Unstable Number of Trinucleotide Repeats
Heritable changes in the number of repeated groups of three nucleotides each (trinucleotide or triplet repeat) represent a newclass of mutations in man for which there is no parallel in other organisms. They either occur within the gene and are translated or occur outside the gene in an untranslated region, and they are unstable during transmission through the germline. Unaffected persons may carry a premutation, whichmay be converted to a full mutation when passed through the germline to the next generation. Therefore, the effects of the mutation differ in severity in affected members within the same family. Occasionally, there is regression and a generation is skipped.

• Genetic diseases with increased numbers of trinucleotides
  
  Some important genetically determined diseases are based on a greater than normal number of trinucleotides: Huntington disease, fragile X syndrome, myotonic dystrophy, spinobulbar muscular atrophy type Kennedy, and spinocerebellar ataxia type 1. A total of 14 defined trinucleotide repeat diseases have been described (see Cummings and Zoghbi, 2000).
  
  
• Huntington disease
  
  Huntington disease is a progressive disease of the brain. Within 5–10 years, it leads to complete loss of motor control and intellectual abilities (1). It usually begins around age 40–50 with uncoordinated movements (chorea, St. Vitus’ dance), excitation, hallucinations, and psychological changes. The disease is transmitted by autosomal dominant inheritance and shows complete penetrance. It presents an affected family with two difficult problems: (i) due to its late onset, carriers of the mutation have usually completed their family planning before the disease is manifest, and (ii) children of affected persons first learn as young adults that they are at a 50% risk of developing the disease later in life. Thus, the introduction of a direct predictive DNA diagnostic procedure is very important. However, before such a genetic test is carried out, it must be established through genetic counseling that the persons at risk have decided for themselves whether they want to have the test performed. The gene is located on the distal short arm of chromosome 4 (2). It spans 210 kb and codes for a protein (called huntingtin) of important function. The 5! end of the gene contains numerous copies of a trinucleotide sequence consisting of cytosine, adenine, and guanine (CAG), a codon for the amino acid glutamine. Normally the gene has 10–34 CAG repeats; in patients there are 42–100. The diagnostic test (3) demonstrates that affected individuals (here, individuals 1, 2, and 4) have enlarged DNA fragments due to expanded CAG repeats. (Findings of the Institut für Humangenetik of the Universität Göttingen with kind permission by Prof.W. Engel; Zühlke et al., Hum. Mol. Genet. 2:1467–1469, 1993).
  
  
• Myotonic dystrophy (MDY1)
  
  Myotonic dystrophy is an autosomal dominant hereditary disease that predominantly affects the central nervous and muscular systems (1). The myotonia causes a masklike facies (2). The disease is very variable and in many families shows increasing severity in consecutive generations (anticipation). An increased number of CTG repeats, more than 50 copies compared with 5–35 in normal individuals (3), is found immediately beyond the 3! end of the gene in affected persons. This is demonstrated in a Southern blot as an enlarged DNA fragment (4). (Schematic representation of a Southern blot at the gene locus D19S95, probe pBBO.7 after DNA cleavage with EcoRI. After Harley et al., Lancet 339:1125–1128, 1992).
  
  
• References
  
  Cummings, C.J., Zoghbi, H.Y.: Fourteen and
  counting: unraveling trinucleotide repeat
  diseases. Hum. Mol. Genet. 9:909–916,
  2000.
  Harley, H.G. et al.: Unstable DNA sequence in
  myotonic dystrophy. Lancet 339:1125–
  1130, 1992.
  Zoghbi, H.Y.: Spinocerebellar ataxia and other
  disorders of trinucleotide repeats, p. 913–
  920, In: Jameson, J.L., ed., Principles of
  Molecular Medicine. Humana Press, Totowa,
  New Jersey, 1998.

• Phenotype
  
  The phenotype is very variable. The mental retardation varies; there is no distinct neurological dysfunction. In adult males, the testes are enlarged (macroorchidism). Affected individuals can usually be integrated well into the family and learn to function in a familiar environment.
  
  
• Fragile site Xq27.3
  
  The gene locus (FRAXA) for the gene (FMR1) is located on the distal long arm of the X chromosome in region 2, band 7.3 (Xq27.3). In this region the great majority of patients and some of the female heterozygotes show a constriction (fragile site) in the affected X chromosome in about 2–25% of metaphases. The constriction must be induced by folic acid deficiency in the culture medium, and it must be differentiated from other fragile sites in this region.
  
  
• Expanded CGG repeats in the fragile X syndrome
  
  The heritable unstable sequences explain two unusual characteristics of the fraX syndrome; (i) the transition from a premutation (about 60–200 CGG repeats) without clinical manifestation into a full mutation (more than 200 CGG repeats) during transmission through the germline, and (ii) differences in the FRAXA locus within a given family. Fragile X syndrome is heritable as an X-linked dominant trait. The risk of transmission and clinical manifestation varies according to the type of mutation (premutation or full mutation), the gender of the patient and of the parent carrying an expanded trinucleotide repeat, and the relationship within the family. Males with the full mutation are mentally retarded and do not reproduce. Heterozygous females for the full mutation have a risk of variable mental retardation of 50%. They transmit the full mutation to 50% of their offspring. A premutation present in amale (“normal male transmitter”) is transmitted to all daughters and none of the sons. Female carriers of the premutation or full mutation have a 50% risk of transmitting the mutant allele. The actual risk of manifest fragile X syndrome depends on the number of CGG repeats and varies between 10% (60–69 repeats) and 50% (more than 100 repeats) for sons (Gene Clinics at http: www.geneclinics.org). The number of CGG repeats is variable within a family. In the pedigree shown (1), individuals II- 3 and III-1 have more than 200 repeats and have fragile X syndrome. Individuals I-2, I-3, II-1, and III-3 are carriers of the premutation with 79–82 repeats. The normal number of CGG repeats is 6 to about 50, the premutation is defined by about 55 to 100, and the full disease-causing mutation by more than 200 repeats (2). The different numbers of CGG repeats can be demonstrated in Southern blots as DNA fragments of different sizes (3). The normal gene is represented by a small DNA fragment (S). A premutation leads to slightly enlarged fragments. The full mutation is characterized by large fragments (L). With this procedure, a reliable diagnosis of the genotype is possible. (Photograph of a Southern blot: HindIII digestion and hybridization with probe Ox1.1; P. Steinbach, Ulm).
  
  
• References
  
  Eichler, E.E., et al.: Length of uninterrupted CGG
  repeats determines instability in the FMR1
  gene. Nature Genet. 8:88–94, 1994.
  Jin, P.,Warren, S. T.: Understanding the molecular
  basis of fragile X syndrome. Hum. Mol.
  Genet. 9:901–908, 2000.
  Nelson, D.L.: Fragile X syndrome, pp. 1063–
  1067. In: J.L. Jameson, ed., Principles of
  Molecular Medicine. Humana Press, Totowa,
  NJ, 1998.

• Two syndromes associated with the same chromosomal region
  
  Prader–Willi syndrome is characterized by neonatal muscular weakness and feeding difficulties, followed in early childhood by reduced or lack of satiation control leading to massive obesity in many patients. Several other, variable features occur, such as mental retardation, characteristic facial features, short stature, hypopigmentation, behavioral problems, and other findings. In Angelman syndrome the developmental retardation is usually very severe. Nearly total lack of speech development, an abnormal electroencephalogram with tendency to seizures, and hyperactivity are almost always present.
  
  
• Parental origin of the deletion
  
  PWS results when the deleted chromosome involves the chromosome 15 of paternal origin (loss of one paternal allele 2 in the diagram of a Southern blot on the left). AS results when the deletion involves the chromosome 15 of maternal origin (loss of one maternal allele 1 in the scheme on the right).
  
  
• Uniparental disomy
  
  Uniparental disomy (UPD) is the presence of two chromosomes or genes from the same parent. The diagram of a Southern blot shows two different types of UPD in PWS: isodisomy and heterodisomy. In isodisomy the two parental alleles are identical (two maternal alleles 1 shown in the diagram). In heterodisomy the two alleles are of the same parental origin, but differ (one allele 1 and one allele 2, both of maternal origin, as shown).
  
  
• Parent-of-origin deletion and uniparental disomy
  
  A deletion and uniparental disomy have the same functional result, i.e., loss of the genetic activity of one parental allele. The frequency of a deletion is about the same for PWS and AS (70% each), whereas the frequency of UPD differs considerably: 29% in PWS, 1% in AS.
  
  
• Chromosomal region 15q11–13 and imprinting center
  
  Five genes known to date are transcribed from the paternal allele only, not from the maternal allele where they are constitutively repressed (blue squares). From the more distal gene UBE3A (a ubiquitin-protein ligase E3) only the maternal allele is transcribed. About 25% of cases of Angelman syndrome are caused by deletion or mutation of this gene. The breakpoints of the common large deletions occur predominantly in three breakpoint cluster regions. The imprinting center (IC) was originally defined by small deletions outside of the known imprinted genes. About 1% of patients with PWS and 4% with AS have imprinting center defects. The imprinted region on 15q11–13 shows a difference in methylation pattern between the maternal and the paternal allele. This is the basis of a diagnostic test. (Data in E. kindly provided by Dr. Karin Buiting). Other imprinted chromosomal regions are also associatedwithhumandiseases, e.g.,Beckwith– Wiedemann syndrome and some patients with Russell–Silver syndrome, among others.
  
  
• References
  
  Buiting, K., et al.: Inherited microdeletions in
  the Angelman and Prader-Willi syndromes
  define an imprinting centre on human chromosome
  15. Nature Genet. 9:395–400,
  1995.
  Gillessen-Kaesbach, G., et al.: DNA methylation
  based testing of 450 patients suspected of
  having Prader-Willi syndrome. J. med.
  Genet. 32:88–92, 1995.
  Horsthemke, B., Dittrich, B., Buiting, K.: Imprinting
  mutations on human chromosome
  15.Human Mutat. 10:329–337, 1997.
  Nicholls, R.D.: Prader-Willi and Angelman Syndromes,
  pp. 1053–1062. In: J.L. Jameson,
  ed., Principles of Molecular Medicine.
  Humana Press, Totowa, New Jersey, 1998.
  Nicholls, R.D., Saitoh, S. , Horsthemke, B.: Imprinting
  in Prader-Willi and Angelman syndromes.
  Trends Genet. 14:194–200, 1998.