific nucleotide sequence. If that sequence is present, the "probe" will "hybridize" with the target and it will be detectable by fluorescent microscopy (or radiation counting). Probes can be specific to one particular region (locus specific probes), identify individual centromeres (centromere probes), or paint entire chromosomes to distinguish one chromosome from another (whole chromosome painting probes). FISH testing is used to complement karyotypes/karyograms, or can be used to probe for specific deletions or duplications. One common use for clinical FISH testing is to probe for the specific deleted region associated with DiGeorge syndrome in fetuses or newborns with certain suspicious features (conotruncal heart defects, cleft palate, micrognathia, etc.).

In the late 1990s, new testing methods that examine DNA or RNA on a molecular level, became more commonly utilized in clinical medicine. One such example is gene sequencing. Gene sequencing can capture data regarding every A, T, G, and C in the area of interest, allowing for very high resolution error detection. It can also be used for RNA. Specific gene sequencing is clinically useful, since many diseases (neurofibromatosis type 1, cystic fibrosis, etc.) are due to single base pair errors that affect protein function. These small single base pair errors cannot be detected by techniques such as karyotyping or FISH since they are so subtle. Gene sequencing is now able to be completed in such a high throughput, automated fashion, that even the 3.2 billion base pairs that comprise the human haploid genome can be sequenced quickly enough to be used clinically. These new techniques are generally referred to as next-generation sequencing or massively paralleled sequencing.

Another very common molecular technique (used in the presenting case above) to look for large and small deletions and duplications of genetic material, is known as chromosome microarray, or array comparative genomic hybridization (array CGH). Array technology uses multiple (sometimes millions) small wells on a chip. Each well has many copies of the same locus specific probe of interest. Separately labeled control DNA (green florescent signal, for instance) and patient DNA (red florescent signal, for instance) are then denatured and allowed to compete for hybridization on the chip. The chip is then analyzed by computer. Equal competition for one locus would lead to, in this case, a well that is read as "yellow" by the computer. If all of the wells are yellow, then the proportion of patient DNA to control DNA is equal (normal) for all loci examined on the chip. If there is over or under competition of patient sample relative to the control, the results would show a respective duplication (favor red signal) or deletion (favor green signal) in the well with the locus discrepancy.

Arrays can be customized to target one specific gene, chromosome, genes within a particular developmental pathway, or can be created with a "back bone" and be used to look at deletions and duplications across the entire genome. Array technology cannot be used in place of gene sequencing as thousands of markers are used, which can result in many discrepant wells due to chance alone. Therefore, only many contiguous deleted or duplicated markers in the same genomic area, or locus of interest, are considered significant for a loss or gain of genetic material. The large number of probes used in chromosomal microarray (CMA) technology allows for higher resolution than karyotype analysis when evaluating for deletions and duplications. Therefore, in 2010, the recommendation was made to use CMA as a first-tier test in place of karyotyping for individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies (2,3). The only types of large-scale genomic errors that may be missed on whole genome arrays are triploidy (69 chromosomes, usually lethal) and balanced rearrangements (used for pregnancy recurrence risk purposes). Karyotypes are still useful for some straightforward common genetic disorders such as trisomies 13, 18, or 21 to confirm the diagnosis in a rapid fashion or to look for specific Robertsonian (or other balanced) structural chromosome rearrangements that could be important for future pregnancy recurrence risk. In the presented case, either a karyotype or CMA could have been used to diagnosis the patient with Turner syndrome. If the clinician was not certain of the Turner syndrome diagnosis prior to testing, then a CMA would be a better choice since it would have the potential to detect smaller imbalances in genetic material.

There are an almost infinite number of other molecular methods used for diagnostic testing purposes. Many of these techniques use variations of polymerase chain reaction (PCR). Many are also proprietary. The main purpose of PCR is to amplify a genomic region of interest. It accomplishes this by first denaturing the double stranded sample DNA into single stranded DNA, annealing specific primers that flank the region of interest, using a polymerase to copy the region of interest, and restarting the cycle all over again. The multiple cycles can be controlled by using chemicals and/or heat. The end product of PCR may contain millions of copies of the area of interest. The isolated, amplified product, known as an amplicon, can then be used for gene sequencing or other probing techniques. PCR can be used to amplify RNA as well. One common type of diagnostic PCR is known as real-time PCR, in which the genetic region of interest for a particular disorder is amplified and probed at the same time, or in real time. Using allele specific primers, which anneal and amplify only the wild type or disease causing allele (a particular disease causing copy of a gene), the amplified product can be detected using probes as the reaction happens, allowing for accurate and rapid results. One example is the use of real time PCR with allele specific primers to only amplify mutant factor V Leiden alleles for evaluation of thrombophilia. Factor V Leiden is a mutation of the normal factor V gene that results in synthesis of an abnormal factor V protein (known as factor V Leiden) that results in a hypercoagulable state. In this case, only patients harboring the factor V Leiden mutation would produce amplified PCR product. Those without the mutation (wild type) would not produce product, allowing easy discrimination between the test results.

A mutation is generally defined as a disease caused by a single base pair change that has a less than one percent frequency in the population. Single base pair changes seen in over one percent of the population are usually referred to as single nucleotide polymorphisms (SNPs). There are multiple different types of genetic mutations that are important to remember. In particular, a silent mutation causes no change in the amino acid coded for by the codon, and therefore usually no functional defect. A missense mutation causes an amino acid substitution due to a change in a single base pair. A nonsense mutation leads to the formation of a premature stop codon. Missense, nonsense, frame shift, insertions, or deletions in DNA can all cause abnormal or truncated proteins. There is specific mutation nomenclature used to help laboratories and physicians communicate in the same language. For instance, nucleotide or amino acid substitutions are preceded by a description of whether they are genomic (g), coding(c), mitochondrial (m), RNA (r), or protein (p). There is then a number to describe where the abnormality exists in the nucleotide sequence (DNA or RNA) or amino acid sequence (transcribed protein) of interest. This is followed by what the change is, such as a "G" was changed to a "T" at position 123 (e.g., c.123G>T), or del, dup, ins; for deletions, duplications, and insertions, respectively. Sometimes the number is written after the wild type nucleotide or amino acid, as in G123T, where "T" denotes the substitution for a wild type "G". In the above factor V Leiden mutation example, the resulting amino acid consequence was an arginine (R) that was changed to a glutamine (Q) at amino acid 506 in the factor V Leiden protein (p.R506Q). In other words, the same mutation could be described in multiple different ways.

In summary, chromosomes, DNA, and RNA are all prone to genetic errors. These errors are the cause of many disorders and conditions. There are multiple types of genetic testing techniques that can detect these errors including: karyotyping, FISH, arrays, sequencing, PCR, and countless other techniques. Once a change is found, it is important to be able to have conventional, proper nomenclature to be able to define the change. Genetic testing is rapidly evolving and will continue to become an increasing part of everyday clinical care.


1. What is the basic difference between cytogenetic and molecular testing?

2. What is the primary use of PCR?

3. True/False: A missense mutation generally causes no change to the final protein product.

4. What would be the best test to order on a newborn infant with dysmorphic features that do not resemble a known clinical syndrome?

5. Roughly how many types of genetic tests exist?

6. True/False: Only a geneticist should order genetic testing.


1. Painter TS. Chromosome numbers in mammals. Science 1925;17;61(1581):423-424.

2. Manning M, Hudgins L for the Professional Practice and Guidelines Committee. Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet Med. 2010;12(11):742-745.

3. Miller DT, Adam MP, Aradhya S, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 2010;14;86(5):749-764.

Answers to questions

1. Cytogenetic testing looks at the structure of chromosomes and molecular techniques examine DNA or RNA.

2. To amplify specific segments of DNA or RNA.

3. False, this statement refers best to silent mutations

4. A whole genome microarray

5. There are countless methods of genetic testing.

6. False, genetic testing is now commonly ordered in all fields of clinical medicine. However, it is fairly easy to order the wrong test or to interpret the test incorrectly. Thus, as with any test, they should be ordered by, or ordered in consultation with clinicians, who have the skill to order and interpret the testing appropriately.

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