Neurocutaneous Diseases: A Practical Approach

1972:1–18.

Chapter 1

Genetic Principles

WILLIAM G. JOHNSON

Publisher Summary

This chapter focuses on understanding of the principles of medical genetics, which is helpful both in diagnosing neurocutaneous diseases, and in working with patients and families after the diagnosis has been made. The genetic basis of a disorder is of critical importance in understanding its cause and developing effective specific treatment. Expressivity is the degree of clinical involvement in an individual with an abnormal gene. Unlike dominant disorders, autosomal recessive disorders have an increased incidence of parental consanguinity. X-linked recessive inheritance somewhat resembles autosomal recessive inheritance especially when only a single sibship is considered. Restriction fragment length polymorphisms (RFLPs) have revolutionized human gene mapping in recent years, yet in principle, the technique is little different from gene mapping using earlier markers. Gene mapping using RFLPs is little different in principle than gene mapping using other kinds of markers.

This work was supported by grants from the National Institutes of Health (NS-15281 and NS-11766), the Muscular Dystrophy Association (H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases), the March of Dimes Birth Defects Foundation, and a generous gift from the Alexander Rapaport Foundation.

An understanding of the principles of medical genetics is helpful both in diagnosing neurocutaneous diseases and in working with patients and families after the diagnosis has been made. In addition, the genetic basis of a disorder is of critical importance in understanding its cause and developing effective specific treatment.

This chapter focuses on the patterns of inheritance that characterize these disorders and on how the clinician can contribute to localization of the genes that cause these disorders.

Inheritance Patterns for Human Genetic Disorders

Autosomal Dominant Inheritance

Pattern of Transmission

Inheritance of a disorder transmitted in autosomal dominant fashion is vertical, that is, through successive generations, from parent to child to grandchild. Dominant inheritance, in the older terminology, is hereditary rather than familial. Males and females are affected with equal frequency and equal severity. There is no increased frequency of parental consanguinity as in autosomal recessive inheritance. Children of an affected parent have a 50% risk of receiving the harmful gene and therefore a 50% risk of being affected (see Penetrance and Expressivity, below). Half sibs through the affected parent have the same risk of being affected as full sibs, in sharp contrast to the situation in autosomal recessive diseases where the risk to half sibs is very small. Male-to-male transmission occurs in autosomal dominant inheritance and should always be looked for. Male-to-male transmission cannot occur in X-linked dominant pedigrees and may not occur in small autosomal dominant pedigrees simply by chance.

Definitions of Dominance–Genotypes vs. Phenotypes

Critical to the understanding of the differences between autosomal dominant and autosomal recessive inheritance is the concept of genotype and phenotype.

Genotype is a shorthand statement of whether an individual’s two gene copies are normal or abnormal. A gene is normally found at a particular spot or place (locus) on a particular choromosome. Genes have different forms (alleles) that may be normal (often called wild type) or abnormal; a gene may have several or even many abnormal forms or abnormal alleles. Since an individual has only two copies of a particular gene (two chromosomes with one copy each), there are only four possible genotypes: (a) the individual has two normal alleles (normal homozygote), (b) the individual has one normal and one abnormal allele (heterozygote), (c) the individual has two abnormal alleles (abnormal homozygote), or (d) the individual has one each of two different abnormal alleles (compound heterozygote or genetic compound).

The phenotype, on the other hand, is a property of the system in which the gene operates: usually this system is the individual patient or family member. The phenotype is either normal or abnormal. For example, the patient may have the abnormal phenotype because he or she complains of symptoms of the disorder, because the patient has the physical findings of that disorder, or because the results of clinical testing methods such as roentgenography or electromyography are abnormal.

The difference between autosomal dominant and autosomal recessive inheritance can be easily explained. In both autosomal dominant and autosomal recessive inheritance patterns, normal homozygotes have the normal phenotype and abnormal homozygotes have the abnormal phenotype. The difference between the dominant and recessive patterns results from the phenotype of the heterozygote: if the heterozygote has the abnormal phenotype, the disorder is dominant; if the heterozygote has the normal phenotype, the disorder is recessive.

Dominant Lethals

This group of dominant disorders is mentioned separately because its inheritance pattern is different. Dominant lethal disorders are those in which an affected individual does not reproduce. The disorder is not necessarily lethal to the patient. However, because the patients do not reproduce, the disease cannot be transmitted from parent to child. Therefore all cases (in fact, nearly all cases) are sporadic and result from new mutation of a gamete. When one searches the literature and finds that all published cases of a disorder are sporadic, it is not reasonable to conclude that the disorder is nongenetic. A fortunate and practical result of this situation, however, is that recurrence risk for the next pregnancy is (nearly) zero for parents who have had a child affected with a dominant lethal disorder.

Penetrance and Expressivity

These features are especially characteristic of dominant rather than recessive inheritance. In individuals who are known to carry the abnormal gene but who have the normal phenotype, the abnormal gene is said to be nonpenetrant. Penetrance refers to the fraction of individuals with an abnormal allele who actually have the abnormal phenotype. The vast majority of individuals affected with dominant disorders in human populations carry a single rather than a double dose of the abnormal gene; that is, they are heterozygotes rather than abnormal homozygotes. Because of this, other factors in addition to the abnormal gene (such as the second, normal, copy of the gene; the individual’s other genes at other loci; and environmental factors) can play a greater role in determining the phenotype. Obviously, whether an individual is affected can be defined or determined in different ways. Different ways of determining the abnormal phenotype will give different numerical values for penetrance. If, for example, in a group of individuals carrying the gene for neurofibromatosis, only individuals with multiple neurofibromas are said to be affected, the penetrance will be rather low. If a careful search for café-au-lait spots is made, additional affected individuals will be uncovered. If axillary freckles and iris Lisch nodules are carefully searched for, additional affected individuals will be found. Finally, if procedures such as magnetic resonance imaging (MRI) of the spinal cord, examination for scoliosis or bony deformities, or complete autopsy are carried out, nearly all the individuals carrying the abnormal gene will be detected and the penetrance of the disorder will be high.

Expressivity is the degree of clinical involvement in an individual with the abnormal gene. It is especially characteristic of dominant disorders that two individuals, perhaps sibs in the same family, who carry the same abnormal gene may vary greatly in the severity of their disease. Obviously, nonpenetrance is an extreme form of variable expressivity.

The Sporadic Case

The most common presentation for genetic disorders in (small) human families is probably the sporadic case. If the clinician waited for a second case in a family before suspecting a genetic disorder, most genetic disorders would escape diagnosis. The following is a partial list of diagnostic possibilities for the sporadic case:

• Autosomal dominant (reduced penetrance).

• Autosomal dominant (new mutation).

• Autosomal recessive.

• X-linked recessive.

• X-linked dominant.

• Multifactorial–threshold inheritance.

• Polygenic inheritance.

• Nonpaternity.

• Adopted child.

• Nongenetic (phenocopy).

Autosomal Recessive Diseases

Pattern of Transmission

Inheritance of a disorder transmitted in autosomal recessive fashion is horizontal rather than vertical. Affected individuals are usually seen only in a single sibship (or family, accounting for the older name familial) in which the parents are unaffected but are both heterozygotes for the harmful gene. Collateral sibships are occasionally affected. Of course, disorders occurring in a sibship are not necessarily genetic; infectious disorders may cluster in a sibship. Males and females are affected with equal frequency and with equal severity in autosomal recessive conditions.

Unlike dominant disorders, autosomal recessive disorders have an increased incidence of parental consanguinity. In general, the rarer the disorder, the greater the fraction of families with parental consanguinity. A corollary of this is that rare recessive disorders are most likely to be found in inbred genetic isolates. Recessive disorders also frequently show striking ethnic predilections, a fact that is helpful in diagnosis. A corollary is that heterozygotes for some autosomal recessive disorders have increased frequency in specific ethnic groups, a fact that has made possible carrier testing for such disorders as Tay-Sachs disease, thalassemia, and sickle cell disease.

Couples at risk, that is, those where both parents are heterozygotes, are usually ascertained only after the birth of the first affected child. Further children of a couple at risk have a 25% chance of being affected and a 50% chance of being heterozygous carriers. Unaffected sibs of an affected individual have a 67% chance of being heterozygous carriers if they are old enough to be sure that they are not themselves affected. Couples at risk for having children affected with autosomal recessive disorders can be detected before the birth of the first child if a carrier test is available and if the heterozygous carriers are common or at least common in a defined ethnic group. Voluntary carrier testing for Tay-Sachs disease in individuals of Ashkenazi Jewish background has been highly successful, and classical infantile Tay-Sachs disease is now rarely seen because of amniocentesis. Half sibs of an affected individual with an autosomal recessive disease have a small chance of themselves being affected, a sharp contrast to the situation in autosomal dominant diseases in which the risk to half sibs through the affected parent is the same (50%) as that of full sibs.

Definitions of Recessive–Genotypes vs. Phenotypes

Definitions of genotypes and phenotypes are the same as those discussed above in Definitions of Dominance—Genotypes vs. Phenotypes for autosomal dominant diseases. However, recessive diseases are those in which the heterozygote is unaffected, that is, the heterozygote has the normal phenotype.

In plant or animal genetics the definitions of dominant inheritance may be somewhat different. Not only must the heterozygote be affected with a trait or disease, but the phenotype of the affected heterozygote and the affected abnormal homozygote must be identical or indistinguishable. As already mentioned, in human populations that are relatively small, abnormal homozygotes are rarely encountered and individuals affected with autosomal dominant diseases are nearly always heterozygotes.

Compound heterozygotes, that is, individuals with two different abnormal alleles at a locus, are particularly important for autosomal recessive diseases. A genetic compound may have a phenotype quite different from that of either corresponding abnormal homozygote. Consequently, in taking the family history of an apparently autosomal recessive disorder, care must be taken not to ignore a cousin or perhaps more distant collateral relative who is affected but appears to have a disorder different from that of the patient; both affected individuals may have different forms of the same disorder, perhaps an enzyme deficiency, one individual being an abnormal homozygote, the other being a genetic compound. A genetic compound may also have a phenotype identical with that of either corresponding abnormal homozygote. In fact, many or even most individuals who are apparently abnormal homozygotes with autosomal recessive disorders may in fact be genetic compounds, except where there is parental consanguinity or where the parents are part of a genetic isolate or members of a defined population where heterozygote frequency for that disorder is high. It is important to recognize genetic compounds, where possible, since carrier testing or prenatal diagnosis in that family may be difficult or impossible without special testing.

Other Features of Autosomal Recessive Disorders

Variable penetrance and variable expressivity are far less important for autosomal recessive disorders than for autosomal dominant disorders. Nonpenetrance is quite unusual, and variation in expressivity tends to be much smaller. When major differences in expressivity are seen, other explanations, such as the presence of a genetic compound, should be considered.

X-Linked Diseases

Pattern of Inheritance

X-Linked Dominant Inheritance

X-linked dominant pedigrees show vertical transmission and look like autosomal dominant pedigrees except that in X-linked dominant pedigrees: (a) male-to-male transmission does not occur, (b) all daughters of an affected male are affected, (c) females are more frequently affected than males, (d) females are less severely affected than males, (e) occasional female heterozygotes show nonpenetrance (probably as a result by chance of preponderant Lyon inactivation of the X chromosome carrying the abnormal gene).

Since X-linked dominant pedigrees look so much like autosomal dominant pedigrees, X-linked dominant inheritance can easily be overlooked unless every apparently autosomal dominant pedigree is carefully examined for the features just mentioned.

X-Linked Recessive Inheritance

X-linked recessive inheritance somewhat resembles autosomal recessive inheritance especially when only a single sibship is considered. However, in larger pedigrees the appearance is different from either [autosomal recessive] or autosomal dominant inheritance. Transmission is diagonal rather than vertical or horizontal; affected males are connected on the pedigree through unaffected females. Only males are affected, but no male-to-male transmission occurs. Occasional female heterozygotes may be affected (probably as a result of preponderant Lyon inactivation by chance of the X chromosome carrying the normal gene).

X-Linked Diseases—Genotypes vs. Phenotypes

Definition of phenotypes is the same for X-linked disorders as for autosomal disorders; and the phenotype is either normal or abnormal (affected). For females, the possible genotypes are the same as for autosomal disorders: the normal homozygote, the heterozygote, the abnormal homozygote, and the genetic compound or compound heterozygote are defined as above. However, definition of the genotypes is somewhat different for males. Since males have only a single X chromosome, they are hemizygotes; depending on whether their X chromosome carries the normal or abnormal allele, the male is a normal or abnormal hemizygote.

The Lyon Hypothesis

A body of knowledge has accumulated about the peculiar behavior of the X chromosome in mammalian females following the hypothesis of Mary Lyon. X-inactivation in the mammalian female has the following features. Early in development (about 15 to 20 days in humans) one of the female’s two X chromosomes is inactivated. The choice of which X chromosome is inactivated is random, although some disease states may affect that choice or at least the final result in the adult (for example, X-autosome translocations and some single genes such as that causing adrenoleukodystrophy). The X-inactivation is stabile, and subsequent progeny of each X-inactivated cell have the same X chromosome inactivated. Germ cell X chromosomes are not inactivated. A small part of the inactivated X chromosome remains active (or not inactivated). This concept of X-inactivation has been well documented by a large body of work. Among other things it explains why some female heterozygotes are unaffected in X-linked dominant diseases, and why some female heterozygotes are affected in X-linked recessive diseases as mentioned in X-Linked Recessive Inheritance, above.

Metabolic Interference

Metabolic interference is a postulated mechanism in which two alleles at a locus or two alleles of genes at different loci cause a harmful effect only when they are present together in the same individual. That is, in the simple form of metabolic interference, only the heterozygote is affected; both of the corresponding homozygotes are unaffected. The pedigrees that result from this are in some instances rather conventional: for example, any apparently autosomal dominant pedigree could in fact result from metabolic interference. In some instances, however, these pedigrees are strikingly unusual: for example, (a) a disorder limited to females, apparently dominant or recessive, especially a disorder passed to affected females through unaffected males; (b) a disorder occurring in all members of a large sibship with normal parents; (c) a disorder occurring in all members of a large sibship with one parent similarly affected; (d) an apparently dominant disorder with females more severely affected than males; or (e) an apparently X-linked dominant disorder in which males are not more severely affected.

A number of pedigrees have been reported that are explained by the metabolic interference hypothesis but are not easily explained by any other known pattern.

Multifactorial–Threshold Diseases

Mechanism of Multifactorial–Threshold Inheritance

These diseases are rather common in the population with frequencies of approximately 1 per thousand (0.1 to 5 per thousand births). However, they are often not recognized to be genetic because recurrence risks in a family are in the range of 0.5 to 20 per thousand, far lower than those for any of the mendelian disorders discussed above.

The mechanism involves the interaction between a continuous variable (the genetic factor) and a discontinuous variable (an environmental factor). The genetic factor may be thought of as a susceptibility to the disease; quantitatively, the degree of susceptibility is distributed in the population roughly according to the normal distribution curve. A threshold then operates on this susceptibility such that if the susceptibility is below the threshold, the phenotype is normal. However, if the susceptibility is above the threshold, the phenotype is abnormal. The presence of a threshold converts the continuous variation into discontinuous variation. Examples of disorders that fit this model are neural tube defects, cleft lip, cleft palate, psoriasis, and pyloric stenosis.

Pattern of Inheritance

As just mentioned, the pattern of inheritance is quite different from the mendelian patterns just discussed. In mendelian pedigrees, the risk to the next child is independent of the number of affected children. However, in a family with a multifactorial disease, the risk that the next child will be affected increases with the number of children in the family who are already affected. The reason is that parents are likely to have higher genetic susceptibility if two children have crossed the threshold and are affected than if only one child has crossed the threshold and is affected. Thus parents with two affected children are likely to transmit a greater genetic susceptibility to their next child than parents with one affected child. In addition, in mendelian pedigrees, the risk to the next child is independent of the disease’s severity in a relative. However, in a family with a multifactorial disease, the risk that the next child will be affected increases if the affected relative is severely affected rather than mildly affected. This is because parents are likely to have higher genetic susceptibility if a child is severely affected than if a child is mildly affected. Thus, parents of a severely affected child are likely to transmit a greater genetic susceptibility to their next child than parents of a mildly affected child.

The frequency of the multifactorial diseases characteristically varies between the sexes, different ethnic groups, and different geographical areas. For example, the incidence of pyloric stenosis is five times as great in males as in females. For anencephaly, the male to female incidence ratio is 0.6. Neural tube defects have increased incidence in Wales and Ireland.

A surprising result of these features is that risk for a multifactorial disease to relatives of a patient with that disease is higher if the patient is of the less frequently affected sex. For example, the risk of pyloric stenosis for the son of an affected father is 5.5 per thousand, while the risk to the son of an affected mother is 18.9 per thousand. The reason is that females, who are less frequently affected, must carry a greater genetic susceptibility than males in order to cross the threshold and have the normal phenotype. Therefore, an affected female is likely to carry a greater genetic susceptibility than an affected male and is likely to transmit a greater genetic susceptibility to her offspring than an affected male.

How the Clinician Can Help to Localize Genes

Molecular genetics is rapidly changing the approach to genetic diseases. This discussion focuses on disorders with unknown gene product since it is relatively straightforward to localize a gene on the genome once the gene product is known.

Gene Mapping by Restriction Fragment Length Polymorphisms

Restriction fragment length polymorphisms (RFLPs) have revolutionized human gene mapping in recent years, yet in principle the technique is little different from gene mapping using earlier markers. The two features that account for the difference are the greatly increased number of informative polymorphic markers that are becoming available for human gene mapping, and the fact that mapping with all of these markers is done with the same methods and the same equipment, that is, in the same laboratory.

Restriction Fragment Length Polymorphisms

Restriction endonucleases are bacterial enzymes that cut DNA in a sequence-specific way. These enzymes, numbering about 300 to date, have nothing to do with human DNA, but seem to be involved with the survival of one bacterial strain versus another. However, these enzymes are able to cut human DNA like other kinds of DNA, and they do this by recognizing a particular DNA sequence that they then cut. Thus these enzymes can recognize and localize certain DNA sequences, usually of four to eight base pairs.

Restriction fragments are the pieces that result after a restriction endonuclease has cut a length of DNA. The DNA that is cut may have been a small DNA fragment, phage DNA, a human chromosome, or the entire human genome. Whatever the starting material, the result is a mixture of DNA fragments of a wide variety of lengths.

Restriction fragment lengths can be determined and restriction fragments of different sizes can be separated by means of sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS–PAGE). Specific DNA sequences can be found on such a gel if the complementary sequence (the probe) is available and is radiolabeled. The gel is blotted to get the restriction fragments out of the Polyacrylamide and onto the flat surface of a nitrocellulose or nylon filter. Then the filter is soaked under specific conditions (hybridized) with a solution containing radiolabeled probe, which will bind to its complementary sequence somewhere on the filter. The location of the probe (determined by autoradiography) on the filter gives the location on the original gel of the DNA fragment it recognizes and therefore the length of the original DNA fragment, since distance of migration on SDS–PAGE is related to the size of DNA.

Restriction fragment length polymorphisms are simply polymorphic variants of restriction fragment lengths. A polymorphism is a common genetic variant that is present in some members of a population but not others. A polymorphism may be a phenotypic variant, a variant in activity or electrophoretic mobility of an enzyme, a variant in DNA sequence, or other genetic variant. A genetic variant is considered common if it is present in 1% of genes; because each individual has two copies of each autosomal gene, a variant present in 1% of genes will be present in 2% of individuals. A polymorphism, then, is a genetic variant present in a least 2% of individuals. However, a useful polymorphism should have a higher frequency with 10 to 40% of individuals carrying the minor allele. Finally, individuals carrying the polymorphic variant will have the normal phenotype in nearly all cases. The reason for this is that harmful alleles will rapidly be eliminated from the population by natural selection and brought down to frequencies of well below 1%. Therefore polymorphic variants are not likely to be harmful. A RFLP results when a polymorphic variant in a specific DNA base sequence eliminates or adds a restriction endonuclease site. If the variant sequence is in the region recognized by a known probe, then the number or position of bands recognized by the probe will be different in the variant sequence than in the normal sequence (by convention, the variant sequence usually is the one with the smaller population frequency).

Use of Restriction Fragment Length Polymorphisms for Gene Mapping

Gene mapping using RFLPs is little different in principle than gene mapping using other kinds of markers. The basic procedure is (a) to collect a kindred affected with the particular disorder, (b) to determine by clinical examination whether each individual carries the disease and by DNA blotting whether each individual carries the normal allele, the variant allele, or both, and finally (c) to compare the distribution in the pedigree of the clinical disorder and in the DNA markers to see if linkage is present.

Each time an individual produces progeny, the two copies of each gene are separated at the first meiotic division of gametogenesis and go into different cells; only one copy from that parent can be transmitted to each child. Genes on different chromosomes assort randomly during meiosis. Because of the crossing-over that takes place at the first meiotic division, genes that are far apart on the same chromosome also assort randomly. Genes that are close together on the same chromosome do not assort randomly, that is, they show linkage. However, before this process can be followed, the two copies of each of the genes being tested for linkage must be marked so that the transmission of each copy from parent to child can be followed. The informative individual is informative precisely because each of the two alleles of the two genes being tested for linkage (the disease gene and the RFLP) has been marked since the individual is heterozygous at each of the two loci.

An informative individual is one whose two alleles can be distinguished for the genes being considered. An affected individual, a heterozygote in the usual situation in which the gene for a dominant disease gene is being mapped, has one abnormal allele (causing the disease) and one normal allele and is therefore informative at the disease gene locus. To be informative for the RFLP being tested for linkage, the individual must be heterozygous at this locus, having one copy of the normal RFLP allele and one copy of the variant RFLP allele.

A final requirement is that the phase be established for that individual: that is, if the disease gene and the marker RFLP are linked, is the variant RFLP allele located on the chromosome that carries the abnormal allele of the disease gene or on the chromosome that carries the normal allele of the disease gene? Knowing the phase makes possible the construction of haplotypes (a shorthand statement of which alleles of the considered genes sit on the same chromosome of a chromosome pair) for the individual; in general, however, this is usually established only by examining the pedigree as a whole.

After the clinical state (affected or not) and the genotype of the RFLP being tested for linkage have been determined for each member of the kindred, the frequency of recombination between the disease gene locus and the RFLP gene locus is determined: 50% recombination is expected if the two gene loci are on different chromosomes or far apart on the same chromosome; 0% recombination would suggest very tight linkage between the two loci, especially if the pedigree were large so that there were many opportunities for recombination to occur.

In practice, tables or a computer program are used to calculate the logarithm of the odds ratio (LOD) at different postulated recombination frequencies from the pedigree data. The logarithm of the odds ratio is the ratio of the odds for linkage to the odds against linkage. Recombination frequency (theta) is successively assumed to be 0%, 5%, 10% … 50%, for example, and LOD is calculated at each theta. The result is a plot whose abscissa is recombination frequency and whose ordinate is the LOD. A plot in which linkage is present gives a curve with a peak at some value of theta. The height of the peak gives the LOD at the most likely recombination frequency. For example, a curve with a peak of 2 at a recombination frequence of 10% is evidence for linkage of the two loci tested at a recombination frequency of 10%; at that recombination frequency, the odds ratio is 100:1 in favor of linkage. This sounds impressive: there seems little likelihood that the result is mere coincidence. Nonetheless a LOD score of 3, an odds ratio of 1,000:1 in favor of linkage, is the usual minimum requirement for general acceptance of a linkage claim.

Since gene loci that are farther apart have a higher recombination frequency, the distance between two gene loci can be measured using recombination frequency; gene loci that have a 1% recombination frequency are 1 centimorgan apart. The distance in centimorgans is approximately linear with recombination frequency for small distances, but not with larger distances because of increasing frequency of double cross-overs. The distance in centimorgans does not show linear correlation with chromosomal length measured morphologically with banded chromosome preparations because cross-overs are more frequent near the ends of chromosomes than near the centromere.

Once linkage testing for a disease locus and an RFLP is completed, it is important to remember that any linkage established is between the two loci and not between any of the specific alleles at these two loci. Association between specific alleles is a different phenomenon called linkage disequilibrium.

If linkage was established, testing can be done with new RFLPs known to be linked to the first RFLP for more accurate mapping. If linkage was not established because the peak of the LOD curve was less than 3, then new families can be studied with the same RFLP. Since the LOD scores from the old and new families are all in the form of logarithms, the old and new data (for the same values of theta) can simply be added to give new LOD scores. When the peak of the LOD curve passes 3, linkage has been established. Of course the new families studied must all have the same disease as the original family; if some of the families appear to have the same disease but in fact have a disease not caused by the same gene, the situation will become more confused rather than more clear with the addition of new families.

If, on the other hand, linkage was not established because there was no peak of the LOD curve, then new RFLPs need to be tested for linkage.

Importance of Large Kindreds

In general, it is easier to do gene mapping with one very large family than with several large families and easier with several large families than with many small families. Therefore, the clinician should be alert for large families. Since large kindreds are relatively uncommon they are a valuable resource for research. When such a large family is ascertained, a laboratory interested in gene mapping should be contacted so that the family may have a chance to participate in research of direct benefit to them.

Gene Mapping by Chromosomal Abnormalities

Patients with genetic disorders have one or more damaged copies of a particular gene. The damage can come about in a number of ways. One way is that a DNA point mutation in a gene’s coding region can lead to an amino acid substitution in the protein gene product. Another is that a small deletion can remove part or all of a gene’s DNA sequence. A larger deletion may remove all of the DNA sequence of the gene in question plus that of one or more neighboring genes. Such a deletion may be large enough to be visible on a banded chromosome preparation. Another possibility is that the gene may be damaged when one breakpoint of a chromosome translocation cuts through the gene’s DNA sequence. Such a translocation is likely to be visible on a banded chromosome preparation.

It is likely that a subgroup of patients with nearly every genetic disease has that disease because of chromosome damage significant enough to be visible in banded chromosome preparations. Although this group is not large, it is extremely important because the site of chromosome damage gives the chromosomal location of the disease gene directly. Moreover, there are strategies for using the abnormal chromosomes from patients to clone the gene for the disease even though the gene product is unknown. Therefore, it is important to study patients with genetic disease for chromosome morphology, especially when there is something atypical about their disease. And it is important to bring such patients to the attention of medical geneticists.

References

1. Emery, AEHAn introduction to recombinant DNA. New York: John Wiley & Sons, 1984.

2. Johnson, WG. Principles of genetics in neuromuscular disease. In: Kelley VC, ed. Practice of pediatrics. New York: Harper & Row; 1979:14.

3. Johnson, WG. Metabolic interference and the +/- heterozygote. A hypothetical form of simple inheritance which is neither dominant nor recessive. Am J Hum Genet. 1980; 32:374–386.

4. Nora, JJ, Fraser, FCMedical genetics: principles and practice. Philadelphia: Lea & Febiger, 1981.

5. Old, RW, Primrose, SBPrinciples of gene manipulation. An introduction to genetic engineering. Berkeley: University of California Press, 1981.

6. Rosenberg, RNNeurogenetics: principles and practice. New York: Raven Press, 1986.

7. Vogel, F, Motulsky, AGHuman genetics. Problems and approaches. New York: Springer-Verlag, 1979.

8. Watson, JD, Tooze, J, Kurtz, DTRecombinant DNA: a short course. New York: WH Freeman, 1983.

PART ONE

DISEASES WITH AUTOSOMAL DOMINANT INHERITANCE

Outline

Chapter 2: Neurofibromatosis

Chapter 3: Tuberous Sclerosis

Chapter 4: Von Hippel–Lindau Disease

Chapter 5: Nevoid Basal Cell Carcinoma Syndrome

Chapter 6: Lentiginosis-Deafness-Cardiopathy Syndrome

Chapter 7: Hypomelanosis of Ito

Chapter 8: Other Autosomal Dominant Diseases

Chapter 2

Neurofibromatosis

VINCENT M. RICCARDI

Publisher Summary

This chapter focuses on neurofibromatosis (NF). NF is the disorder par excellence to introduce and focus attention on neurocutaneous disorders. NF in the broadest sense has come to mean the presence of multiple neurofibromas or multiple café-au-lait spots, and is to be distinguished from solitary neurofibromas and nonspecific hyperpigmentation. The specific type of NF may be apparent from the timing of the appearance of neurofibromas or their total number and distribution over the body, although often, especially in younger patients, the presence of other features, especially various types of tumors, will indicate the type of NF that is present in an individual or a family. The features that characterize NF-I are best considered from two vantage points: (1) structural or anatomic and (2) functional. Neurofibromas can occur in all parts of the body and anywhere on the skin surface, except perhaps for the glans penis. Iris Lisch nodules are pigmented hamartomas that appear to be unique to NF-I. Neurofibromas of the skin can be of minimal or trivial importance or their numbers or location can cause a variety of problems, the most common one being cosmetic. Other types of benign tumors, particularly other types of neural crest-derived tumors such as ganglioneuromas and glomus tumors may occur as a part of NF-I.

Neurofibromatosis (NF) is the disorder par excellence to introduce and focus attention on neurocutaneous disorders. On the positive side, NF epitomizes the combined involvement of the skin and various parts of the nervous system, central and peripheral, and it exemplifies general features shared with other neurocutaneous disorders, such as a genetic etiology, a frequent concern for the embryonic neural crest having a role in pathogenesis, and a frequent, if not consistent presence of benign or malignant tumors during the course of the disease. On the negative side, however, emphasis on neurocutaneous as such tends to detract from the fact that these disorders are almost always much more pleiotropic, requiring a pathogenetic explanation that goes far beyond the skin and nervous system.

In the following discussion, the emphasis is on a practical approach, in keeping with the title and intention of this book. For a more encyclopedic and general discussion, the reader is referred to the recent work of Riccardi and Eichner [1]. The material presented here derives in large part from the experiences recorded in that book, as well as from previous publications, particularly reviews such as those by Borberg [2], Crowe et al. [3], Brasfield and Das Gupta [4], Wander and Das Gupta [5], Holt [6], Riccardi [7], Samuelsson [8], and Sorensen et al. [9], plus additional data from the entire array of some one thousand patients with or at risk for NF seen through the Baylor College of Medicine NF Program from March 1978 through January 1986.

Definition and Heterogeneity

Before proceeding to what NF is, however, it must be made clear the one thing NF is not. Neurofibromatosis is not a single disorder. That is, the unqualified use of the term neurofibromatosis designates a spectrum of disorders that share many features, although no one feature or set of features is shared by all of them. On the other hand, the term neurofibromatosis is often taken to specify the disorder described in 1882 by von Recklinghausen [10]. This latter, more specific syndrome will be referred to here as NF–I [1, 7, 11], and it will be given the most attention, because it accounts for at least 85% of all NF cases. The converse is equally important: with as many as 15% of NF cases not being adequately characterized by the details of NF–I, any reasonably complete discussion must consider all types or forms of the disorder. Put another way, heterogeneity is a cardinal element of NF.

Neurofibromatosis in the broadest sense has come to mean the presence of multiple neurofibromas or multiple café-au-lait spots, and is to be distinguished from solitary neurofibromas and nonspecific hyperpigmentation (although either of these two distinctions may not always be easy or straightforward). The specific type of NF may be apparent from the timing of the appearance of neurofibromas or their total number and distribution over the body, although often, especially in younger patients, the presence of other features, especially various types of tumors, will indicate the type of NF that is present in an individual or a family. Iris Lisch nodules [12], optic gliomas (and other intracranial astrocytomas), or pseudarthroses appear to be reasonably specific for NF–I. Intracranial and spinal schwannomas and meningiomas ordinarily indicate another form of NF, the prototypic one being the acoustic form reported on extensively by Eldridge [13]. The point here is that any realistic discussion of NF immediately engenders the notion of heterogeneity. However, the pitfall in dealing with heterogeneity prematurely is that the distinctions seem to be mere hairsplitting. Thus, while heterogeneity is critical in the definition of NF, this is largely a function of the consistency of the most common form (regarding progression with age and largely unknown factors that determine expressivity). Thus, at this point we specifically address NF–I, and only later consider the other forms.

Itemization of NF–I Features

The features that characterize NF–I are best considered from two vantage points, structural (or anatomic) and functional.

Structural NF–I Features

Benign Tumors and Pigmentation

Neurofibromas can occur in all parts of the body and anywhere on the skin surface, except perhaps for the glans penis. They may also arise at sensory and autonomic ganglia, and on dorsal nerve roots, primary and secondary plexus radicles, and major, intermediate, minor, and terminal nerve components. There are basically three varieties: (a) cutaneous tumors (that is, tumors that move when the skin is moved) that are often violaceous or red in color at the surface, soft and fleshy, and sessile or pedunculated (Figure 2.1); they vary in size from several millimeters to well over a meter in circumference; they are generally not painful or tender; (b) subcutaneous neurofibromas (that is, ones that do not move when the skin above is moved) that are oval in shape (Figure 2.2), have the consistency of a hard rubber eraser, and may be painful or tender; they vary in size from several millimeters to 3 to 4 cm in their longest diameters; (c) plexiform neurofibromas, which combine elements of cutaneous and subcutaneous elements, and characteristically insinuate into adjacent normal tissue as the tumor grows, not infrequently to massive proportions; often the overlying skin demonstrates hyperpigmentation (distinct from café-au-lait spots) [14, 15], or hypertrichosis (Figure 2.3). Neurofibromas may be present at birth and develop at any time thereafter, generally with a steady increase in number and size with age. Both adolescence and pregnancy appear to increase the likelihood of new neurofibromas appearing and previous ones growing. The sexually mature adult female nipple and areola are especially likely to manifest cutaneous neurofibromas (at least 85% at age 21). (More will be said about neurofibromas below.)

Figure 2.1 Cutaneous neurofibromas on the trunk.

Figure 2.2 Subcutaneous neurofibromas on the trunk.

Figure 2.3 A diffuse plexiform neurofibroma with overlying hyperpigmentation on the arm.

Café-au-lait spots are one of the characteristic types of NF–I hyperpigmentation (Figure 2.4). Size varies with age, but they are present at almost all ages, though they often are not as readily visible in infancy as they are later. Ordinarily, by 1 year of age at least six or so café-au-lait spots are apparent, ranging in size from 15 to 150 mm or more in diameter. Most often the edges are well defined and the pigmentation is more or less uniform throughout the lesion. Freckling, that is, very small (1 to 3 mm) hyperpigmented macules present at birth, is indistinguishable by color from café-au-lait spots; congenital freckling is most commonly seen in the axillae (Figure 2.5). Freckling may also develop after birth in any of the intertrigenous regions (including the axillae), and anywhere that there is skinfold apposition (for example, in the inframammary region and in obesity skinfolds) or constant rubbing, as from underwear or persistent scratching of itching. Hyperpigmentation overlying and contiguous with the borders of a plexiform neurofibroma [15] may be the earliest indication that the neurofibroma is present. When such hyperpigmentation approaches or crosses the midline, neurofibroma involvement of the neuraxis should be presumed unless proved otherwise. In some NF–I patients the entire skin surface manifests a distinctive, somewhat darker hue than is expected on the basis of the ethnic background and skin coloration of the parents, siblings, or offspring.

Figure 2.4 Typical NF–I café-au-lait spots about the neck.

Figure 2.5 Axillary freckling typical of NF–I.

Iris Lisch nodules [12, 16] are pigmented hamartomas [17] (Figure 2.6) that appear to be unique to NF–I (with the exception of at least one patient with a more localized form of NF [18]). Their presence and number is a function of age: at age 6 years only about 10% of NF–I patients manifest them; at 29 years the proportion reaches 50%, and by the mid-sixties 100% of patients show them. Other than indicating the presence of the mutant NF gene, Lisch nodules are uninformative about the course of the disease in general. At least one case has been reported with Lisch nodules as the only expression of the mutant gene [19]. Certainly, every adult at risk for NF must have a slit-lamp ocular examination to look for these lesions before a conclusion is drawn that the gene is not present. For children, the absence of Lisch nodules is less compelling as evidence against the diagnosis because of the age differential noted above.

Figure 2.6 Iris Lisch nodules seen in an adult with NF–I.

Neurofibromas of the skin can be of minimal or trivial importance, or their numbers or location can cause a variety of problems, the most common one being cosmetic. Neurofibromas, particularly the plexiform variety, occurring elsewhere than on the skin can cause a number of problems, reflecting both the size of the tumor and disruption of the integrity of adjacent normal tissue. Discrete neurofibromas of larger nerves may lead to peripheral neuropathies. Oral (including lingual) neurofibromas may disrupt phonation, respiration, and deglutition. Diffuse plexiform neurofibromas of the face tend to be of two distinct types, though occasionally there are patients with a combination of distributions. About 5% of NF youngsters show an orbital/periorbital plexiform tumor with resultant proptosis and, sometimes, visual compromise. Other facial plexiform neurofibromas tend to have an alternative distribution, that is, below the zygomatic arch, with invasion into the buccal pad, gingivae, lingual, pharyngeal, retropharyngeal, and laryngeal regions. Infants with this type of tumor may develop sudden respiratory compromise, even if there are few or no outward signs of the tumor. For this reason, all infants with, or at risk for, NF–I should have a radiographic examination of their cervical and mediastinal anatomy. Paraspinal neurofibromas can cause problems from the pressure exerted on adjacent structures, direct invasion of the thecal space about the spinal cord, compromise of dorsal and ventral nerve roots, and erosion of vertebrae, with a resultant collapse and further disruption of spinal cord and nerve root function. Paraspinal neurofibromas in the cervical region can invade the retropharyngeal/laryngeal regions; in the thoracic region, compromise of mediastinal and other intrathoracic structures is possible, if not likely; in the lower thoracic, lumbar, and sacral regions, extensive growth in the retroperitoneal space is a potential complication; in additon, sacral neurofibromas may extend into the perineal region, distorting both the appearance and function of the internal and external genitalia, the ureters, bladder, and terminal bowel. Plexiform neurofibromas of the limbs may assume gigantic proportions, even to the point of requiring surgical amputation as the only reasonable treatment approach. Neurofibromas of the viscera may also occur, the most frequent sites being at the renal hilum, leading to one form of renovascular compromise, and the entire length of the bowel, leading to bowel obstruction or gastrointestinal bleeding. Schwannomas may rarely occur as part of NF–I, and when they do, they ordinarily only involve peripheral nerves.

Central nervous system tumors tend to be restricted to astrocytomas, the most common one being optic glioma (or pilocytic astrocytoma). Optic gliomas appear to occur in more than 15% of NF–I patients, but cause problems in only about one-third (5% of the total)[l]. Optic gliomas as part of NF–I warrant special emphasis: they result in a series of serious complications that can be prevented by the use of routine computerized radiographic (CT) or magnetic resonance (MRI) scanning and they appear to be a hallmark of this particular form of NF (Figure 2.7). That is, I am totally unaware of any patient with NF–I having an acoustic neuroma (schwannoma) or meningioma, which are the key features of other forms of NF. Simply put, we have never seen an instance of an optic glioma (or any other intracranial astrocytoma) in association with an acoustic neuroma or meningioma. Optic gliomas may be confined to either or both of the optic nerves in their intraorbital segments, or may extend into the optic chiasm. Alternatively, the chiasm may be involved and the intraorbital components spared. Chiasmal involvement may also extend into the optic tracts and radiations. Although the observations are currently incomplete, non-optic-pathway intracranial astrocytomas may be more common among patients with optic gliomas [9]. This possibility is distinct from the relatively frequent, though unaccounted-for, abnormal findings (suggestive of multiple astrocytomas) seen on MRI scans of patients studied because of an optic glioma. Central nervous system spinal cord tumors, distinct from paraspinal neurofibromas, are most unusual among NF–I