Many of the neurological disorders discussed in this book have a genetic etiology that can be diagnosed and/or confirmed by chromosomal or molecular testing. Due to advances in genetic technology, the range of testing options has increased dramatically in the last few years. Deciding which test to perform and how to interpret the results can be daunting. Accordingly, in this chapter we review the different types of clinically available genetic tests. These can be employed for general screening or for confirmation of a clinically diagnosed condition. The general portfolio of such testing is illustrated in Table 21-1, and this chapter will review these types of tests.

A routine chromosome study (karyotype) involves evaluating the chromosomes during the metaphase period of cell mitosis, and the test is usually performed on blood cells. Lymphocytes are grown in culture and stained with a dye such as Giemsa to obtain a distinct pattern of light and dark bands. This banding pattern is unique to each of the 46 chromosomes, and helps to identify them on microscopic examination (Figure 21-1). A routine chromosome study can identify extra or missing chromosomes (eg, Down [47, XY+21] and Klinefelter [47, XXY] syndromes) as well as detect large inversions, translocations, deletions, duplications, and extra fragments (eg, marker chromosomes). The visual resolution of this study, however, is only at the 10 to 20 million base pair level (Table 21-2). Higher-resolution tests are available (as discussed below in Array-Based Comparative Genomic Hybridization) that detect much smaller changes in the chromosome.

Individuals who present with developmental delay and/or mental retardation should prompt the clinician to consider ordering at least a routine chromosome study. A chromosome study is especially indicated when a developmentally delayed child has any of these associated conditions:

• Single or multiple malformations
  
• Seizures
  
• Dysmorphic facial features
  
• Short stature
  
• Autism
  
• Family history of mental retardation

Fluorescence in-situ hybridization (FISH) detects the presence or absence of a large DNA sequence involving a particular gene or chromosome region. In this technique, test DNA is first separated into single strands and then hybridized with a fluorescent-labeled DNA probe (usually 0.3-1.0 Mb in size) that is complementary to the region of interest. If the area under study is present, the labeled DNA will bind to it. This can be visualized as a fluorescent spot under the microscope, as illustrated in Figure 21-2 for the 1p36.3 microdeletion condition. This deletion is usually not detected on a routine chromosome study.1 During the last decade, the development of FISH led to the identification of various “microdeletion syndromes” (Table 21-3).2-4 However, to be able to judiciously order the appropriate FISH study, one must be aware of the clinical phenotypes of the specific microdeletion syndromes. To overcome this limitation, FISH screening panels have been developed to test simultaneously for multiple microdeletion syndromes.5 An example of this is the subtelomere FISH panel that can detect minute rearrangements in the subtelomeric regions of all the chromosomes. Subtelomeric deletions or duplications are responsible for some cases of unexplained developmental delay that were otherwise not diagnosed by a routine karyotype.6

Array-based comparative genomic hybridization (array-CGH) can detect alterations in copy number of DNA sequences for specific chromosome regions.7 In this technique, the DNA of the patient and a normal control are labeled with fluorescent dyes (green and red, respectively) and both DNAs are hybridized to normal cloned DNA fragments embedded in a silicon target chip. The exact locations of these DNA fragments on the chromosomes are known. The DNA sequences from both sources compete for their targets on the chip, and images of both fluorescent signals are then captured. Regions equally represented in both samples appear yellow (because both the red and green fluorochromes are detected), deleted regions appear red, and amplified regions are green (Figure 21-3). In this way, a global overview of gains and losses throughout the entire test genome is obtained.8 Microarray analysis can identify deletions and duplications of the loci represented on the microarray chip, with the resolution being a function of the number and size of target fragments used and the genomic distance between each of the fragments.9 Using array-CGH, detection rates for chromosome abnormalities range from 5% to 17% in individuals with developmental delay who have had prior routine cytogenetic testing.9-11Figure 21-4 illustrates the utility of this test in a case of identical twins that otherwise had normal chromosome study and extensive FISH testing. Because of the high sensitivity of array-CGH, false positives can occur due to the presence of genomic regions of duplications and deletions that are evolutionarily derived and are of no clinical consequence (often termed copy number variants). In such cases, the parents must be tested to see if they too carry the copy number variation. Array-CGH will not be able to detect balanced translocations because in these situations there is no loss or gain of chromosome material.

Molecular genetic testing reviewed herein includes tests to detect mutations, gene deletions, abnormal number of nucleotide repeats, abnormal DNA methylation, and uniparental disomy. A mutation in a gene can have different effects, and some of the common changes are illustrated in Figure 21-5. A pathogenic mutation either eliminates the protein that the gene codes for, or affects the structure and ultimately the function of the translated protein. Gene mutations can be identified either by targeted mutation analysis (searching for common known mutations) or by sequence analysis of the entire coding region of the gene. These can be achieved by using techniques such as Southern blot analysis or polymerase chain reaction (PCR). In Southern blot analysis, restriction fragment enzymes are used to break the DNA into fragments of different size. The DNA fragments are loaded onto an agarose gel and separated by applying an electric current. If the test DNA has mutations, bands with abnormal lengths are formed. PCR is a method that allows the DNA region of interest to be amplified in an exponential manner using a DNA polymerase enzyme and an amplification reaction that is repeated over and over again, producing multiple copies of the DNA region of interest (eg, 20 cycles will produce over 100,000 copies). The PCR product can be analyzed by several methods. For sequence analysis of the gene, nucleotides tagged to fluorescent dyes are added to the PCR reaction, and a laser within an automated DNA sequencing machine is used to analyze the DNA for sequence changes.