American Journal of Medical Genetics 29517422 (1988)

Deletion Mapping of the ,&Glucuronidase Gene

Judith E. Allanson, Robert M. Gemmill, Barbara K. Hecht, Stanley Johnsen, and David A. Wenger

The Genetics Center, Southwest Biomedical Research Institute, Scottsdale, Arizona (J. E. A., R. M. G., B. K. H.), Barrow Neurological /nstitute, Phoenix, Arizona (S. J.), and Jefferson Medical College, Department of Medicine (Medical Genetics) and Biochemistry and Molecular Biology, Philadelphia, Pennsylvania (D.A. W.)

GUSB, the gene for @-glucuronidase, has been localized to the proximal long arm of chromosome 7 between 7ql1.2 and 7q22. Deficiency of @-glucuronidase results in mucopolysaccharidosis type VII (MPS VII, Sly syndrome). The enzymatic defect has been demonstrated in cultured skin fibroblasts, leukocytes and serum of affected patients.

An 8-yr-old boy presented with manifestations similar to MPS VII (mental retardation, short stature, “coarse” facial appearance, mild skeletal involvement and recurrent lower respiratory tract infection) but other, discrepant abnormalities, e.g., bilateral iris colobomata and cleft palate. Normal activity of 6-glucuronidase was found in the patient’s leukocytes. Chromosome analysis disclosed an interstitial deletion of 7q with one breakpoint at the interface between bands 1 1.22 and 1 1.23 and the other breakpoint within band 21.1. DNA from this patient’s leukocytes was analyzed for dosage of GUSB sequences. This locus appeared to be present a t the normal diploid level.

These findings suggest that GUSB is not in the portion of chromosome 7 deleted in our case, narrowing the smallest region of overlap to 7q21.1 - 7q22. We therefore assign the P-glucuronidase gene to 7q21.1 - 7q22.

Key words: interstitial 7q deletion, Sly syndrome, autosomal recessive inheritance

INTRODUCTION

Interstitial deletions of chromosome 7q are relatively rare; less than 50 cases have been reported. Gibson et al. [ 19821 reviewed most of these cases and divided the patients

Received for publication April 25, 1987; revision received August 31, 1987.

Address reprint requests to Dr. Judith E. Allanson, The Genetics Center, Southwest Biomedical Research Institute, 6401 East Thomas Road, Scottsdale, AZ 85251.

0 1988 Alan R. Liss, Inc.

518 Allanson et al.

into three groups according to the locations of the breakpoints. Patients in the first group had proximal deletions in band 7ql l - q21. The second group included intermediate deletions involving band 7q21 - q32. In the third group a terminal deletion of band 7q32 - qter was observed. A consistent phenotype was noted only in the latter group. The critical region for expression of this phenotype appeared to be 7q35 - qter.

The gene coding for P-glucuronidase (GUSB) is assigned to chromosome 7 [Grzeschik, 1976; Lalley et al., 19771; until recently the smallest region of overlap was 7cen - q22 [Meera-Khan and Robson, 19791. Regional assignment of the gene to band 7q22 was suggested by Danesino et al. [ 198 1 1. Recently Frydman et al. [ 19861 reported a 14-yr-old severely retarded boy with a deletion of band 7cen - ql I .2 who had normal activity of 0-glucuronidase, narrowing the smallest region of overlap to 7q11.2 - q22. We describe a patient with a proximal interstitial deletion of 7q which allows more precise mapping of the @-glucuronidase gene.

CLINICAL REPORT

The patient was born after a pregnancy complicated by deficient antenatal care, vertex presentation, vaginal delivery. Birth weight was 28508, length 48.2 cm. The newborn period was complicated by polycythemia, transient hypoglycemia and hyperbili- rubinemia.

Family history shows that the natural parents are a healthy nonconsanguineous couple who had previously lost two sets of twins due to prematurity. They had had one spontaneous miscarriage. They have an older healthy son.

At age seven wk, length and weight were below the 3rd centile; occipitofrontal circumference (OFC) was at the 25th centile. The patient was noted to have a hoarse cry, hypertonia, a right inguinal hernia, umbilical hernia, heart murmur and abnormal facial appearance. Investigations at that time showed a diffusely abnormal electroencephalo- gram, bilaterally dislocated hips, a right-sided partial ureteropelvic junction obstruction and spina bifida occulta at S1.

At age eight yr, the child was referred to our center because of profound mental retardation, seizure disorder and minor anomalies (Fig. 1). His OFC was 53.5 cm (50th-98th centile), weight 24 kg (25th-50th centile), height 120 cm (25th centile), lower segment 53 cm, upper segment: lower segment ratio was abnormal. There was pronounced dolichocephaly with a prominent forehead, a frontal upsweep and coarse hair. Palpebral fissures were level, outer canthal distance 9 cm (75th-97th centile), inner canthal distance 3.5 cm (97th centile). Bilateral iris colobornata were noted. The brows were heavy. The nose was short with a small, upturned tip and depressed nasal root. The philtrum was long and wide. There were thick protuberant lips and a wide mouth. Malar hypoplasia was present, the alveolar ridges were thickened and a cleft palate was noted. Liver and spleen were not palpable. There was no evidence for a congenital heart defect. He had “spatulate” thumbs with short, broad distal phalanges and short, wide nails; markedly tapering fingers with flexion contractures principally of index fingers; bilateral fifth digit clinodactyly. Severe scoliosis and elbow contractures were present. The patient was cortically blind and deaf.

Results of chromosome studies obtained in infancy were reportedly normal. Because of the nature of the clinical picture, chromosome studies were repeated at age eight yr. Peripheral blood lymphocytes were cultured with phytohemagglutinin and harvested using standard methods. Careful scrutiny of G-banded metaphases showed an interstitial

Mapping P-Glucuronidase Gene 519

Fig. 1. The patient, age 8 yr.

Fig. 2. The normal and deleted chromosome 7 from 3 separate metaphases. Arrows denote breakpoints.

deletion of 7q in all cells analyzed. At the 550 band level, the deletion appeared to be between a breakpoint at the interface of bands 11.22 and 11.23 and a breakpoint within band 21.1 (Fig. 2,3). Since the patient is adopted and the natural parents are not available for study, it cannot be determined whether the child’s chromosome abnormality was the result of crossing-over within a parental inversion loop or occurred de novo.

Biochemical Studies

P-glucuronidase activity was measured in sonicated leukocytes as described previ- ously [Wenger et al., 19761. Briefly, 1&20 pg protein in the total leukocyte sonicate was incubated with 4-methylumbelliferyl-P-D-glucuronide ( 5 mM final concentration in 0.05 M sodium acetate buffer, pH 4.8) in a total volume of 0.2 ml. After incubation at

520 Allanson et al.

f

Chr Normal

'ornosome 7 / Q /

Chromosome 7 With Deletion

Fig. 3. An ideogram of the derivative 7.

TABLE I. Comparison of Manifestations Associated With Deletions of 7q and MPS MI

7 q l l - q22 7q21+ q32 7q32 + qter MPS VII Our patient

Growth retardation Mental retardation + Microcephaly + Short/bulbous nose Malformed ears + Long philtrum + Large mouth Cleft palate CHD Genital abnormality Inguinal hernia

+ + + + + + + + + + +

+

+ + + +

+ + + +

t

+ + Hypotonia + + H ypertonia + + Seizures + + Recurrent infection + + +

37OC for 20 min, the resulting fluorescence was read on a Perkin-Elmer spectrofluorom- eter at an excitor wavelength of 365 nm and an analyzer wavelength of 448 nm. The values obtained were compared to a standard solution of recrystallized 4methylumbelliferone. The 6-glucuronidase activity in our patient was found to be entirely normal at 384 nmol/hr mg protein (normal mean 396 * 90 nmol/hr/mg protein; range 300-500 nmol/hr/mg protein). A patient with MPS VII had a value of 30.5 nmol/hr/mg protein; two obligate heterozygotes had values of 172 and 238 nmol/hr/mg protein.

Mapping P-Glucuronidase Gene 521

7 Fig. 4. Smallest region of overlap: progressive localization of GUS 9. 1. Meera-Khan and Robson, 1979. 2. Frydman et al., 1986. 3. Present study.

Molecular Studies

A lymphoblastoid cell line was established from peripheral blood lymphocytes from this patient by Epstein-Barr virus transformation. DNA isolated from this cell line was studied by Southern blot analysis for possible changes in dosage of the P-glucuronidase gene. The probe used, pHUG60.2, is a cDNA clone of human P-glucuronidase (kindly provided by Roy Gravel). Hybridization of this probe to DNA from a control and from our patient showed a large number of homologous DNA fragments in Eco RI, Hind 111 and Pst I digests. These fragments derived from a large number of 8-glucuronidase pseudo- genes of unknown location. The fragments that derive from the GUSB locus on chromosome 7 are not currently known. Because the pseudogene-derived fragments cannot be isolated from those fragments derived from the gene coding for GUSB, the interpretation of results is difficult. None of the detected bands showed any consistent dosage effect, however. This suggested that the GUSB locus was probably not deleted in our patient.

DISCUSSION

Remarkable phenotypic variability is seen in patients with proximal deletions of 7q. No syndrome characteristic of proximal deletion of 7q has yet been defined, although Gibson et al. [ 19821 commented on the consistent association in these patients of profound mental retardation, microcephaly, malformed ears, a long, simple philtrum and hypotonia. All of these findings are fairly general anomalies seen in many different aneuploidy syndromes. One patient with a deletion of 7qll- q21 reportedly had a normal phenotype (Seabright and Lewis, 19781, whereas the patient described by Gibson et al. [1982] shares

522 Allanson et al.

many phenotypic and cytogenetic characteristics with our patient. A P-glucuronidase assay was apparently not done in the case of Gibson et al. [1982]. Patients with intermediate deletions involving band 7q21 - q32 have some manifestations in common. Patients with terminal deletions have a fairly consistent phenotype/karyotype correlation (Table I). Our patient shares many anomalies with other patients with proximal 7q deletions. He also has many manifestations in common with patients who have p- glucuronidase deficiency (MPS VII, Sly syndrome) including mental retardation, short stature, coarse facies, mild skeletal involvement and recurrent lower respiratory tract infections. The presence of bilateral iris colobomata and cleft palate serve to distinguish this boy both from patients with B-glucuronidase deficiency and other cases reported with proximal 7q deletions.

The gene coding for P-glucuronidase (GUSB) has been assigned to chromosome 7 [Grzeschik, 1976; Lalley et al., 19761. Initially, the smallest region of overlap was 7cen - q22 [Meera-Kahn and Robson, 19791. A regional assignment of the gene to band 7q22 was proposed by Danesino et al. [1981]. Recently, Frydman et al. [1986] reported a severely retarded male with a deletion of chromosomal band 7cen - q l 1.2. This narrows the smallest region of overlap to 7q11.2 - q22.

Normal activity of P-glucuronidase in our patient and the presence of a deletion running from subband 7q11.22 to 7q21.1 further narrows the smallest region of overlap. It leaves band 7q21.1 to 7q22 as the most likely location of the gene. This is consistent with the results of Danesino et al. [ 198 11. Although the molecular studies are not definitive due to the presence of autosomal pseudogenes, the absence of a dosage effect on Southern blot analysis is consistent with retention of both GUSB genes. We therefore assign GUSB to 7q21.1 - 7q22 (Fig. 4).

ACKNOWLEDGMENTS

We are very grateful to Dr. Roy Gravel for providing the P-glucuronidase probe. We thank Helen Bixenman for excellent cytogenetic analysis, Fred Flohrschutz for photogra- phy and graphics, and Donna Packard for preparing the manuscript.

REFERENCES

Danesino C, Gimelli G, Cuoco C, Ciccone MO (1981): Triplex gene dosage effect for 0-glucuronidase, and possible assignment to band q22 i n a partial duplication 7q. Hum Genet 56:371-373.

Frydman M, Steinberger J, Shabtai F, Steinherz R (1986): Interstitial 7q deletion [46,XY,de1(7) (pter 4 cen::qll2 + qter)] in a retarded quadriplegic boy with normal beta-glucuronidase. Am J Med Genet 25:245-249.

Gibson J, Ellis PM, Forsyth J S (1982): Interstitial deletion of chromosome 7: A case report and review of the literature. Clin Genet 22256265.

Grzeschik KH (1976): Assignment of structural gene for beta-glucuronidase to human chromosome C7. Somat Cell Genet 2:401410.

Lalley PA, Brown JA, Eddy RL, Haley LL, Byers MG, Goggin AP, Shows TB (1977): Human beta- glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids. Biochem Genet 15:367-382.

Meera-Khan P, Robson EB (1979): Report of the committee on genetic constitution of chromosomes 7 , 8 and 9. Cytogenet Cell Genet 25:3946.

Seabright M, Lewis GM (1978): Interstitial deletion of chromosome 7 detected in 3 unrelated patients. Hum Genet 42223-226.

Wenger DA, Sattler M, Clark C, Wharton C (1976): I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates. Life Sci 19413420.

Edited by John M. Opitz and James F. Reynolds