Reverse genetics and human disease

Reverse genetics and human disease

Cell, Vol. 47, 845-850, December 26, 1986, Copyright 0 1988 by Cell Press Reverse Genetics and Human Disease Stuart H. Orkin Division of Hematolo...

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Cell, Vol. 47, 845-850,


26, 1986, Copyright

0 1988 by Cell Press

Reverse Genetics and Human Disease

Stuart H. Orkin Division of Hematology/Oncology Children’s Hospital and Dana Farber Cancer Institute Howard Hughes Medical Institute Harvard Medical School Boston, Massachusetts 02115

Until recently, molecular analysis of inherited disorders of man has proceeded largely through identification and characterization of specific proteins and their corresponding genes. This approach has been highly successful, as best exemplified by the precise molecular descriptions of thalassemia syndromes (reviewed by Orkin and Kazazian, 1984) and familial hypercholesterolemia (reviewed by Brown and Goldstein, 1986). Although these disorders (among others) have provided important insights into RNA metabolism and receptor function, respectively, progress in understanding these processes rested on recognition of the affected proteins-globins and the LDL-receptorprior to molecular analysis. Many inherited disorders of man display interesting phenotypes for which adequate biochemical explanations are lacking. Often there are no animal models that faithfully mimic the human disease. In these situations the application of “reverse genetics,” the isolation of a gene without reference to a specific protein or without any reagents or functional assays useful in its detection, may permit access to complex cellular systems. Of late, much has been written regarding the theoretical and practical aspects of identifying a gene responsible for an inherited disorder by this approach. The logic is simple in principle. First, establish the map position of the gene and then identify a specific gene within this region in which mutations are strictly correlated with the disease. Restriction fragment length polymorphisms (RFLPs) (Botstein et al., 1980) in combination with cytogenetic methods, provide the key to map assignment with a resolution of roughly several million base pairs (White et al., 1985a; 1986). Identifying the gene of interest is the practical issue. For purposes of discussion, I consider identification of an RNA transcript (or cDNA) and its disruption (or altered expression) in a disorder to be essential elements of a genetic argument that a disease-gene locus has been found. Here I review recent results on three human disorders for which this general approach appears to have achieved initial success, and then discuss specific aspects that relate more generally to the analysis of other disorders. Duchenne



Duchenne muscular dystrophy (DMD), an X-linked disorder of unknown etiology affecting about 1 in 3000 males, is characterized by progressive muscle wasting. Based on cytologically detectable chromosome abnormalities, X-autosome translocations giving rise to the disease in females, and linkage analysis using RFLPs, the DMD gene


has been assigned to the short arm of the X chromosome at band 21 (Xp21) (Murray et al., 1982; Davies et al., 1985; lngle et al., 1985; de Martinville et al., 1985). Two approaches have forged a path to the DMD gene. First, very closely linked RFLP markers have been sought among cloned, anonymous DNA fragments in the hope that structural changes in the DMD gene would become evident as the locus was entered. Second, the breakpoint of an X-autosome translocation has been cloned in the belief that the translocation interrupts the DMD gene. Ingenious use of material from a rare patient who was affected with several diseases--MD, chronic granulomatous disease (CGD), retinitis pigmentosa, and McLeod syndrome-and who carried an interstitial deletion in Xp21 (Francke et al., 1985), led Kunkel and his associates (Kunkel et al., 1985) to the DMD locus. Normal human DNA digested with the restriction enzyme Mbo I was reassociated in the presence of a vast excess of sheared patient DNA, and then cloned in an effort to enrich for DNA sequences encompassed by the deletion; this represented about 0.1% of the human genome, or ~3000-5000 kb (van Ommen et al., 1986). One of six fragments derived from Xp21 (designated pERT87) detected DNA deletions in about 5% of classical DMD patients and was tightly linked to the disease in family studies (Monaco et al., 1985). Approximately 200 kb of contiguous DNA in the PERT 87 region (now termed the DXS164 locus) was isolated by chromosome walking procedures. Deletion breakpoints in DMD patients occur heterogeneously over the entire expanse (Kunket et al., 1986). Concurrently, Worton and colleagues isolated the breakpoint region (called XJ) from an X-autosome translocation by relying on the observation that a ribosomal RNA gene cluster on chromosome 21 was split in the process (Worton et al., 1984; Ray et al., 1985). The XJ probe, like pERT87, detected deletions in some classical DMD patients and was linked tightly to the disease. Interestingly, recombination between XJ and pERT87, and between the disease and these markers, is seen occasionally (4%-80/o of meioses). Recent data demonstrate specific deletion of the XJ region in some DMD patients and suggest that the XJ and pERT87 regions lie, at most, 400 kb apart (R. Worton, personal communication). To search for RNA transcripts of the DMD gene, Kunkel and associates identified nonrepetitive DNA segments within DXS164 that are highly conserved across species (Monaco et al., 1986). One particular segment (pERT8725) located 70 kb distal to the original pERT87 clone, hybridized at high stringency with chicken, as well as rodent and primate, DNA. Sequencing of the homologous human and mouse segments suggested the existence of an exon bounded by RNA splicing signals. The pERT87-25 probe detected a large (~16 kb) RNA species in human fetal muscle that was absent from several other tissues (lymphocytes, fibroblasts). Partial cDNA about 1 kb in length was derived from more than 100 kb of genomic DNA in the DXS164 locus. If this intron/exon ratio were maintained

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throughout the entire DMD gene, the transcribed region might span more than 1000 kb of Xp21. The evidence strongly suggests that the partial cDNA obtained represents a portion of the authentic DMD transcript. In particular, the unusually large size of the transcript and its corresponding gene are consistent with the huge size of the DMD gene, as defined by heterogeneous DNA breakpoints throughout the DXS164 and XJ regions and recombination of the disease with DNA segments within the gene itself. The identification of exons in the XJ region that are contained within the 16 kb RNA transcript from the DXS164 locus is needed to support the contention that the DMD gene is directly interrupted by X-autosome translocations. Demonstration that the 16 kb RNA transcript is structurally abnormal in the muscle of an affected patient with a deletion entirely contained within the defined DXS164 region would provide additional direct evidence for the relevance of the transcript to the disorder. No data exist to relate the DMD transcript to a specific protein. Plausible candidate proteins, however, are two very large skeletal muscle polypeptides, titin (2000 kd) and nebulin (600 kd), which appear to be important for stability of the sarcomere during contraction (Horowits et al., 1986). Chronic Granulomatous


Chronic granulomatous disease (CGD) is an inherited disorder that affects the bacteriocidal capacity of phagocytic cells (granulocytes, monocytes, and eosinophils) (Tauber et al., 1983). Affected individuals have greatly impaired host defenses against a variety of microorganisms. In contrast with the normal situation, phagocytes from affected patients fail to generate superoxide on ingestion of microbes, due to an unknown defect in a plasma membraneassociated NADPH-oxidase system. The most common form of the disorder is X-linked. Despite nearly two decades of effort in many laboratories, the components required for normal function of the oxidase system remain incompletely characterized. One candidate protein for the X-linked form of the disorder, a low potential b-type cytochrome, emerged on the basis of its consistent spectral absence from phagocytes of individuals with the X-linked disease, but not the recessive form (Segal, 1985; Segal et al., 1983; Harper et al., 1985). The extent to which this association is causal genetically has been uncertain, since an intimate relationship between the cytochrome and an authentic product of the X-CGD gene might be required for the integrity and functioning of the oxidase system. In view of the difficulties inherent in dissecting this complex system by conventional methods, the cloning of the X-CGD gene was approached by genetic means. At the outset, the position of the gene on the X chromosome was uncertain, although a distal Xp location had been proposed. Cytogenetic and molecular study of a female patient with an X chromosome deletion (Francke, 1984) and the Xp21 DMDlCGD patient described above (Francke et al., 1985) suggested a more proximal location, that is, within Xp21. Study of a second DMD/CGD patient with a

deletion within Xp21 and formal linkage analysis of classical CGD patients established an Xp21 location, centromere proximal to the DMD locus (Baehner et al., 1986). A search for the transcript of the X-CGD gene was performed in a different manner from that described above (Royer-Pokora et al., 1986). As part of the expansion of Xp21 PERT clones, bacteriophage clones hybridizing with segments other than pERT87 were obtained which encompassed at most 10% of the entire region deleted in the DMDlCGD patients. Since one of these might fall by chance within the X-CGD gene, an enriched cDNA pool was hybridized directly to a Southern blot of bacteriophage DNAs. Constitutive and non-Xp21 sequences were removed from cDNA prepared from phagocytic cells (granulocyte-like, chemically induced HL60 leukemic cells) by subtractive hybridization with RNA from a 6 cell line of a DMDlCGD Xp21-deletion patient. The subtracted cDNA, representing about 500 mRNA sequences, identified a DNA segment common to overlapping bacteriophage clones within a region defined by pERT379. The hybridizing segment of the bacteriophage detected a phagocytespecific 4.7 kb mRNA that was absent from the monocytes of 3 of 4 classical X-CGD patients and that was structurally abnormal in the remaining patient due to a small interstitial deletion encompassing the C-terminal 41 amino acids of the predicted protein. Absence of expression and disruption of the 4.7 kb RNA species in affected patients provide direct evidence that the RNA is relevant to the disorder. The predicted X-CGD protein derived from the cDNA sequence is 54-56 kd (468-486 amino acids), depending on which of two potential in-frame AUGs is used in vivo for initiation of translation (Royer-Pokora et al., 1986). If the more 5’ proximal AUG were used, a cleavable twenty amino acid signal sequence would be present for intracellular targeting (T Creighton, personal communication). Several potential N-glycosylation sites are present in the predicted protein. Two other features of the deduced protein are striking. First, it does not resemble other known proteins, including reported cytochromes. The presence of weak homology between a region near the C-terminus with a cytochrome P450 (Ashworth et al., 1986) is of uncertain significance. Therefore, there is no evidence that the predicted X-CGD protein corresponds to the candidate b-cytochrome. Second, the X-CGD protein sequence is highly basic (calculated pl = 9.5). Whether the predicted X-CGD protein corresponds to a cluster of basic polypeptides of m48 kd that are phosphorylated upon treatment of granulocytes with phorbol ester (Hayakawa et al., 1986) is under study. Alternatively, the X-CGD protein may serve as the membrane-anchoring subunit of a multicomponent cytochrome b complex. These issues will be resolved once antibody reagents to the X-CGD protein are prepared. Retinoblastoma Retinoblastoma (Rb) is a rare childhood malignancy that exhibits both sporadic and hereditary forms (Knudson, 1978). The hereditary form is transmitted as an autosomal dominant trait. Deletion and family studies have shown

Review: Reverse Genetics a47


of Genetic

and Human Disease

Loci for Duchenne

Affected tissue Search for aene Localized to chromosomal deletion Linkage to region of deletion “Random” clone detected deletions at increased frequency Chromosome walking of >lOO kb Search for RNA transcript Via interspecies sequence conservation Via enriched cDNA RNA transcript Tissue specificity Abundance Size of transcribed region Patients carry internal deletions within transcribed region Encoded protein Predicted size Potential for functional proof by phenotypic correction



Chronic Granulomatous

Disease, and Retinoblastoma







a+ +

+ +

+ + + -

+ +


+ + muscle


O.Ol%-0.1% >150 kb

0.05% ~30 kb

absent from retinoblastomal osteosarcoma very low >70 kb




54-56 +


a The gene for DMD has also been localized to chromosomal

kb +


that the enzyme esterase D is tightly linked to the retinoblastoma locus, on chromosome 13, band q14 (Sparkes et al., 1983). Two observations-that deletion of this chromosomal segment predisposes to retinoblastoma and that loss of heterozygosity at 13q14 occurs somatically in tumor tissue (Cavenee et al., 1983)-have led to the proposal that the Rb locus encodes a recessive oncogene. Mutations at the Rb locus are also thought to underlie the development of osteosarcoma (Hansen et al., 1985). Isolation and characterization of the Rb gene should permit a direct test of this model and may provide novel insights into cancer. The focus of most efforts has been to acquire DNA markers within 13q14 (Cavenee et al., 1984; Lalande et al., 1984). Recently, Dryja et al. (1986) noted that a sequence termed H3-8, previously isolated from a flow-sorted chromosome 13 bacteriophage library (Lalande et al., 1984) detected DNA deletions in a subset of retinoblastomas. Two patients had somatically occurring, homozygous deletions extending for greater than 25 kb. Other previously isolated, anonymous DNA fragments from 13q14 did not detect deletions in those patients deleted for H3-8. These observations suggested that H3-8 was within or near the Rb locus. Friend et al. (1986) observed that a single-copy DNA fragment in the immediate vicinity of H3-8 hybridized with a 4.7 kb RNA species that was present in low abundance in retinal cells (immortalized with adenovirus) and in all other tissues, but was absent from all retinoblastomas and osteosarcomas examined. The RNA transcript is derived from more than 70 kb of genomic DNA. Somatically arising deletions (some interstitial and some homozygous) and rearrangements within the transcribed region were seen in 30% of retinoblastomas and osteosarcomas. Deletions entirely contained within the tran-

scribed region most strongly suggest that the Rb gene has been identified. Taking advantage of the presumed proximity of the esterase D locus to the Rb gene, other investigators elected to clone esterase D cDNA as a directed approach to the Rb locus (Lee and Lee, 1986; Squire et al., 1986). Ongoing chromosome walking from the esterase D gene may soon provide a physical estimate of its distance from the region defined by H3-8. No data are available on the nature of the protein encoded by the Rb gene transcript. Although the genetic evidence very strongly implicates the loss of this transcript in tumor formation, it is not yet known whether acquisition of function will revert tumor cells to a normal phenotype. The extent to which this can be achieved may depend on the nature of additional events that intervene in the formation and progression of this cancer. Common Themes What common themes and lessons emerge from the above? Features of the DMD, CGD, and Rb loci are presented in the table. The contribution of cytological deletions that pinpointed the chromosomal location of each gene cannot be overstated. These deletions served not only to localize the genes, but were essential reagents in enriching for specific genomic fragments in the work of Kunkel et al. (1985) and in choosing anonymous chromosome 13 probes within a subregion of the chromosome (Dryja et al., 1986). Similarly, the availability of a translocation into Xp21 offered an alternative route to the putative DMD gene (Worton et al., 1984). Perhaps somewhat surprising is that without application of new technology such as pulsed field gradient electrophoresis and extended

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chromosome cloning (Collins and Weissman, 1984; Smith et al., 1986) essentially randon DNA clones (pERT87, pERT379, and H3-8) fell within each locus, rather than a “hop” away. Although this certainly reflects some element of chance, the rather large sizes of the Rb gene and the DMD megagene makes this occurrence more likely. In the search for RNA transcripts, anticipated patterns of tissuespecific expression and exon conservation across species have been particularly useful. The relevance of each RNA transcript to its respective disorder is directly established by deletions and rearrangements contained entirely within the transcribed regions. For the present, these constitute the most direct evidence of success. Formal proof must ultimately rest, where possible, on functional assays based on transfer of the normal version of a gene into phenotypically abnormal cells. For CGD and retinoblastoma, the availability of fulllength cDNA and appropriate target cells makes this a realistic goal; whereas for DMD, given the very large size of the mRNA, the uncertainty about what phenotype to score upon attempted correction of deficient cells, and limited availability of target cells, this approach may be restricted. That the first disorders for which success has been achieved include two X-chromosome conditions and an inherited cancer may not be strictly fortuitous. New mutations in X-chromosome lethals (DMD and CGD), and strong selection for phenotypically important somatic mutations in the Rb gene during genesis of retinoblastoma, may greatly contribute to the extensive diversity of structural gene defects among affected patients. Recognition of this diversity has been instrumental in identifying each locus. Other Disorders The three conditions described here are but a subset of the many inherited disorders of man for which molecular genetic approaches are being applied. Those highlighted by cytological deletions or translocations, associated with unusually large genes, or frequently disrupted by structural aberrations appear especially tractable, while a complete human gene map is being developed (White et al., 1986). For example, Wilms’ tumor (WT), which is associated with deletion of 11~13 in the rare aniridia-WT syndrome and reduction to homozygosity in sporadic cases (Fearon et al., 1984; Koufos et al., 1984; Orkin et al., 1984) bears striking similarities with Rb in its genetics. Additional evidence suggests that other embryonal tumors, such as hepatoblastoma and rhabdomyosarcoma, result from mutations on chromosome 11 (Koufos et al., 1965) presumably at the WT locus. The recent recognition that the gene for the 8-subunit of follicle-stimulating hormone lies near the putative WT locus (Glaser et al., 1986) should encourage direct efforts to identify the relevant region. Possible homology of the Rb gene with sequences within 11~13 will surely be examined in this regard. Localization of a testis-determining gene to interval 1 by deletionmapping of the Y-chromosome (Vergnaud et al., 1986; Page, 1986) suggests that identification of this fascinating

gene and its product should also be feasible. Finally, use of a deletion syndrome (Wolf-Hirschhorn syndrome) to assign an anonymous probe linked to Huntington disease to distal 4p (Gusella et al., 1983; 1985) implies that a more limited chromosomal region can be the focus of further investigation of that disease. For the common disorders polycystic kidney disease and cystic fibrosis (CF), prospects for molecular resolution in the near future are less certain, although recent history suggests that predictions of this kind may be short-lived. DNA segments tightly linked to both inherited disorders have been identified (Reeders et al., 1985; Tsui et al., 1985; Knowlton et al., 1985; White et al., 1985b; Wainwright et al., 1985). For CF, the most severe common disorder among Caucasians, the search has been particularly intense. At least two (possibly flanking) markers within 1% recombination of the gene are known (Beaudet et al., 1986). These might lie within several million bp of the locus, if recombination is not unduly suppressed in the region of chromosome 7q in which the CF gene is thought to reside. Without sizable DNA deletions or rearrangements in CF patients (neither of which appear to be forthcoming), identification of the gene may rely on saturation cloning of the DNA between flanking markers and analysis of all RNA transcripts derived from this region. Careful attention to specific expression in affected tissues (sweat glands, lungs, and pancreas) and comparison of cDNA sequences from normal and CF patients will be required. Given recent data that suggest abnormal regulation of epithelial cell chloride channels in CF, rather than a defect within the channel itself (Frizzell et al., 1986; Welsh and Liedtke, 1986), molecular approaches to the CF gene product seem the most promising route at present. Recent experience underscores the enormous contribution of cytogenetics, careful pedigree analysis, and cooperation among investigators to successful identification of a disease gene without reference to specific protein products. In each of the instances described above, close collaboration and sharing of resources between laboratories was a prominent feature. A new phase in the application of molecular genetics to the dissection of human inherited disease has begun. Beyond direct clinical application of newly derived DNA probes, the success of “reverse genetics” in the analysis of human disease will be measured largely by what can be learned about normal cellular biology and physiology. The question of the hour now shifts from “how do we get there or know when we are there?” to “what can we learn once we have arrived?”

I am thankful to the numerous individuals who provided preprints and discussed their findings prior to publication. Without their cooperation a timely review would not have been possible. I am also most grateful to Lou Kunkel for his close collaboration and interaction during the development of the CGD project in my laboratory. Work from my laboratory was supported by agrant from the NIH. S. H. Orkin is an Investigator of the Howard Hughes Medical Institute. References Ashworth, A., Shephard, E. A., and Phillips, I. R. (1986). Disease gene relationship seen. Nature 322, 599.

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and Human Disease

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