Laboratory Diagnosis in Neurology, 1 Ed.


Basic Terms

Coding and noncoding sequences. The genetic information of the DNA strand is organized in the form of approximately 30 000 to 40 000 genes (i. e., DNA sequences coding for proteins). The actual information-carrying (“coding”) sequences of individual genes are usually several hundred to a few thousand base pairs in length. All the coding areas taken together make up only a small fraction of the total DNA. They are separated by often very much longer DNA sections that do not code for protein sequences. Some of these sequences have regulatory functions, but for many of them no function has as yet been clearly defined. Individual genes usually consist of several short coding sections of several dozen to a few hundred base pairs (exons) separated from one another by noncoding sequences (introns) (Fig. 8.1).

Although the sequence is now known for most of the human genome, we are still a long way from knowing the functions of all the genes. Up-to-date information on the state of the International Human Genome Project can be found on the Internet (

Gene products. The information stored in the base sequence of a gene is translated in the cell into the amino acid sequence of a protein. These proteins are therefore also called gene products. Changing the gene sequence (mutation) may result in alteration or absence of gene products, which in turn can lead to a particular disease. If a mutation occurs in the germ line, it is inherited from generation to generation, as is the risk of developing the disease associated with it.


Fig. 8.1 By linkage analysis, a gene site on a chromosome having a total length of about 100 million base pairs (A) can be narrowed to a section of a few million base pairs (B). This section contains several genes (blue rectangles). The gene in question (C) can be identified and sequenced using molecular genetic methods. Sections of the gene carrying the information for the corresponding protein (exon sequences) alternate with sections that will be spliced out prior to the final protein synthesis (intron sequences).

Monogenic inheritance. When changes in a single gene lead to disease, this is known as monogenic inheritance. The disease may be due to loss of the normal function of the encoded gene product (“loss-of-function mutation”), or it may develop because the altered gene product has gained a new, harmful function that is independent of the original function (“gain-of-function mutation”).

Polymorphism. When mutations take place outside the coding sequence, they often have no effect on gene function. These harmless variations in the base sequence are common (about 1 in every 1000 bases in the human genome) and are referred to as polymorphisms.


Fig. 8.2 Structure of the CAG trinucleotide repeat sequence in exon 1 of the huntingtin gene. The base triplet CAG is translated into the amino acid glutamine. The CAG repeat thus codes for a polyglutamine domain within the protein.

Molecular Diagnosis

Allelic and Genetic Heterogeneity

The possibilities of molecular diagnostic testing of genetic neurological disorders are very much determined by the genetic complexity of the disease in question. Some diseases, such as Huntington's disease (HD, formerly known as Huntington's chorea), are practically always caused by a single genetic change that is relatively easy to identify—in this case, the expansion of a repetitive trinucleotide sequence in the HD gene (or huntingtin gene, see below). Often, however, a disease is caused by different mutations at quite different sites within a gene (“allelic heterogeneity”). Depending on the size of the gene, this may make molecular diagnostic testing technically much more difficult to carry out. In other cases again, mutations in several different genes can lead to clinically identical or very similar diseases (“genetic heterogeneity”). This, too, can complicate molecular diagnostic testing.

Objective and Prerequisites

Objective. The objective of molecular diagnostic testing is to help individual patients or individual families.

Prerequisites. Prerequisites for carrying out molecular diagnostic testing are (Statement of the Practice Committee, 1996):

• Provision of comprehensive information to the patient or other person seeking advice.

• Voluntary and informed consent of the same.

• Absolute confidentiality of the results.

Genetic Counseling

Genetic counseling is an essential part of the diagnostic procedure for genetic diseases. Before any diagnostic procedures are carried out, the patient is informed not only about the nature and course of the disease, but also about potential consequences for his or her family with reference to the most important genetic features of the disease, such as mode of inheritance and penetrance. This patient information process will increasingly frequently have to be provided at least in part by the patient's physician. Nevertheless, genetic counseling at a qualified department of human genetics should be offered to every patient seeking molecular diagnostic testing of a hereditary disease. The patient may be under a moral duty to share the genetic knowledge with other family members. The counseling physician, however, should not actively inform family members on his or her own initiative.

Seeking diagnosis and genetic counseling is entirely voluntary.

In many cases, identification of the molecular causes of a disease allows a presymptomatic diagnosis to be made, i. e., identification of a genetic predisposition in a clinically healthy person. The reasons for requesting a presymptomatic diagnosis must be thoroughly reviewed in each case. This kind of diagnosis should therefore only be carried out within a framework of competent genetic counseling at a qualified department of human genetics.


Direct detection of mutations. These days, molecular diagnostic tests are usually based on direct detection of a mutation (“direct DNA diagnosis”). This is easiest when a change in a particular base sequence leads to a particular corresponding disease, as is the case, for example, with trinucleotide expansions (Figs. 8.2 and 8.3). Also relatively easy to detect are large duplications or deletions, like those found in many cases of Charcot-Marie-Tooth disease or tomaculous neuropathy. The detection of point mutations, by contrast, is very time-consuming and costly when the gene is large and the mutations are not clustered in specific regions.

Source of the DNA. The starting point of molecular diagnostic testing is always genomic DNA. Since every cell of the body contains the entire genetic information, the source of the DNA is irrelevant. DNA is usually isolated from the most accessible cells, i. e., leukocytes. Exceptions to this rule are diseases caused by mutations in mitochondrial DNA. Mito-chondria possess their own small genome consisting of a circular molecule 16 569 base pairs in length. Since the distribution of mitochondria varies from tissue to tissue, it can happen that a mutation is not detected in every cell type of the body.


Fig. 8.3 Electrophoretogram of CAG repeats within the huntingtin gene. The DNA fragment containing the repeat is amplified by PCR and separated by size using electrophoresis. The fluorescence-labeled fragments are detected by an automated fluorescent DNA sequence analyzer. The longer the fragment, the more slowly it migrates in the electric field and the later it is detected (further to the right in the electrophoretogram).

Polymerase chain reaction. The starting material for genetic analysis is isolated DNA. Currently, the DNA segment of interest is almost always amplified using the polymerase chain reaction (PCR). The method makes it possible to copy short pieces of DNA (usually several hundred base pairs) many million times. The PCR products are then either separated by size using gel electrophoresis and visualized by fluorescence (Fig. 8.3), or they are analyzed for their base sequences (DNA sequencing) (Fig. 8.4).

Microarrays. In the near future, microarray technology will probably permit a much wider application of molecular diagnostic tests. About 100 000 or more oligonucleotide spots are applied onto a solid support measuring only a few centimeters (DNA chip). These chips are hybridized with the DNA sample and then read by special scanners. This method will soon make it possible to screen a DNA sample for thousands of possible mutations.


Fig. 8.4 Electrophoretogram of an automatic sequence analysis. The arrow marks a heterozygous mutation: at a specific site in the genome on the two corresponding chromosomes, there is one T (gray) and one C (blue). The overlapping bases are not recognized by the automated detection program and are read as “N” (not recognized).