Proc. Natl. Acad. Sci. USA Vol. 83, pp. 1817-1821, March 1986 Genetics Sialidosis and galactosialidosis: Chromosomal assignment of two genes associated with neuraminidase-deficiency disorders (sialidase/gene mapping/complementation analysis) 0. THOMAS MUELLER, W. MICHAEL HENRY, LINDA L. HALEY, MARY G. BYERS, ROGER L. EDDY, AND THOMAS B. SHOWS Department of Human Genetics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, NY 14263 Communicated by Victor A. McKusick, November 14, 1985 ABSTRACT The inherited human disorders sialidosis and galactosialidosis heterozygotes have not consistently shown galactosialidosis are the result of deficiencies of glycoprotein- a reduction in either ,B-galactosidase or a-neuraminidase (2, specific a-neuraminidase (acylneuraminyl hydrolase, EC 9, 13-16). Complementation studies from a number of labo- 3.2.1.18; sialidase) activity. Two genes were determined to be ratories show that the primary defect is different from that necessary for expression of neuraminidase by using human- causing single deficiencies of P-galactosidase (GM1- mouse somatic cell hybrids segregating human chromosomes. gangliosidosis) or neuraminidase (7, 17-19). The residual A panel of mouse RAG-human hybrid cells demonstrated a 3-galactosidase activity in galactosialidosis fibroblasts exists single-gene requirement for human neuraminidase and allowed entirely as a monomer with an absence of a large multimer assignment of this gene to the (pter-_q23) region of chromo- form found in normal cells (20). The mutation causing some 10. A second panel of mouse thymidine kinase (TK)- galactosialidosis also causes a decrease in turnover time due deficient LM/TK-human hybrid cells demonstrated that to an increased susceptibility of,-galactosidase to proteo- human neuraminidase activity required both chromosomes 10 lytic degradation (20, 21). The P-galactosidase deficiency, but and 20 to be present. Analysis of human neuraminidase not the neuraminidase deficiency, can be corrected by the expression in interspecific hybrid cells or polykaryocytes addition of protease inhibitors (21-24). This could be accom- formed from fusion of mouse RAG (hypoxanthine/guanine plished also with the addition of a "corrective factor" phosphoribosyltransferase deficient) or LM/TK- cell lines concentrated from cell culture medium, which has charac- with human sialidosis or galactosialidosis fibroblasts indicated teristics of a glycoprotein (23-25). Recent evidence suggests that the RAG cell line complemented the galactosialidosis that the mutation causing galactosialidosis results in an defect, but the LM/TK- cell line did not. This eliminates the absence of a 32-kDa glycoprotein in anti-p-galactosidase requirement for this gene in RAG-human hybrid cells and immunoprecipitates, and this protein was postulated to be the explains the different chromosome requirements of these two primary defect in galactosialidosis (23). hybrid panels. Fusion of LM/TK- cell hybrids lacking chro- We present evidence for the chromosomal assignment of mosome 10 or 20 (phenotype 10+,20- and 10-,20+) and two genes required for the expression of human neuramini- neuraminidase-deficient fibroblasts confirmed by complemen- dase in somatic cell hybrids. In addition, our evidence tation analysis that the sialidosis disorder results from a indicates that the sialidosis and galactosialidosis diseases are mutation on chromosome 10, presumably encoding the caused by mutations in these genes located on chromosomes neuraminidase structural gene. Galactosialidosis is caused by a 10 and 20, respectively. A portion of this work has been mutation in a second gene required for neuraminidase expres- presented in abstract form (26). sion located on chromosome 20. MATERIALS AND METHODS Several inherited diseases have been found to be associated with a deficiency of glycoprotein-specific N-acetyl-a- Fibroblasts and Hybrid Cells. The fibroblasts used were neuraminidase activity (acylneuraminyl hydrolase, EC GM1718, derived from an infantile-onset variant of sialidosis 3.2.1.18; sialidase). These disorders are typically classified as type II (mucolipidosis I), and GM806, derived from a subject the sialidoses, which have only a neuraminidase deficiency, with an early-onset form of galactosialidosis. Hybrid cells and the galactosialidoses, which have a coexistent deficiency were made by fusing normal human cells or cells with of P-galactosidase (1, 2). The sialidosis disorder, originally balanced chromosomal translocations with either mouse termed lipomucopolysaccharidosis (3), includes several var- LM/TK- [thymidine kinase (TK) deficient] or mouse RAG iants with different degrees of clinical severity, including an (hypoxanthine/guanine phosphoribosyl transferase defi- adult-onset form known as sialidosis type I and the infantile- cient) cells and selecting clones in hypoxanthine/aminopter- onset variant known as mucolipidosis I or sialidosis type II (4, in/thymidine selection medium as described (27). The pres- 5). The galactosialidosis disorder, which has also been ence of human chromosomes was determined in each hybrid termed the Goldberg Syndrome (6), GM1 gangliosidosis type clone by scoring for previously mapped human enzyme 4 (7), the cherry-red-spot-myoclonus syndrome with demen- markers and by karyotype analysis with trypsin/Giemsa tia (2), and the juvenile-onset form of sialidosis type II (8), is banding (28). also clinically heterogeneous (9, 10). Neuraminidase Assays. Hybrids used for chromosomal The primary defect in the sialidoses is thought to be a localization studies were analyzed for human neuraminidase mutation in the neuraminidase structural gene. Obligate expression as freshly harvested cultures due to the lability of heterozygotes show a gene-dosage effect and have approxi- this enzyme to freezing. Hybrid harvests were scored for mately halfthe normal neuraminidase levels (11, 12). Obligate neuraminidase and analyzed karyotypically on the same passage. Hybrid cultures were rinsed in Dulbecco's phos- The publication costs of this article were defrayed in part by page charge phate-buffered saline and scraped from flasks with a rubber payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: TK, thymidine kinase. 1817 Downloaded by guest on September 30, 2021 1818 Genetics: Mueller et al. Proc. Natl. Acad. Sci. USA 83 (1986) policeman. Cell pellets were homogenized gently in cold Table 2. Human-mouse hybrid cell neuraminidase activity distilled water by using a Dounce type homogenizer with a Teflon pestle. Neuraminidase activity was determined by a RAG Neuraminidase LM/TK- Neuraminidase procedure modified from that of Warner and O'Brien (29). hybrids Activity Score hybrids Activity Score Homogenates (20 ,ul) were incubated with 5 .ul of 1 M sodium ATR-22 6.0 - ICL-6 10.8 + acetate (pH 4.2) and 5 ALI of 7.1 mM 4-methylumbelliferyl-a- DUA-1 CH 2.0 - NSL-16 5.1 - neuraminide (Koch-Light Laboratories, Colnbrook, U.K.) at DUA-5 BA 4.0 - TSL-2 10.3 + 370C for 30 min. Reactions were terminated by adding 2 ml of DUM-13 29.8 + TSL-2 CF 3.7 - 0.85 M glycine (pH 10), and the fluorescence was determined DUM-23 36.3 + TSL-6F 6.0 - with an Aminco fluorimeter. The inclusion of bovine serum JSR-2 6.7 - VTL-6 10.3 + albumin at 1 mg/ml or 1 mM CaCl2 in assays or the JSR-6C 24.5 + VTL-11 4.1 - preparation of homogenates in the presence of 1 mM JSR-6D 21.2 + VTL-13 4.4 - phenylmethylsulfonyl fluoride had no effect on activity. JWR-22H 57.3 + VTL-14 4.5 - Protein concentration was measured by using a modification JWR-26C 25.3 + VTL-15 5.8 - of the Folin phenol procedure (30). RAS-M3 26.0 + VTL-16 10.6 + Complementation Analysis. Each of the two cultures used REW-4 8.0 - VTL-17 4.3 - in complementation experiments was seeded at half the REW-8I C4 5.0 - VTL-18 4.4 - confluent cell density, and the mixture was cultivated over- REW-11 5.5 - VTL-19 3.2 - night. These cultures were fused by using 42% (wt/vol) REX-11 BF 20.5 + VTL-21 4.2 - polyethylene glycol 1000 (Koch-Light Laboratories) contain- REW-13 57.0 + VTL-22 5.0 - ing 7% (vol/vol) dimethyl sulfoxide (31), and the resulting REW-15 34.8 + VTL-23 3.4 - polykaryocytes were enriched by sedimentation velocity REX-26 6.3 - WIL-2 3.5 - under sterile conditions with a Sta-Put apparatus (Johns REX-33 5.0 - WIL-2 C 3.9 - Scientific, Toronto) as described (19). This polykaryocyte REX-57 BB 5.0 - WIL-6 10.4 + fraction was cultured for 10-14 days, and the neuraminidase SIR-1 4.0 - WIL-8 12.9 + activity was determined along with cultures of each parental XER-8 21.3 + WIL-8S 9.6 + cell type and a cocultivated mixture. In complemnentation XER-11 27.6 + WIL-8Y 11.9 + experiments involving human fibroblasts and RAG or XER-15 21.3 + WIL-li 14.5 + LM/TK- cells, the polykaryocytes were cultivated in XTR-1 27.0 + WIL-13 4.0 - hypoxanthine/aminopterin/thymidine selection medium in XTR-1 BD 35.7 + WIL-14 5.7 - order to inhibit growth of the unfused mouse parental cells. XTR-3 BH 20.8 + WIL-14 C 6.1 - A significant increase in the neuraminidase activity of the XTR-22 26.3 + polykaryocyte fraction over that of the cocultivated mixture The neuraminidase activity of hybrid cells is the mean of at least and parental cell activity range, as determined with Student's three separate harvests and is expressed in nmol/hr per mg of t test (at the 1% confidence level), was assumed to indicate homogenate protein. Neuraminidase scores are based on statistically complementation. significant increases over the activity of parental mouse cells (Table 1). Hybrid clone XTR-3 BH contains del(10)(q23--qter) (deletion in RESULTS chromosome 10 in region q23--qter). Somatic Cell Hybrid Mapping of Neuraminidase. The pres- ence of human neuraminidase activity in human-mouse mg for LM/TK- hybrids) were scored negative for human somatic cell hybrids that segregate human chromosomes activity.