Linkage to Fucosidosis - NCBI - NIH

Am. J. Hum. Genet. 43:749-755, 1988

Restriction Analysis of the Structural a-L-Fucosidase Gene and Its Linkage to Fucosidosis John K. Darby, ' t Patrick J. Willems, $' § Phil Nakashima, * Jeff Johnsen, * Robert E. Ferrell, Ellen M. Wijsman,* Daniella S. Gerhard,# Nicholas C. Dracopoli,# David Housman,# Jurgen Henke, * * Michael L. Fowler,I' t Thomas B. Shows,t T John S. O'Brient and Luca L. Cavalli-Sforza* Departments of *Genetics and tNeurobiology, Stanford University School of Medicine, Stanford; tDepartment of Neurosciences, University of California School of Medicine, San Diego; §Department of Medical Genetics, University of Antwerp; IIDepartment of Biostatistics, University of Pittsburgh; #Center for Cancer Research, Massachusetts Institute of Technology, Cambridge; **Laboratorium fur Forensische Blutgruppenkunde, Dusseldorf; and ttDepartment of Human Genetics, Roswell Park Memorial Institute, Buffalo

Summary Human a-L-fucosidase is a lysosomal enzyme responsible for hydrolysis of a-L-fucoside linkages in fucoglycoconjugates. A single gene, FUCA 1, located on chromosome lp34.1-lp36.1 encodes for a-L-fucosidase activity. To gain insight into the nature of the molecular defects leading to fucosidosis, we have characterized the genomic structure of FUCA 1. Restriction-endonuclease analysis suggests that at least seven exons dispersed over 22 kb are present in genomic FUCA 1. Two restriction-fragment-length polymorphisms (RFLPs) have been identified in the Caucasian population. The PvuII and BgII RFLPs each have two codominant alleles in Hardy-Weinberg equilibrium. Allele frequencies for the PvuII RFLP are .70/.30, and those for the BgdI RFLP .63/.37. Both RFLPs are in strong linkage disequilibrium with each other, with a correlation coefficient of .94. The polymorphism information content (PIC) of the combined DNA markers is .38, high enough to be useful in the prenatal diagnosis of fucosidosis. The combined lod score for linkage between the fucosidosis mutation and FUCA 1 markers in two families was significant at a recombination fraction of 0. This suggests that the fucosidosis mutation resides in FUCA 1.

Introduction

Human a-L-fucosidase (a-L-fucoside fucohydrolase; E.C.3.2.1.51) is a lysosomal enzyme that hydrolyzes a-fucose from glycolipids and glycoproteins (Warner and O'Brien 1983). We have previously cloned and sequenced several cDNAs for the structural gene encoding human a-L-fucosidase (FUCA 1) (de Wet et al. 1984; Fukushima et al. 1985). The compiled sequence (1,829 bp) encodes approximately 90% of the mature enzyme. It is complete at the 3' end with a polyadenylation sequence and a poly (A) + tail but lacks sequences encoding the NH2 terminus of fucosidase at the 5' end Received November 5, 1987; revision received May 16, 1988. Address for correspondence and reprints: Dr. John K. Darby, Department of Genetics, Stanford University, School of Medicine, Stanford, CA 94305. i 1988 by The American Society of Human Genetics. All rights reserved. 0002-9297/88/4305-0023$02.00

(O'Brien et al. 1987). Using this cDNA we assigned FUCA 1 to lp34.14p36.1 by in situ hybridization and Southern blot analysis of somatic cell hybrids (Fowler et al. 1986). In addition, a region with homology to the fucosidase gene was identified on chromosome 2 and designated FUCA 1L (Fowler et al. 1986). This was confirmed by Carritt and Welch (1987), who showed that the FUCA 1 cDNA is homologous to at least 650 bp of the genomic FUCA 1L sequence. The function of this FUCA 1L region on chromosome 2 is not clear, but it does not encode for a-L-fucosidase enzyme activity (Turner et al. 1978; Carritt and Welch 1987). A third locus, FUCA 2, perhaps linked to the plasminogen gene (Eiberg et al. 1984) on chromosome 6 (Murray et al. 1987), is thought to regulate the fucosidase activity in plasma (Willems et al. 1981) and fibroblasts (Van Elsen et al. 1983), although it apparently does not encode for the fucosidase enzyme (Turner et al. 1978). 749

750

Deficient activity of a-L-fucosidase (Van Hoof and Hers 1968) leads to fucosidosis (Durand et al. 1966, 1969; Kousseff et al. 1976). The fucosidosis null allele Fuo was shown to be coallelic with the electrophoretic fucosidase alleles Fui and FU2 (Turner et al. 1975a), which were mapped to chromosome 1 (Carritt et al. 1982), in close linkage with Rh (Corney et al. 1977). Therefore, it seems probable that the fucosidosis mutation resides in FUCA 1. We present here two FUCA 1 RFLPs, demonstrate their linkage to fucosidosis, and suggest their usefulness as prenatal diagnostic markers. Material and Methods 1. Southern Blot Analysis

Buffy coats were prepared from whole blood obtained from normal Caucasian individuals, Caucasian families affected with melanoma, and two Caucasian families affected with fucosidosis. Informed consent was obtained for all samples. Cultured fibroblast cell lines from 10 fucosidosis patients were obtained from referring physicians (Willems et al. 1988a). The buffy coats and fibroblast pellets were suspended in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 7.5), 100 mM NaCl, 0.5 % SDS, and 100 gg proteinase K/ml. This mixture was incubated at 55 C overnight, followed by treatment with 100 gg RNase A/ml for 1 h at 37 C and by phenol, phenol:chloroform (1: 1), and chloroform extractions. DNA was precipitated with 0.3 M sodium acetate in ethanol, collected by centrifugation, and resuspended in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Samples of DNA (10-15 gg) were digested with a panel of restriction enzymes and electrophoresed in 0.85% agarose in 89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA at 0.5 V/cm. Capillary transfer of DNA fragments to Nytran (Schleicher and Schuell) filters was performed according to the method of Southern (1975). The filters were hybridized to three adjacent DNA fragments (probes A-C) of a cDNA encoding the human structural a-L-fucosidase gene FUCA 1 (O'Brien et al. 1987). The probes were radiolabeled to specific activities of 3-5 x 108 cpm/pg (Feinberg and Vogelstein 1983). Prehybridization, hybridization, washing, and autoradiographs were performed as described elsewhere (Feder et al. 1985). 2. Restriction-Endonuclease Analysis of Genomic FUCA I DNA was digested with BglI, BglII, EcoRI, HincII, HindIII, Hinfi, MboI, MspI, PvuII, RsaI, Sau3A, SacI, TaqI, TthIII, and XbaI. The Southern blots were suc-

Darby et al.

cessively hybridized to each of the three adjacent cDNA probes A-C. 3. RFLP Search

Genomic DNA of eight unrelated Caucasians was digested with AvalI, BamHI, BglI, BglII, EcoRI, EcoRV, KpnI, MboI, PstI, PvuII, RsaI, SacI, TaqI, and XmnI. The Southern blots were hybridized to probe B. Because of a shortage of DNA, fucosidosis patients and their families were typed only for PvuII (shown to be polymorphic in the normal population) and for EcoRI (shown to be polymorphic only in fucosidosis patients; Willems et al. 1988a). Frequency estimates were obtained by screening 80 independent controls for EcoRI, 122 controls for PvuII, and 46 controls for BglI. 4. Chromosomal Localization of the Pvull RFLP

Twenty-five human-mouse hybrid cell lines from 14 different human cell lines and four different mouse cell lines were analyzed for their specific human chromosome content (Shows and Brown 1975). DNA isolated from those hybrid cell lines was digested with PvuII and subjected to Southern blotting and subsequent probing by DNA fragment B. 5. a-L-Fucosidase Isoelectric Focusing

Leucocytes were harvested from heparinized blood samples by the dextran method. The cell pellets were lysed by repeated freeze/thawing cycles prior to neuraminidase treatment, isoelectric focusing, and staining for a-L-fucosidase activity, as described elsewhere (Turner et al. 1975b; Henke and Netzer 1982). 6. Linkage Analysis between Fucosidosis and FUCA I

The linkage studies were performed on two Caucasian families affected with fucosidosis. Family M consisted of 14 individuals of whom two were affected and whose pedigree is cited by Turner et al. (1975a). Family LB consisted of 63 individuals of whom three were affected and whose pedigree is cited by Willems et al. (1988b). FUCA 1 PvuII and EcoRI RFLPs, Ful-2, Rh, and PGM1 typing was done on most but not all individuals, and this was limited owing to sample availability. The genotype with respect to the fucosidosis mutation in the two fucosidosis families was ascertained through pedigree analysis and results of the isoelectric phenotyping of Q-L-fucosidase. An electrophoretic phenotype, Fu1 2, excludes heterozygosity for fucosidosis, as the silent Fuo is coallelic with the Fui and F2 alleles (Turner et al. 1975a). Linkage analysis between the fucosidosis mutation and the relevant protein and

Gene Structure of a-L-Fucosidase

751

DNA markers was performed using the LIPED program (Ott 1974, 1976). Results 1. Restriction-Endonuclease Analysis of Genomic FUCA I

Restriction-endonuclease mapping was used to construct an approximate physical map of genomic FUCA 1. The Southern blots were successively hybridized to the three adjacent probes (A-C) composing the available cDNA (1,829bp) for FUCA 1. The number and localization of the different exons was estimated by comparison of the DNA fragments obtained by digests with 15 different restriction endonucleases. This analysis indicates that genomic FUCA 1 has at least seven exons dispersed over 22 kb. 2. RFLP Search

Using probe B we observed in genomic DNA from eight normal unrelated Caucasians no polymorphic bands for AvalI, BamIII, BgIH, EcoRI, EcoRV, KpnI,

MboI, PstI, RsaI, Sad, Stul, TaqI, and XmnI. In addition, none of 80 controls had the EcoRI polymorphism described in the patient population (Willems et al. 1988a). PvuII identified a two-allele polymorphism (Darby et al. 1986) with bands at either 7.0 kb (Pi) or 6.0 kb (P2) and four invariant bands at 3.0, 4.5, 8.6, and 19 kb (fig. 1). The differential 1-kb fragment was not detected, thereby indicating that it is composed of intron DNA sequence. BglI identified (1) a two-allele polymorphism with either one band at 12 kb (B1) or two bands at 6.5 and 5.5 kb (B2) and (2) two invariant bands at 3.2 and 7.2 kb (fig. 1). Both RFLPs are in Hardy-Weinberg equilibrium with X2 values of 1.94 (P > .1) and 0.02 (P > .5), respectively. Codominant inheritance for the PvuII and Bg1I RFLPs was seen in six families (96 individuals). Allele frequencies for the PvuII RFLP studied in 122 random unrelated Caucasians were .70 (Pi) and .30 (P2). The Bg1I RFLP had allele frequencies of .63 (B1) and .37 (B2) in 46 random, unrelated Caucasians. In six families (96 individuals) informative for both the PvuII and Bg1I RFLPs the maximum lod score in favor of linkage between both

Pvu II size (kb)

F

C1 C2

Bgl I

M

F

C1 C2

M

size (kb)

19.0 12.0

8.6 7.0 6.0

7.2 6.5 5.5

4.5

3.0

Figure I

Autoradiograph of Southern blots of genomic DNA digested with PvuII or BglI and hybridized to probe B. Sizes of the fragpolymorphic bands are 6.0 or 7.0 kb. BglI detects one band ot 12 linkage disequilibrium of these RFLPs can be seen in this family. mother, F = father; C1 and C2 = children.

ments are given in kilobases, as judged by log-scale graphs. The PvuII kb or two bands of 5.5 and 6.5 kb each. Mendelian inheritance and

M

=

3.2

Darby et

752 RFLPs was 8.29 at a recombination fraction (0) of .001. Both RFLPs were in strong linkage disequilibrium with each other (D = .208), with a correlation coefficient (r) of .94 (X2 = 67.07; P < .001) (table 1). The two RFLPs are < 1 kb apart. THE PIC value (Botstein et al. 1980) of the two RFLPs was .33 for PvuII and .36 for BglI. The PIC for the combined RFLPs was .38. Linkage disequilibrium was also detected between the PvuII RFLP and the a-L-fucosidase protein polymorphism (P = .008 with the Fisher exact test). The PvuII 6-kb allele was associated with Fu2, and the PvuII 7kb allele was associated with Ful.

bined maximum lod score was 0.95 at 0 = 0 (table 2). If the EcoRI RFLP lod score in family M is summated with the PvuII RFLP lod score in family LB, then the maximum lod score for the two families rises to 1.35 at 0 = 0. Haplotype analysis did not add to these lod scores, as the larger family, LB, did not have the EcoRI RFLP. Since the only hypotheses of interest in this example are r = .0 and r = .5, the "classical" lod score of 3.0 is not relevant, as that was based on random searching for linkage. Wald's (1947) lemma shows that the P value corresponding to the observed lod score of 1.35 is 1/22.4 = 0.045 when 22.4 is the antilog of 1.35, and linkage is therefore significant at the P < .05 level. The maximum lod score of 2.4 at 0 = 0 was obtained in family M, between Fu12 and fucosidosis, by use of a standard recessive matrix for LIPED. The PvuII-versus-Fuv2 lod score was 0.49 at 0 = 0, and the EcoRI-versus-Fulv2 lod score was 0.60 at 0 = 0 in family M. Use of the PvuII/EcoRI haplotype in family M causes this lod score to rise to 1.43 at 0 = 0. Corroborative results between Rh and FUCA 1 markers and fucosidosis were also obtained, and these results support FUCA 1 and fucosidosis linkage. Results are shown in table 2. PGM1 and FUCA 1 markers were also tested and showed loose linkage (results not shown).

3. Chromosomal Localization of the Pvull RFLP

Cosegregation of the polymorphic 6.0- or 7.0-kb PvuII bands with specific human chromosomes in 25 human-mouse hybrid cell lines was demonstrated. One hundred percent concordance with chromosome 1 was observed. this assigns the polymorphic PvuII bands to chromosome 1. Further localization to the 1p21-lpter region was obtained by analysis of two cell hybrid lines, JWR-22 R and JWR-266, which contain a t(1:2) reciprocal translocation (Fowler et al. 1986). The 19-, 4.5-, and 2.8-kb bands always cosegregated with the polymorphic 6.0- or 7.0-kb band. Discordancy of all above-mentioned bands with chromosome 2 (50%) and chromosome 6 (52%) excluded linkage to the fucosidase homologous site (FUCA 1L) on chromosome 2 (Fowler et al. 1986; Carritt and Welch 1987) and the FUCA 2 locus on chromosome 6 (Eiberg et al. 1984; Murray et al. 1987), respectively. The weak signal of 8.6 kb probably maps to the FUCA 1L region with 8% discordancy (results not shown).

5. Linkage Disequilibrium in Fucosidosis

Twelve unrelated fucosidosis patients from four ethnic origins and 122 controls differed significantly (X2 = 10.2; P< .005) in the frequencies of the PvuII alleles (table 3A). The frequency of the 6.0-kb allele was .62 in the patient population and .30 in the control population. Patients and controls also differed in the frequency of the EcoRI polymorphism (table 3B; P = .00001 with Fisher exact test). Within the control population it is not possible to estimate linkage disequilibrium for the EcoRI and PvuII

4. Linkage between Fucosidosis and FUCA I

The PvuII RFLP segregated concordantly with the fucosidosis mutant gene in two different families (families M and LB) affected with the disease. The comTable I

x2 Test of Linkage Equilibrium for Pvull and BgIl Alleles BgII ALLELE

(kb)

(kb)

Observed

Expected

x2

12.0

50

.12.0

0

33.52 14.36 19.68

67.07 (df = 1; P < .001)

6.0 7.0 6.0

HAPLOTYPES

PvuII ALLELE

7.0 ............. ........ ............

Total

6.5-5.5 6.5-5.5

al.

2 24

76

8.44

76

753

Gene Structure of a-L-Fucosidase Table 2 Lod Scores for Recombination Fractions between FUCA I Linked Polymorphisms and Fucosidosis

RECOMBINATION FRACTION MARKERS (Family)

.00

.05

.10

.20

.30

.40

Fucosidosis and PvuII (2) ........ Fucosidosis and EcoRI (M) ...... ....... Fucosidosis and Ful-2 (M) FU1-2 and PvuII (M) ............ FU1-2 and EcoRI (M) ..... ...... FU1-2 and PvuII/EcoRI (M) ...... Rh and fucosidosis (2) ..... ..... ....... Rh and PvuII (LB) ...... ....... Rh and EcoRI (M) ...... ........ Rh and Ful-2 (M) ......

.95 .60 2.41 .49 .60 1.43 1.37 1.67 .30 .60

.80 .55 2.17

.66 .51 1.92 .34 .49 1.10 .91 1.22 .21 .43

.44 .41 1.40 .19 .39 .77 .52 .80 .13 .27

.26 .29 .86 .09 .28 .46 .23 .42 .06 .13

.12 .16 .34 .02 .16 .20 .05 .12 .02 .03

alleles, because EcoRI is monomorphic in the controls. However, within the patient population, there is significant linkage disequilibrium between these two markers (table 3C; P = .019 with the Fisher exact text). The correlation between these two markers is .38, as estimated from the gametic association D = .109 (Cavalli-Sforza and Bodmer 1971, p. 285). Discussion

Preliminary restriction-endonuclease analysis of genomic FUCA 1 suggests the presence of at least seven exons dispersed over 22 kb. It is likely that the 5' end of genomic FUCA 1 contain additional ex3ns, as the cDNA used in the restriction-enzyme mapping is incomplete. The compiled total length of the isolated cDNAs is 1,829 bp, including the poly (A)+ tail, whereas the FUCA 1 mRNA is 2.3 kb (O'Brien et al. 1987). Consequently this cDNA lacks approximately 500 bp at the 5' end, which likely represent additional exons in genomic FUCA 1. A precise picture of the genomic structure will have to await cloning and sequencing of exons and exon-intron junctions. Carritt and Welch (1987) have presented evidence that the FUCA 1L region on chromosome 2 is homologous to at least 650 bp at the 5' end of the FUCA 1 cDNA. This suggests that FUCA 1L shares at least three exons with genomic FUCA 1, but it is not interrupted by introns in this homologous region. For this reason, new a-L-fucosidase RFLPs need to be individually mapped so as to exclude the chromosome 2 location. The function of FUCA 1L is unclear, but it does not encode for fucosidase enzyme activity (Turner et al. 1975a; Carritt et al. 1982).

.41 .54 1.26 1.13 1.45 .25 .51

Two RFLPs in FUCA 1 were identified in the control population. The PvuII and BglI RFLPs are common in the normal population, with allele frequencies of .70/.30 for PvuII and .63/.37 for BglI, respectively. Both RFLPs are in strong linkage disequilibrium (r = .94), which is consistent with their close physical proximity. One hundred percent concordance with chromosome 1 in somatic cell hybrids assigns the PvuI RFLP to the structural fucosidase gene locus (FUCA 1) on chromo-

Table 3 Linkage Disequilibrium in Fucosidosis

A. Control vs. Patient Gametes with PvuII Alleles

PvuII GROUP

Patients Controls

....

6 kb

15 74

7 kb

9 }

X2 TEST

10.2 (P