Molecular Basis and Approach to Gene Cloning

Cong. Anorn., 30: 317-333, 1990 Review Contiguous Gene Syndromes as Multiple Anomalies Syndromes: Molecular Basis and Approach to Gene Cloning Norio NIIKAWA Department of Human Genetics, Nagasaki University School of Medicine, Sakamoto- Machi 12-4, Nagasaki 852, Japan ABSTRACT Recent knowledge on molecular basis of several contiguous gene syndromes as multiple anomalies syndromes, such as Prader-Willi syndrome (PWS), Angelman syndrome (AS), Beckwith-Wiedemann syndrome (BWS), tricho-rhino-phalageal syndrome types I (TRPS I) and I1 (TRPS I1 or LGS), and complex glycerol kinase deficiency (CGKD), are reviewed. Based on the results of DNA deletion studies and on the evidence for the genomic imprinting mechanism of both PWS and AS, a model for the occurrence of the two syndromes is proposed. Also, a strategy of the microdissection/microcloning technique as a reverse genetics technique, i.e., direct cloning of chro- mosomal DNAs from a defined region of human chromosome, particularly for the cloning of the exostosis gene in TRPS, is presented. Key words: contiguous gene syndromes, Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome, tricho-rhino-phalangeal syndromes, complex glycerol kinase deficiency, genomic imprinting, microdissection/microcloning technique, reverse genetics, exostosis gene A number of congenital malformation syndromes are known. In the London Dysmorphology Data Base (Winter et al., 1987), more than 1,700 different syndromes have been registered, and most of them are genetic diseases. To understand the fundamental causes, cloning of the genes responsible for the syndromes is essentially necessary. However, although their pathogenesis has been studied, in almost all the syndromes the molecular basis remains unknown. Therefore, as far as following the conventional “forward genetics” techniques for gene cloning, it is almost impossible to isolate such genes. On the other hand, the “reverse genetics” techniques have recently been developed (Kunkel et al., 1985; Liidecke et al., 1989). The reverse genetics implies the direction of genetics study, from a gene locus through gene isolation toward function of its product, that is the reversal direction of orthodox genetics. Theoretically, this strategy makes the isolation of the gene for the malformation syndromes possible, if the gene locus is known. Contiguous gene syndromes as malformation syndromes have advantages, because most of them represent minute chromosome deletions in defined chromosome regions in which each gene responsible for each clinical manifestation is thought to be located. In this paper, current advancements on several contiguous gene syndromes, Received August 23, 1990 Presented in the invited lecture at the 30th Annual Meeting of the Japanese Teratology Society, Miyazaki, July 12, 1990. %JIIZ%, A~k~~~~~~~~R~B~WRll~~~~~~~,t852 Al%fmthi*~lZ-4 318 N. Niikawa especially on cytogenetic and molecular-genetic findings, are reviewed. Also, a strategy to clone DNAs from the chromosome regions responsible for the syndromes by the use of chromosome microdissection- microcloning technique as one of the reverse genetics techniques is presented. CONTIGUOUS GENE SYNDROMES Contiguous gene syndromes are defined as a group of clinical entities due to comprehensive mutations of genes located contiguously in the genome [Schmickel, 1986; Emanuel, 1988; Niikawa, 19891. There- fore, they represent several clinical manifestations together, which are more than those explainable by pleiotropism of a single gene. Each manifestation is usually related to each gene mutation. Microdeletions or microduplications of chromosomes are often observed in patients with this kind of disorders. In this case, multiple anomalies are the main manifestations. Several possible contiguous gene syndromes with malformations have been known. They include tricho-rhino-phalangeal syndrome type I (TRPS I) and Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome type 11) (TRPS 11) both due to microdeletion of 8q23-q24 band, Beckwith-Wiedemann syndrome probably due to duplication of I lp15.5, aniridia- Wilms tumor association due to deletion of 1 lp13, retinoblastoma-malformation complex due to deletion of 13q14, Prader-Willi syndrome and Angelman syndrome both due to deletion of 15qll-ql2, Miller-Dieker syndrome due to deletion of 17~13.3,DiGeorge syndrome due to deletion of 22q11.2, Rud syndrome due to deletion Xp22.3, and complex glycerol kinase deficiency due to deletion of Xp21.2. Chromosome microrearrangements observed in these syndrome not only give useful information for understanding their pathogenesis, but also clues of isolations of genes responsible for the syndromes. Prader-Willi syndrome (PWS), Angelman syndrome (AS), and genomic imprinting PWS is a multiple congenital anomalies and mental retardation (MCA/MR) syndrome characterized by severe hypotonia and feeding difficulty in infancy, polyphagia leading to obesity beginning in child- hood, craniofacial dysmorphism, short-stature, hypopigmented skin and hair, small hands and feet, hypogonadism, and mental retardation. A half or more of PWS patients have microdeletion at 15qll-q12, most likely at band 15q11.2 (Fig. l), while the other half have normal karyotype (Ledbetter et al., 1982). DNA deletions are also detected with DNA probes derived from the 15qll-q12 region (Fig. 2) (Donlon et al., 1986; Kamei et al., 1988; Donlon, 1988; Nicholls et al., 1989a; 1989b; Gregory et al., 1990; Hamabe et al., 1990a). In our recent study (Hamabe et al., 1990a), 32 (61.5%) of the 52 patients studied showed such molecular deletions, but deletion ranges differed among them (Fig. 3). Patients in whom no molecular deletions were detected often lack some phenotypes, such as “small hands and feet” and “hypopigmented skin and/or hair”. Furthermore, Angelman syndrome, a clinically distinct syndrome from PWS but represents del(15) (qllq13), shows several features overlapping the PWS phenotype, such as hypotonia, a jolly mood, hypopigmentation of the skin, obesity, short stature, and mental retardation (Fig. 4). These findings may support that PWS, and possibly AS, are contiguous gene syndromes. Parental origin of de novo chromosome deletions have been studied, and the origin is preferentially paternal (Table 1) (Butler and Palmer, 1983; Niikawa and Ishikiriyama, 1985). Curiously, a recent molecular study with RFLPs showed that the chromosomes 15 in patients with normal karyotypes are both derived from their respective mothers, i.e., uniparental hetero- or homodisomy (Nicholls et al. 1989b). We also con- firmed the uniparental homodisomy in two PWS patients (Fig. 5) (Hamabe et al., 1990a). From these find- Contiguous Gene Syndromes as Multiple Anomalies Syndromes 319 normal del td55) inv invdup mos Fig. 1 Various chromosome abnormalities associated with Prader-Willi syndrome (PWS) and Angelman syndrome. PWS AS WSB 1 2 3 4 5 1415161718 123 4 5 6 1 1 C kb 6.4 w 4.6 pPAl 2.2 p3-21 11111 22222112222222 Fig. 2 Molecular deletions in patients with PWS or with Angelman syndrome (AS). The numbers at the top and at the bottom of the Southern blots are case numbers and gene-copy numbers, respectively. Probes, pML34 and p3-21, are located at 15q11.2, and a probe, pPAl (internal control) is at 18qll-qI2. ings in PWS patients, a hypothesis was induced that a loss of the paternally-derived alleles at 15qll-q12 region may lead to the PWS phenotype (Nichols et al., 1989b). By contrast, microdeletions of the same region at both the chromosome and the molecular levels were 320 N. Niikawa Chromosome 15 Probe Gene locus Fig. 3 Deletion map in PWS patients. The numbers on the left are the number of patients examined. Closed bars and open areas depict the presence and the absence of DNA, respectively, thick bars the duplicated DNA, dot- ted bars the DNA not examined, and stippled bars uniparetal disomy. also found in Angelman syndrome (AS) patients (Donlon, 1988; Magenis et al., 1990; Knoll et al., 1990; Imaizumi et al., 1990; Hamabe et al., 1990b). The frequency and the deleted chromosome sizes are almost comparable between the two syndromes (Gregory et al., 1990; Knoll et al., 1989; 1990, Hamabe et al., 1990a; 1990b). However, the parental origin of the deleted chromosomes is contrary between the two; the origin in AS is almost exclusively maternal (Table 1) (Knoll et al., 1989; Magenis et al., 1990; Williams et al., 1990). An interesting “genomic imprinting” hypothesis has recently been proposed that parental genes of the same locus are differently marked (modified through methylation?) and gene expressions in offspring are different between the paternally-derived and the maternally-derived alleles (Reik, 1989; Hall, 1990). Evi- dence for the imprinting has been provided in the rodent (Swain et al., 1987). Applying this hypothesis Contiguous Gene Syndromes as Multiple Anomalies Syndromes 32 1 pter I other PWS features I hypogonadism hypopigmented skin feeding difficulty in infancy pol y -hyperphagia obesity scoliosis small hands and feet mild short stature hypotonia mental retardation jolly mood microcephaly seizure I other AS features I qter chromosome 15 Fig. 4 A hypothetical model for overlapping clinical manifestations between PWS and AS. to PWS and AS, the occurrence of PWS could be explained by the loss of a paternally-derived allele, while that of AS by the loss of a maternally-transmitted allele (Fig. 6) (Nicholls et al., 1989b; Williams et al., 1990). This model was based on the assumptions that the genes for the two syndromes are same, and the alternative phenotype for PWS is AS. However, our recent molecular studies on PWS and on AS provided evidence against this model (Hamabe et al., 1990a; 1990b). Hamabe et al. (1990a; 1990b) showed that a common site of molecular rearrangements among PWS patients is confined to the segment between the two loci, D15S9 and D15S12 (Fig. 3), that is different from the common site (the segment between D15S11 and D15S10) of deletion among AS patients (Fig. 7), indicating that the gene loci for the two syndromes are different. Moreover, RFLP analysis on one AS family revealed that all the 3 children with AS, their mother, and a maternal grandfather all have deletions at the locus D15S10 (Fig.

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