The term genetic disease is not a single concept. It can be applied to diseases in which there is just a genetic component to susceptibility - for instance to coronary artery disease or to diabetes, it can even be used to consider resistance to infectious disease. It can be applied to cancer and to the ultimate killer - old age. However, in this lecture I want to consider briefly two examples of straightforward, Mendelian conditions which no self respecting Introduction to Human Genetics course could possibly fail to mention.
In this course we have already casually mentioned many different Mendelian diseases such as alkaptonuria, phenylketonuria, sickle cell disease, elliptocytosis, tuberous sclerosis, cystic fibrosis, Hereditary hemorrhagic telangiectasia, osteogenesis imperfecta, alpha thalassemia, X linked ichthyosis, and others. You can be reminded of why and how they were mentioned by clicking on their names when the previous lecture notes should appear in a new window. In general, for any genetic disease which you want to find out more about, I strongly recommend that you consult Online Mendelian Inheritance in Man (OMIM). Click here to open an OMIM window.
The following information about this X linked recessive disease is quoted almost directly from the thorough review in OMIM. If you wish to find the quoted references go directly to the OMIM review.
Dystrophin-associated muscular dystrophies range from the severe Duchenne to the milder Becker muscular dystrophy (DMD and BMD). Mapping and molecular genetic studies indicate that both are the result of mutations in the huge gene that encodes dystrophin. Approximately two-thirds of the mutations in both forms are deletions of one or many exons in the dystrophin gene. Although there is no clear correlation found between the extent of the deletion and the severity of the disorder, DMD deletions usually shift the frame. The most distinctive feature of Duchenne muscular dystrophy is a progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves. The bulbar muscles are spared but the myocardium is affected. There is massive elevation of creatine kinase levels in the blood, myopathic changes by electromyography, and myofiber degeneration with fibrosis and fatty infiltration on muscle biopsy. The onset of Duchenne muscular dystrophy usually occurs before age 3 years, and the victim is chair ridden by age 12 and dead by age 20. The onset of Becker muscular dystrophy is often in the 20s and 30s and survival to a relatively advanced age is frequent.
Burn et al. (1986) reported monozygotic twin girls, one of whom had typical clinical features of DMD despite a normal female karyotype and the second of whom was normal. They proposed that differences in lyonization accounted for the findings. Hybridization of fibroblasts from each twin with RAG-mouse cell line deficient in HPRT showed that in the affected twin it was the mother's X chromosome that was predominantly the active one, whereas in the normal twin it was the father's.
Greenstein et al. (1977) found DMD in a 16-year-old girl with a reciprocal X;11 translocation. The mother was thought not to be a carrier. Possibly the break at Xp21 caused a null mutation; the normal X chromosome was inactivated. Verellen et al. (1978) reported the same situation with X;21 translocation and break at Xp21. Canki et al. (1979) described similar findings in a girl with X;3 translocation with break at Xp21. The mother was thought to be heterozygous.
Lindenbaum et al. (1979) found DMD with X-1 translocation and suggested that the DMD locus is at Xp11 or Xp21. A number of females with X-autosome translocations with the breakpoint in the Xp21 band have shown Duchenne muscular dystrophy. One interpretation is that the gene locus is in that region and that the locus on the normal X is inactivated. Murray et al. (1982) found linkage of DMD with a restriction enzyme polymorphism at a distance of about 10 cM. The cloned DNA sequence bearing the polymorphism (lambda RC8) was assigned to Xp22.3-p21 by study of somatic cell hybrids.
Wieacker et al. (1983) studied the linkage between the restriction fragment length polymorphism defined by the cloned DNA sequence RC8 and X-linked ichthyosis. At least 2 crossovers were found among 9 meioses in an informative family, suggesting that RC8 and STS may be about 25 cM apart. Since STS is 15 cM proximal to the Xg locus and since the RC8 and Duchenne muscular dystrophy are closely linked, DMD may be 50 cM or more from Xg. Worton et al. (1984) studied a female with DMD and an X;21 translocation which split the block of genes encoding ribosomal RNA on 21p. Thus, ribosomal RNA gene probes can be used to identify a junction fragment from the translocation site and to clone segments of the X at or near the DMD locus.
Francke et al. (1985) studied a male patient with 3 X-linked disorders: chronic granulomatous disease with cytochrome b(-245) deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa. A very subtle interstitial deletion of part of Xp21 was demonstrated as the presumed basis of this 'contiguous gene syndrome.' That this was a deletion and not a translocation was demonstrated by the absence of 1 DNA probe from the genome of the patient. Since this probe (called 754) was clearly very close to DMD and recognised a RFLP of high frequency, it proved highly useful for linkage studies of DMD.
Kunkel et al. (1985) used a method of subtractive hybridization for cloning the specific DNA fragment absent in patients homozygous or hemizygous for chromosomal deletions. They applied the method to the DNA of the patient with a minute interstitial deletion of Xp who was reported by Francke et al. (1985).
Bodrug et al. (1987) cloned, restriction-mapped, and sequenced the exchange points from the case of X;21 translocation reported by Verellen-Dumoulin et al. (1984). The translocation was found to be reciprocal but not conserved. A small amount of DNA was missing from the translocated chromosomes; 71 or 72 basepairs from the X chromosome and 16 to 23 basepairs from the 28S ribosomal gene on chromosome 21. Although a number of tumor-associated translocations had been studied in molecular detail, this was probably the first instance in which a constitutional translocation had been studied at the nucleotide level. By the time of this report, there were 20 known cases of DMD-BMD females with X-autosome translocations with breakpoints at Xp21.
Tennyson et al. (1995) stated that the DMD gene has 79 exons spanning at least 2,300 kb (2.3 Mb). This is equivalent to approximately 0.08% of the human genome and one-half of an E. coli genome.
They monitored transcript accumulation from 4 regions of the gene following induction of expression in muscle cell cultures. Quantitative RT-PCR results indicated that approximately 12 hours are required for transcription of 1,770 kb (at an average elongation rate of 0.24 kb per 6 seconds), extrapolating to a transcription time of 16 hours for the complete gene. Accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5-prime end before transcription is complete, providing strong evidence for cotranscriptional splicing.
England et al. (1990) demonstrated that a family segregating for a very mild BMD (1 affected member was still ambulant at age 61) had a mutation that removed the central part of the dystrophin gene encompassing 5,106 bp of coding sequence, almost half the coding information. Immunological analysis of muscle from one of the patients showed that the mutation resulted in the production of a truncated polypeptide localised correctly in the muscle cell. Immunostaining with antibody against the central part of the DMD molecule resulted in no staining of muscle membranes; immunostaining with antibody against the N- and C-terminal portions did yield muscle membrane staining. They concluded that the findings are meaningful in the context of gene therapy which would be facilitated by the replacement of the very large dystrophin gene with a more manipulatable mini-gene construct.
In a review, Ahn and Kunkel (1993) pointed out that expression of the large DMD gene is under elaborate transcriptional and splicing control. At least 5 independent promoters specify the transcription of their respective alternative first exons in a cell-specific and developmentally controlled manner. Three promoters express full-length dystrophin, while 2 promoters near the C-terminus express the last domains in a mutually exclusive manner. Six exons of the C-terminus are alternatively spliced, giving rise to several alternative forms. Genetic, biochemical, and anatomic studies of dystrophin suggested a number of distinct functions are subserved by its great structural diversity.
At least 5 different promoters drive the transcription of tissue-specific dystrophin isoforms. Three promoters are located at the 5-prime end of the gene and give rise to 427-kD brain and muscle isoforms. Two other promoters drive the expression of alternate first exons spliced to distal parts of dystrophin sequence: these protein products are named (by their molecular weight) Dp116 and Dp71. The transcription of Dp116 is initiated at an alternate first exon located upstream to exon 56. Dp116 is exclusively expressed in Schwann cells as a thin rim of immunoreactivity located around the outside of the myelinated peripheral nerve fibers.
Many DMD patients have rare staining dystrophin-positive fibers. The possibility of somatic mosaicism can be raised, but somatic reversion/suppression is another possibility. Indeed, the dystrophin-positive fibers have been referred to as 'revertants.' The revertants are found in both familial and nonfamilial cases. Klein et al. (1992) found that in patients with deletions, revertants did not stain with antibodies raised to polypeptide sequences within the deletion. These results indicated that positively stained fibers were not the result of somatic mosaicism in deletion patients. Klein et al. (1992) concluded that the most likely mechanism giving rise to positively staining fibers is a second site in-frame deletion. Thanh et al. (1995) used exon-specific monoclonal antibodies to determine which exons are removed in order to correct the reading frame in individual revertant muscle fibers. They showed that 15 revertant fibers in a DMD patient with a frameshift deletion of exon 45 had correction of the frameshift by the additional deletion of exon 44 (or perhaps exon 46 in some fibers) from the dystrophin mRNA, but not by larger deletions.
Beggs and Kunkel (1990) presented a flow diagram illustrating procedures for the molecular diagnosis of DMD or BMD. For males with consistent clinical features, CPK levels, and muscle biopsy, they suggested that Western blot testing for dystrophin be done first. If this is normal, the patient should be studied for other neuromuscular diseases. If dystrophin is of reduced or increased size, with or without reduction in the amount of dystrophin, BMD should be suspected. If dystrophin is absent, DMD should be suspected. Thereafter, PCR testing and Southern blot analysis should be done, looking for deletion/duplication. These procedures detect about 65% of patients, and the Southern blot permits prognostication of severity by distinguishing in-frame versus frameshift mutations in over 90% of cases. If no deletion or duplication is found, it is then necessary to resort to RFLP-based linkage studies, which unfortunately are laborious and time consuming. Once the diagnosis has been made, the information can be used for carrier detection and prenatal diagnosis. In females who are having symptoms of muscular dystrophy, immunohistochemistry for dystrophin in muscle showing a patchy loss of dystrophin can be used, and when abnormality is found, the same procedures of PCR, Southern blot, and linkage studies can be pursued. If the immunohistochemistry is normal, the female can be studied for other neuromuscular diseases. (Abnormality is indicative of the manifesting carrier state.) Beggs and Kunkel (1990) provided useful illustrative case histories as well as a hypothetical case in which a newborn male was found to have elevated CPK on a screening program but normal physical examination and negative family history. If Western blotting revealed absence of detectable dystrophin in the muscle and the PCR analysis detected a deletion which was confirmed by Southern blotting, his mother might carry the deletion or be normal. Even if normal, prenatal diagnosis could be offered her because of the significant probability that she was a germline mosaic. The usefulness of such screening programs for diagnosing DMD at a stage when diagnosis can be useful to the parents in the planning of other pregnancies is worthy of consideration.
Cystic fibrosis is an autosomal recessive genetic disease which, untreated, leads to death in infancy but with modern therapy sometimes allows a good quality of life at least into the fourth decade and possibly beyond. Babies with this condition are usually born healthy but show a failure to thrive because they are unable to digest their food properly. Their sweat is also much more salty than normal and this is the basis of a diagnostic test. The Scientific American article above quotes an adge from European folklore
Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die.
Whether this is a real adage or not I don't know. I've certainly never heard it. However, it certainly fits the disease symptoms. The digestive problems stem from a lack of pancreatic enzymes in the gut. The name cystic fibrosis is actually short for cystic fibrosis of the pancreas. The duct leading from the pancres to the gut is blocked by mucus and the pancres can self destruct as a consequence. Diabetes is a frequent secondary complication. If the digestive problems are overcome by giving a capsule of pancreatic enzymes with each meal, then the next problem is that the lungs become clogged with sticky mucus and frequent respiritory infections occur. About 1 baby in 2000 of Northern European racial origin is affected with the disease. Making the assumption that the Hardy Weinberg equilibrium will approximately hold true enables us to deduce that the carrier frequency in this population is about 1 person in 22. There is no biochemical or physiological test which can distinguish carriers so most cases occur suddenly in families with no previous history of the disease. For many years, the only advice which could be given to parents with one affected child was that every subsequent pregnancy was at a 25% risk of producing another affected child.
gene cloning: With few clues from the symptoms as to the nature of the protein affected by the mutation, cystic fibrosis provides a paradigm of the process known now as positional cloning. The mutant gene was identifed primarily on the basis of its genetic map position which was discovered through genetic linkage analysis. By now this should be familiar to you. The sequence of the gene was examined and much of it was related to other proteins whose structure had already been worked out. Together with information which had by then been discovered that chloride ion transport was defective in the disease this enabled predictions to be made as to its role as a regulated ion channel. The gene was named CFTR for Cystic Fibrosis Transmembrane Regulator.
I have borrowed here the excellent image from the Walsh and Smith article in Scientific American which shows what the product of the CFTR gene is predicted to look like and how it is now thought to act.
mutations: About 70% of the mutant alleles in the european population are of one type, a deletion of three nucleotides which leads to the loss of a single aminoacid from the CFTR polypeptide. This is enough to prevent the polypeptide from being properly processed in the golgi apparatus and it is not transported to the cell membrane. A small number of mutations account for about 15% of mutant alleles but the remaining 15% of mutant alleles are composed of several hundred different, individually rare, alleles.
Why are mutant alleles maintained at such a high level in the population? One possible reason is that heterozygous carriers are more resistant to fluid loss caused by enteric bacterial infection.