American Journal of Medical Genetics 85:361364 (1999)

Revertant Mosaicism in Human Genetic Disorders

Marcel F. Jonkman*
Department of Dermatology, University Hospital Groningen, Groningen, The Netherlands

Somatic reversion of inherited mutations is known for many years in plant breeding, however it was recognized only recently in humans. The concept of revertant mosaicism is important in medical genetics. Am. J. Med. Genet. 85:361364, 1999
1999 Wiley-Liss, Inc.

KEY WORDS: reverse mutation; mosaicism; review; skin

INTRODUCTION A revertant is a mutant that has regained, partially or completely, the wild-type phenotype by either genetic or nongenetic mechanism of reversion [Rieger et al., 1976]. Genotypic revertants fall into two classes: 1) true revertants represent a genuine reversal of the original mutational event. A so-called back or reverse mutation changes the original forward mutation either back to the original base or to a third one, thereby restoring the amino acid sequence of the wild-type polypeptide. The genetic mechanisms involved in reversion are true back mutation (reverse point mutation), crossing-over, and gene conversion [Green, 1959] (Fig. 1). 2) Revertants resulting from second site mutations, which take either place inside or outside the mutated gene, without changing the nucleotide of the original mutation. Such mutations restore the activity of the mutated gene but frequently lead only to partial reversion. The mechanism of second site mutations include base pair addition or deletion, suppressor mutation, and chromosomal loss or gain. If the reversal occurs in cells of the germ line carrying a mutation, the revertant allele may be transmitted to the offspring that will not express the trait caused by the mutation, e.g., germ-line revertant mosaicism. Reversion in germ line cells is encountered in genetic dis-

orders caused by increased numbers of short tandem repeats in the untranslated 3 region of the RNA molecule, such as myotonic dystrophy [Brunner et al., 1993], in unstable duplicated genes, such as the mouse pink-eyed unstable mutation [Schiestl et al., 1994], and in transgene inserts of transgenic mouse lines [Trudel et al., 1994]. If the reverse mutation occurs in a somatic cell, only part of the organism will be revertant. This is called somatic revertant mosaicism. In contrast, somatic forward mosaicism, a forward mutation in a somatic cell resulting in an organism part of which is mutant, is the classical nevus type of mosaicism [Hall, 1988; Happle, 1993; Paller et al., 1994]. This review describes revertant mosaicism of genetic disorders caused by reversion in somatic cells. REVERTANT MOSAICISM Somatic reversion of gene expression in plant tissue has been known for centuries by horticulturists, for example in purple sectors on white flowers of ornamentals. Even then, it was recognized as a genetic trait in that variegating lines could be bred as such [Darwin, 1868; De Vries, 1910]. Molecular evidence of somatic reversion has been demonstrated in maize [McClintock, 1948; Peterson, 1953] in which mobile elements, called transposons, are recognized as the driving force [McClintock, 1987]. In Drosophila, somatic reversion accounts for wild-type sectors recurring in mutant strains [Green, 1967]. In animals, somatic reversion was first encountered in hypothalamic neurons of the diabetes insipidus Brattleboro rat (di/di), which has impaired vasopressin synthesis because of a recessive single-base deletion in the vasopressin gene [Van Leeuwen et al., 1989]. The revertant neurons had switched to the heterozygous di/+ phenotype, their number increasing from 0.1 to 3% with age. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes. Reverse mutation has also been documented in dystrophin deficient mdx mice [Hoffman et al., 1990] and in humans with Duchenne muscular dystrophy [Klein et al., 1992]. In the latter, the mechanism was demonstrated to be an in-frame skipping of the exon containing the frameshift mutation (transcriptional gene deletion) [Sherratt et al., 1993]. The mosaic phenotype in dystrophin deficiency was not clinically rec-

Contract grant sponsor: Jan Kornelis de Cock Stichting. In part presented at the International Symposium Mosaicism in Human Skin on the occasion of the 60th birthday of Rodolph Happle, Marburg, Germany, 2223 May 1998. *Correspondence to: M.F. Jonkman, M.D., Ph.D., Department of Dermatology, University Hospital Groningen, Hanzeplein 1, P.O.Box 30.001, NL-9700 RB, Groningen, The Netherlands. E-mail m.f.jonkman@derm.azg.nl Received 11 August 1998; Accepted 23 November 1998

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Fig. 1. Diagram of possible mechanisms of reverse mutation during mitosis in a diploid cell that is compound heterozygous for an autosomal recessive mutation. Homologous chromosomes carry the paternal (light gray) or maternal (dark gray) mutation. In each situation, loss of heterozygosity homozygosity or hemizygosity is reached along the altered tract. Cells devoid of the protein are depicted white, and cells with a revertant phenotype are depicted black solid for producing functional protein and hatched for producing semifunctional protein. Note, gene conversion and double crossingover may result in the same revertant genotype, however in the latter the revertant cells may theoritically be accompanied by unique sister cells. It can be anticipated that these cells will disappear because of selective disadvantage. Adapted from Jonkman et al. [1997] with permission of the publisher.

ognizable and only demonstrable by immunohistochemistry. The second site mutation that caused the reversion led to a shortened protein, the immunoreactivity of part of which was preserved, but which was functionally impaired and thus remained clinically unnoticed. Table I summarizes recently discovered somatic reversion events in human genetic disorders. A true back mutation was demonstrated to cause reversion to normal of a deleterious missense mutation on the maternal allele in a severe combined immunodeficiency (SCID) patient who was a compound heterozygote for autosomal recessive adenosine deaminase (ADA) deficiency [Hirschhorn et al., 1996]. According to

the authors, the patients condition progressively improved because of the selective growth advantage of the revertant lymphocytes in vivo. Somatic reversion has also been reported in liver nodules in patients with autosomal recessive disorder, tyrosinaemia type I caused by fumarylacetoacetate hydrolase (FAH) deficiency [Kvittingen et al., 1994]. The molecular mechanism underlying the reverse mutation remained unresolved, although in some patients it could not be on account of intragenic gene conversion because the mutation was homozygous. The first sample of somatic reversion by (single) cross-over of the affected gene is in Bloom syndrome, an autosomal recessive disorder of

TABLE I. Revertant Mosaicism in Human Genetic Disorders Genetic disorder Duchenne muscular dystrophy Tyrosinemia type I Trisomic fertilization rescue Monosomy X rescue Bloom syndrome Severe combined immunodeficiency syndrome Generalized atrophic benign epidermolysis bullosa Fanconi anemia Gene DMD FAH BLM ADA COL17A1 FAC Genetic mechanism In-frame exon skipping by Unknown mechanism Unknown Somatic loss of chromosome Somatic gain of chromosome Intragenic recombination True back mutation Gene conversion In-frame 2-nucleotide insertion Gene conversion Cell type Myocyte Hepatocyte Zygotic cell Zygotic cell Lymphocyte Lymphocyte Keratinocyte Keratinocyte Lymphocyte Reference Klein et al., 1992, Sherratt et al., 1993 Kvittingen et al., 1994 Robinson et al., 1995 Robinson et al., 1995 Ellis et al., 1995 Hirschhorn et al., 1996 Jonkman et al., 1997; Darling et al., 1998 Lo Ten Foe et al., 1997

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Fig. 2. Clinically unaffected patches of skin in a patient with generalized atrophic benign epidermolysis bullosa. Patches of skin that never blister (outlined with a solid line) show normal pigmentation. Adapted from Jonkman et al. [1997] with permission of the publisher.

chromosomal stability characterized by high rates of exchange of genetic material between sister chromatids [Ellis et al., 1995]. In patients with Fanconi anemia, somatic reversion is manifested by the presence of two populations of lymphocytes, one of which is hypersensitive for cross-linking agents (mitomycin C), whereas the other behaves normally in response to these agents [Lo Ten Foe et al., 1997]. The molecular basis for the revertant phenotype was recently disclosed in three Fanconi patients who were compound heterozygous for the FAC gene; in one patient intragenic cross-over had occurred between the paternal and maternal allele and in the other two the mechanism appeared to be gene conversion [Lo Ten Foe et al., 1997]. The hematological symptoms in these mosaic Fanconi patients were relatively mild. REVERTANT MOSAICISM IN SKIN The first clinically visible revertant mosaic in humans was described by Jonkman et al. [1997] in a 28year-old women with generalized atrophic benign epidermolysis bullosa (GABEB) (McKusick #22665), a form of autosomal recessive junctional epidermolysis bullosa characterized by continuous blistering since birth with alopecia, dental anomalies, and nail dystrophy [Jonkman et al., 1996]. The patient had mosaic patches of clinically normal skin in which blisters could not be evoked even after vigorous rubbing (Fig. 2). The authors found that the patient was a compound heterozygote for truncation mutations in COL17A1, the gene for type XVII collagen (BPAG2, BP180): a cell-matrix adhesion molecule located in the hemidesmosomes of stratified squamous epithelia. The affected skin of the patient was completely devoid of type XVII collagen, whereas in the normal skin patches type XVII collagen was present in clusters of basal cells. The authors demonstrated that the mosaic phenotype in this patient was caused by reversion of one of the mutations in the COL17A1 gene. The mechanism behind the reversal was gene conversion: the nonreciprocal transfer of part of one parental allele for the other. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381

base pairs in revertant keratinocytes not in fibroblasts derived from the normal mosaic skin patches; the paternal mutation (R1226X) remained present in all cell samples. Loss of heterozygosity by single crossover or chromosomal nondisjunction were excluded by demonstrating heterozygosity of flanking polymorphic markers. Revertant mosaicism is an example of natural gene therapy and validates the potential success of the gene correction approach by recombinational repair [Cole Strauss et al., 1997]. In the revertant mosaic GABEB patient reversion of the affected genotype to carrier genotype of approximately 50% of the basal keratinocytes appears to be sufficient to normalize skin function locally. The GABEB patient noticed that some of the normal skin patches were new and that they slowly grew bigger over the years, whereas other patches had been present as long as she could remember and remained stationary. In contrast, the normalization with age of the phenotype in patients with SCID or Fanconi anemia with somatic reversion [Hirschhorn et al., 1996; Lo Ten Foe et al., 1997] suggest that selective growth advantage of revertant hematological cells plays a major role in the prognosis of mosaic conditions in these genetic hematological disorders. Recently, revertant mosaicism was demonstrated again in a patient with GABEB [Darling et al., 1998]. The patient homozygously inherited a 2-nucleotide deletion mutation of COL17A1 that shifts the reading frame leading to an upstream premature termination codon [McGrath et al., 1996]. Although the whole skin of the patient was affected and no mosaic skin patches were clinically apparent, immunoreactive type XVII collagen was present in clusters of basal cells in skin specimens [Pohla-Gubo et al., 1995]. Analysis of the revertant keratinocytes by laser capture microdissection of the revertant epidermal cells demonstrated a second 2-nucelotide insertion mutation 3 of the deletion mutation, which had restored the reading frame in these cells [Darling et al., 1998]. It was deduced that the revertant type XVII collagen polypeptide contained a unique stretch of 26 amino acids corresponding to the coding region between the inherited deletion mutation and the somatic insertion mutation. This phenomenon was an example of a revertant by intragenic second site mutation. Because mosaicism was not perceived clinically, the revertant type XVII collagen polypeptide was apparently nonfunctional, similar as in revertant mosaicism caused by second site mutations in Duchenne muscular dystrophy. COMMENT For the medical geneticist and dermatologist it is important to consider revertant mosaicism in cases with extended nevoid skin lesions. One also should look for patches of normal skin in generalized inherited pathological skin conditions. If mutational analysis is possible for the condition under examination then DNA samples of several cell types should be compared for presence of the mutation. If the mutation is present in cell types not related to the phenotype (e.g., peripheral blood cells) while in the affected skin a revertant and a

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1996. Generalized atrophic benign epidermolysis bullosa: Either 180kD bullous pemphigoid antigen (BP180) or laminin-5 is deficient. Arch Dermatol 132:145150. Jonkman MF, Scheffer H, Stulp R, Pas HH, Nijenhuis M, Heeres K, Owaribe K, Pulkkinen L, Uitto J. 1997. Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 88:543551. Klein CJ, Coovert DD, Bulman DE, Ray PN, Mendell JR, Burghes AHM. 1992. Somatic reversion/suppression in Duchenne muscular dystrophy (DMD): Evidence supporting a frame-restoring mechanism in rare dystrophin-positive fibers. Am J Hum Genet 50:950959. Kvittingen EA, Rootwelt H, Berger R, Brandtzaeg P. 1994. Self-induced correction of the genetic defect in tyrosinemia type I. J Clin Invest 94:16571661. Lo Ten Foe JR, Kwee ML, Rooimans MA, Oostra AB, Veerman AJP, Van Weel M, Pauli RM, Shahidi NT, Dokal I, Roberts I, Altay C, Gluckman E, Gibson RA, Mathew CG, Arwer F, Joenje H. 1997. Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance. Eur J Human Genet 5:137148. McClintock B. 1948. Mutable loci in maize. Carnegie Inst Wash Year Book 47:155169. McClintock B. 1987. The discovery and characterization of transposable elements: The collected papers of Barbara McClintock. New York: Garland Pub. McGrath JA, Darling T, Gatalica B, Pohla-Gubo G, Hintner H, Christiano AM, Yancey KB, Uitto J. 1996. A homozygous deletion mutation in the gene encoding the 180-kD bullous pemphigoid antigen (BPAG2) in a family with generalized atrophic benign epidermolysis bullosa. J Invest Dermatol 106:771774. Paller AS, Syder AJ, Chan Y-M, Yu Q-C, Hutton E, Tadini G, Fuchs E. 1994. Genetic and clinical mosaicism in a type of epidermal nevus. N Eng J Med 331:14081415. Peterson P. 1953. A mutable pale green locus in maize. Genetics 38:682 683. Pohla-Gubo G, Lazarova Z, Giudice GJ, Liebert M, Grasegger A, Hintner H, Yancey KB. 1995. Diminished expression of the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) in the epidermal basement membrane of patients with generalized atrophic benign epidermolysis bullosa. Exp Dermatol 4:199206. Rieger R, Michaelis A, Green MM. 1976. Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th edition. Berlin, Heidelberg, New York: Springer. p 478. Robinson WP, Binkert F, Bernasconi F, Lorda-Sanchez I, Werder EA, Schinzel AA. 1995. Molecular studies of chromosomal mosaicism: Relative frequency of chromosome gain or loss and possible role of cell selection. Am J Hum Genet 56:444451. Schiestl RH, Khogali F, Carls N. 1994. Reversion of the mouse pink-eyed unstable mutation induced by low doses of X-rays. Science 266:1573 1576. Sherratt TG, Vulliamy V, Dubowitz V, Sewry CA, Strong PN. 1993. Exon skipping and translation in patients with frame shift deletions in the dystrophin gene. Am J Hum Genet 53:10071015. Trudel M, Chretien N, DAgati V. 1994. Disappearance of polycystic kidney disease in revertant c-myc transgenic mice. Mamm Genome 5:149152. Van Leeuwen F, Van der Beek E, Seger M, Burbach P, Ivell R. 1989. Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat. Proc Natl Acad Sci USA 86: 64176420.

mutant cell population are found, than revertant mosaicism is imposed. DNA analysis is not possible for genetic disorders of which only the chromosomal locus is known because studies of DNA polymorphism cannot be conclusive as far as reversion of the mutation is concerned. The most powerful argument, which enables the clinician to establish the diagnosis of reversion, is that in revertant mosaicism the parents carry the mutation and other sibs are affected. ACKNOWLEDGMENTS This work was supported by the Jan Kornelis de Cock - Stichting. I thank Dr. Clifford Weil (Moscow, ID) for his helpful comments on revertants in botany. REFERENCES
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