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 chromosomal 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 confirmed 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. 8). The findings in this family supported the genomic imprinting hypothesis but provided evidence against the previous model that PWS is the alternative phenotype, since the mother and the grandfather are both phenotypically normal 3 22 N. Niikawa

kb 1.2 1.0

pIR4-3R/RsaI

1. 50

0.86 0. 64

9.0 8. 2

4. 1 pTD3-2 1 /TmI

Fig. 5 Uniparental disomy in two PWS patients.

(not PWS!). Thus, we propose here a new model explaining the occurrence of PWS (Fig. 9) (Hamabe et al., 1990a). Our proposed model requires the following two assumptions: (1) The maternal gene localizd et 15q11.2 is genomic-imprinted; (2) For normal development of a child, the existence of intact (not imprinted) paternal gene is essential and the gene number ratio of the paternally-derived to the maternally-derived allele should be even or more. The latter assumption is not impractical, since in Drosopila, sex is determined by a number ratio of X chromosome to autosomes (Baker, 1989). Accepting these assumptions, our model could explain the occurrence of PWS with all the variety of chromosome abnormalities reported so far. Deletion or point mutation (normal karyotype with biparental disomy) of a paternally derived allele (Fig. 9c, d), and maternal uniparental disomy (Fig. 9e, g) would lead to PWS, while deletion or mutation (Fig. Contiguous Gene Syndromes as Multiple Anomalies Syndromes 323

Table 1 Results of studies on the parental origin of abnormal or normal chromosome(s) 15 in PWS and in AS patients

~ Parental origin

Syndromes Markers used Del (15q)/mol dela Normal 15s or t (15; 15) Totalb Paternal Maternalb Uniparental Biparental

PWS Cyto 19 3 - - 22 RFLP 4 - 8 4 16

23 3 8 4 38

AS Cyto 1 12 - - 13 RFLP 1 7 (8) - - 8 (9) CytoIRFLP - 1 - - 1

2 20 (21) - - 22 (23)

Cyto: chromosomal heteromorphisms or other cytogenetic markers; RFLP: restriction fragment length polymorphisms; a: molecular deletion; b: the numbers in parentheses are those including a pair of sibs. Data are from this paper, Butler and Palmer [1983], Mattei et al. [1984], Niikawa and Ishikiriyama [1985], Kamei et al. 119881, Donlon [1988], Knoll et al. [1989], Pembrey et al. [1989], Williams et al. [1989], Nicholls et al. [1989a,b], Magenis et al. [1990], Williams et al. [1990], Gregory et al. [1990], and Harnabe et al. [1990].

MM MP PP MP 0.tiii iiii Y PWS AS

Fig. 6 Previous model of genomic imprinting for the occurrence of PWS and AS. M and P are maternally and paternally derived chromosomes 15, respectively. 324 N. Niikawa

Chromosome 15 @'IvmIlmI I1 I)

Probe Gene locus n n n n U U U

Fig. 7 Deletion map in AS patients. The meanings of numbers and bars are same as those in Fig. 3

9a) at the same locus in the maternally derived chromosome would result in a normal phenotype. In case of molecular duplication (Fig. 9f), or an additional inverted duplication of 15q if it is derived from mother (Fig. 9h), an excess of maternally-derived alleles would disturb harmonious development, resulting in PWS. On the other hand, inv dup (15q) derived from father would proceed to develop normally, or if it involves more extended chromosome segments (Fig. lOj), it may result in abnormal phenotype other than those of PWS. In case of AS, the mechanism would be completely contrary. Although our proposed model is not absolutely simple and needs independent assumptions, it has an advantage to be able to explain all the karyotypes reported in PWS patients. Anyway, further studies are necessary to understand the molecular basis of genomic imprinting on these peculiar syndromes. Thus, a competition for gene-cloning with the use of reverse genetics techniques have just begun (Liidecke et al., 1989; 1990; Senger et al., 1990).

Beckwith-Wiedemann syndrome (BWS)

BWS (EMG syndrome) is characterized by ~exomphalos, -macroglossia, - gigantism, ear lobe grooves, craniofacial dysmorphism, and occasional hypoglycemia in infancy. Patients with BWS frequently develop Wilms tumor, the gene of which is now symbolized as WT2 different from WT1. Familial occurrence was reported in 24 families, and in these families, the disorder was transmitted in an autosomal dominant fashion of inheritance but usually through females (Niikawa et al., 1986). Duplication of llp15.5 band or seem- ingly balanced translocation with a break point of 15q15.5 have rarely been observed in sporadic patients (Fig. 10) (Waziri et al., 1983). The insulin gene (INS), the insulin-like growth factor I1 (IGF2) gene and the H-ras oncogene (HRAS) are located on 1lp15.5 (Fig. 11). Although a role of the excess genes due to dupli- Contiguous Gene Syndromes as Multiple Anomalies Syndromes 325

111

4.1

Fig. 8 A family of Angelman syndrome. All affected children (111-1,2,3), their mother (11-2), and the maternal grandfather (1-1) all lack an allele at the locus D15S10. The lack of the gene is confirmed by RFLP and densitometric analyses (a: D15SIO/Alu I; b: D14SlO/Taq I).

cation in each manifestation, such as hypoglycemia, over-growth, and the occurrence of Wilms tumor, was first doubted (Spritz et al., 1986), but it has still remained obscure. Since most BWS patients have normal karyotypes (two copies of each gene), and a few have balanced translocations (two copies) or duplication (three copies or more), the fundamental mechanism causing BWS seems similar to that presumed for PWS. A preliminary result of molecular analysis by Tonoki et al. (personal communication, 1990) on familial BWS patients with normal karyotypes suggested three copies of INS. Therefore, further studies are necessary to analyze BWS patients from the point of genomic imprinting.

Tricho-rhino-phalangeal syndrome type I (TRPS I), Langer-Giedion syndrome (LGS or TRPS II), and the exostosis gene TRPS I is an autosomal dominant disorder characterized by sparse hair, hypoplastic alae nasi, clinodac- 326 N. Niikawa

GENOMIC I IMPRINTING i

MP MP MP MM MP agr

111h

iali) ii I] I NORMAL-' PWS I!' N9,MAL OTHER FEATURES

Fig. 9 Hypothetical model to explain the occurrence of PWS with various chromosome abnormalities.

11 der(14) der(l1) der(l2) Patient 1 Patient 2

Fig. 10 Two different karyotypes in BWS patients. Patient 1 has partial trisomy for 1 lp15, and patient 2 a translocation between chromosomes 11 and 12 with a breakpoint of llp15.4.

tyly of fingers with early fusion of epiphyses, and short stature. In addition to the manifestations in TRPS I, LGS represents exostoses and mental retardation. Patients with LGS have microdeletions or translocations always involving 8q23.3-q24.13 (Fig. 12) (Buhler et al., 1980; Fukushima et al., 1983). There is another dominant disorder, the multiple cartilaginous exostoses syndrome. Three to ten percent of patients with the multiple exostoses syndrome develop chondrosarcoma after middle age. Thus, it is plausible that LGS is a contiguous gene syndrome which involves both the TRPS I gene and the exostosis gene, due to chro- Contiguous Gene Syndromes as Multiple Anomalies Syndromes 321 IIGFZHRAS1 INS I D11S12 ~HBBC CALCA PT Ii 15 3

15.1

14 3 LDHA 14 2 14 1 FSH B CAT

Fig. 11 Gene map on llp.

23 1

2: 12 TRPS I1 21 13 21 21 2122 21 23 213 ( J 8

Fig. 12 Deletion at 8q23-q24 in patients with Langer-Giedion syndrome. Karyotypes were provided by Dr. Yoshimitsu Fukushima and by Dr. Yoshikazu Kuroki. 328 N. Niikawa

Table 2 Predicted signals in TRPS I and I1 patients

8q24 clones Cells a b C

Mouse - Human + Hybrid TRPS I + Hybrid TRPS I1 +

Normal control 2 2 2 TRPS I 2 1 2 TRPS I1 2 1 1

When clones from 8q24 library are hybridized to different cells, "c" clone will be a candidate of the exostosis gene. + , - : positive and negative signals; number in each column: copy number for the clone

ptor

22.33 22.32

GiC AriC GK 22.13

22.11

21 2 .~~21 1

X del(X) DXSBL DXSlL8 OT C

i I I1 111 IV v

Fig. 13 Deletion at Xp21.2 in a carrier mothter of a patient with complex glycerol kinase deficiency, and deletion map in 5 patients. Contiguous Gene Syndromes as Multiple Anomalies Syndromes 329

A B

Fig. 14 Chromosome microdissection. Chromosome 8 (arrowhead) before (A), during (B), and after (C) dissection.

(IM-6. Narishige)

Inverted microscope (IMT-2. Olympus)

Fig. 15 Inverted microscope equipped with micromanipulators and a glass needle. 330 N. Niikawa

mlcrodissection 'of chromosome

MboI (Sau3AI) digestion

20mer primer 1 1 lher linker I I MboI fragment

I primer/linker ligation

-GACATGGATC-ATCCATGTC 3' CTGTACCTAGFTAGGTACAG- 5 '

filling recessed 3' ends tI

5 ' -GACATG---.---CATGTC------3' 3' ------CTGTAC GTACAG- 5'

1 PCR

3' 5' 3' 5'

Fig. 16 Strategy of PCR of DNA dissected from chromosome 8.

mosome microdeletions. Moreover, it is likely that the exostosis gene itself is an onco-suppresser gene, and a hemizygous condition may lead to multiple exostoses, a nullizygous condition may result in chondrosarcoma. It is thus essential to clone this gene to know the mechanism of its occurrence. The cell line from a TRPS I patient who has deletion at 8q23-q24 which seems identical to that in LGS is available (Yamamoto et al., 1989). The existence of such a patient has an advantage for gene cloning, because it implies that the deletion in this patient is expected not to involve the exostosis gene but the TRPS gene (Fig. 12). Therefore, if the 8q23-q24 band-specific DNA libraries can be made from the cells of both patients, and if clones representing one copy in LGS patients but two copies in the TRPS I patient are select- ed, these clones would be candidates for the exostosis gene or flanking DNAs to the gene (Table 2). A strategy of the cloning with microdissection/microcloning as a reverse genetics technique will be described blow. Contiguous Gene Syndromes as Multiple Anomalies Syndromes 331

Complex glycerol kinase deficiency (CGKD) CGKD is an X-linked recessive disorder characterized by hyperglycerolemia and hyperglyceroluria due to a deficiency of an enzyme, glycerol kinase, severe myopathy, congenital adrenal hypoplasia (AHC), gonadotropin deficiency (GTD), and mental retardation. CGKD is sometimes associated with osteoporo- sis. Glycerol kinase deficiency (GKD) itself is a single gene defect (McCabe, 1983). Clinical course of the myopathy in CGKD suggests that it is indistinguishable from Duchenne muscular dystrophy (DMD) (Mat- sumoto et al., 1988). Also interesting is that adrenal hypoplasia is another X-linked single gene defect. When these findings are combined, it is quite reasonable to presume that CGKD is an X-linked contiguous gene syndrome, involving all of the genes responsible for GTD, GKD, AHC, DMD. Chromosome microdeletion at Xp21.2 was found in the X chromosome of CGKD patients and in their carrier mothers (Fig. 13) (Saito et al., 1986). Molecular analyses also showed deletions in CGKD patients (Fig. 13) (Francke et al., 1987; Matsumoto et al., 1988). The deletions almost always involve the 3’ end portion of the DMD gene. However, the deletion ranges differ in different patients, being consistent with different phenotypes among patients (Fig. 13). A common deletion site among patients with molecular deletions are between the 3’ end of the DMD gene and DXS68, where the genes for glycerol kinase and CAH are expected to be located. From these data, the genes involved are presumed to be ordered as pter- GTD-AHC-GK-DMD-cen. Chromosome walking on the basis of the gene order is now proceeding.

APPROACH TO THE ISOLATION OF THE GENES RESPONSIBLE FOR CONGENTAL MALFORMATION SYNDROMES

Microdissection of chromosome and microcloning of DNA Since one human chromosome is composed, in average, of 10-100 Mb of DNA, the cloning of DNA in a defined region is tough to work. Microdissection/microcloning may overcome this problem. One or two chromosome bands at 800-1,000 band stage are dissected (Fig. 14) with a fine glass needle attached to a micromanipulator under an inverted microscope (Fig. 15). The chromosome preparation for dissection should be made with the avoidance of long-exposure of chromosomes to acetic acid which is used as one component of fixative, because the acetic acid exposure may result in depurination in the chromosomal DNA. Ten to 20 dissected chromosome pieces are collected into a 1 nl water ball under a microscope, and then digested with proteinase K. The DNA extracted with phenol-chroloform method within the water ball is digested with an endonuclease, Sau3AI. The Sau3AI digestion protrudes four-bases [(5’) GATC (3’)] at the 5’ end. The 5’ end is ligated to a 20mer oligonucleotide through a lOmer linker as shown in Fig. 16. Then, the ligation product undergoes polymerase chain reaction (PCR) using the 20mer oligonucleotide as a universal primer (Fig. 16). The PCR product is then cut with EcoRI, ligated to the EcoRI site of pUC19, and cloned. Some several thousands to several hundred thousans DNA clones are obtained from one fine chromosome band. These are so-called chromosome band-specific DNA library, from which the gene isolation starts.

ACKNOWLEDGEMENTS

I express my gratitude to Drs. Yoshikazu Kuroki, Kiyoshi Imaizumi, Tateo Sugimoto, Tomoko Hasega- wa, Kohji Narahara, Tetsuo Miki, Yoshimitsu Fukushima, Nobutake Matsuo, Toshiro Nagai, Akira Yoshio- 332 N. Niikawa ka, Hidefumi Tonoki, Ryuichi Tsukino, Atsuko Yamaguchi, Yoshinori Izumikawa, and to many other doctors for providing blood materials of patients. I aslo debt to late Professor S.A. Latt and American Type Culture Collection (ATCC) for providing probes. Finally, I thank staffs of our laboratory, Drs. Jun- ichi Hamabe, Naoki Harada, Kyhoko Abe, Deng Han-Xiang for their helps for the studies.

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