Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2968-2972, July 1977 Genetics

Mannosidosis: Assignment of the lysosomal a-mannosidase B gene to chromosome 19 in man (gene mapping/cell hybrids/inherited storage disease) M. J. CHAMPION AND T. B. SHOWS Biochemical Genetics Section, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 Communicated by James V. Neel, April 28, 1977

ABSTRACT Human a-mannosidase activity (a-D-mannos- genetic control (12). Residual acidic a-mannosidase activity, ide mannohydrolase, EC 3.2.1.24) from tissues and cultured skin purified from mannosidosis tissues, shows abnormal metal ion fibroblasts was separated by gel electrophoresis into a neutral, cytoplasmic form (a-mannosidase A) and two closely related activation (13), altered thermostability, and increased Km (14), acidic, lysosomal components (a-mannosidase B). Human demonstrating a structural change in the enzyme. Such a mannosidosis, an inherited glycoprotein storage disorder, has structural change implicates a mutation in a structural gene been associated with severe deficiency of both lysosomal a- whose product is common to both forms of acidic a-mannos- mannosidase B molecular forms. Chromosome assignment of idase. A similar deficiency of acidic a-mannosidase has been the gene coding for human a-mannosidase B (MANB) has been seen in inherited mucolipodisis II, but this disorder, in contrast, determined in human-mouse and human-Chinese hamster somatic cell hybrids. The human a-mannosidase B phenotype appears to result from a defect in enzyme processing (15). showed concordant segregation with the human-enzyme glu- Human-rodent somatic cell hybridization has been used to cosephosphate isomerase (GPI) (D-glucose-6-phosp ate ketol- investigate the genetic, linkage, and structural relationships of isomerase, EC 5.3.1.9) but discordant segregation with 30 other several acid hydrolases involved in inherited lysosomal storage enzyme markers representing 20 linkage groups. The glucose- diseases (16, 17). We have investigated the expression of human phosphate isomerase gene has been assigned to chromosome a-mannosidase in somatic cell hybrids to assign the gene coding 19 in man. This MANB-GPI linkage and confirming chromo- some studies demonstrate assignment of the a-mannosidase B for the lysosomal a-mannosidase associated with mannosidosis structural gene to chromosome 19 in man. Since mannosidosis to a specific chromosome and to determine the genetic and is believed to result from a structural defect in a-mannosidase possible structural relationships of the a-mannosidase molecular B, these findings suggest that the mannosidosis mutation is lo- forms. A gel electrophoretic procedure was developed to sep- cated on chromosome 19 in man. arate human lysosomal a-mannosidase (MANB) in human- mouse and human-Chinese hamster somatic cell hybrids. Ev- Mannosidosis is a rare lysosomal storage disease associated with idence is reported for the linkage of the MANB gene to the gene deficient activity for the acid hydrolase a-mannosidase (a- coding for human glucosephosphate isomerase (GPI) (D-glu- D-mannoside mannohydrolase, EC 3.2.1.24). Clinical features cose-6-phosphate ketolisomerase, EC 5.3.1.9) and the assign- of this disorder include early onset, mild physical deformities, ment of the MANE structural locus to human chromosome 19. progressive mental and psychomotor retardation, and death These findings, together with previous biochemical evidence in early childhood (1, 2). Affected children show less than 10% demonstrating that the enzyme defect in mannosidosis is a a-mannosidase activity, leading to impaired glycoprotein ca- structural mutation of MANB, suggest that the mannosidosis tabolism and massive accumulations in nervous tissues and defect exists at the MANE locus assigned to chromosome 19. urinary excretion of mannose-rich oligosaccharides (3, 4). The genetics of this disorder remain unclear, although existing ev- MATERIALS AND METHODS idence suggests an autosomal mode of inheritance. The enzyme defect of mannosidosis in humans has been Human and Rodent Parental Cells. Human cells for fusion identified in tissues and cultured fibroblasts as deficient activity were GM 1006 fibroblasts (mucolipidosis II), JoVa fibroblasts, for acidic a-mannosidase (MANB), while neutral a-mannosidase DUV fibroblasts, AnLy fibroblasts, SH 421 fibroblasts, AlTr (MANA) is unaffected (5, 6). Acidic and neutral forms of a- leukocytes, PeLa leukocytes, and CaVa leukocytes (15, 17-20). mannosidase have been separated by DEAE-cellulose chro- Human mannosidosis fibroblasts (GM 654) and mucolipidosis matography (5) and cellulose acetate electrophoresis (7). The II fibroblasts (GM 1006) were obtained from the Human Ge- acidic MANB activity (optimum pH 4.0-4.5) consists of two netic Mutant Cell Repository, Camden, NJ. The human fi- closely related molecular forms which show similar thermo- broblasts were maintained on Eagle's basal medium (diploid) stability, enzyme kinetics, and molecular weight, while the (GIBCO), 10% fetal calf serum, and antibiotics. Rodent parental single neutral MANA enzyme (optimum pH 6.0-6.5) is ther- lines with selected markers were mouse A9, LM/TK-, and molabile and enzymatically distinct (8-10). The two acidic RAG; and Chinese hamster lines were CHW-1 102 and A3 a-mannosidase enzymes are distributed in the lysosome while (17). the neutral form shows highest activity in the cytoplasm (8, 9). Human-Rodent Cell Hybrids. Human and rodent cells were The two purified acidic, lysosomal forms interconvert sponta- fused in suspension or as monolayers with inactivated Sendai neously (9, 10) or with neuraminidase treatment (11); they are virus, and hybrid cells were cloned and maintained on HAT immunologically identical (12) and appear to be structurally (hypoxanthine/aminopterin/thymidine) selection medium and genetically interrelated. Neutral a-mannosidase activity consisting of Dulbecco's modified Eagle's medium (GIBCO), is immunologically distinct and is probably under separate HAT, 10% fetal calf serum, and antibiotics (18). Primary hybrid clones established from 14 separate fusion experiments with 8 Abbreviations: MANA and MANB, neutral and acidic a-mannosidase, different human parental cells were analyzed. Hybrid sets respectively; GPI, glucosephosphate isomerase. consisted of the human-mouse hybrids DUA (DUV X A9), ICL 2968 Downloaded by guest on September 28, 2021 Genetics: Champion and Shows Proc. Natl. Acad. Sci. USA 74 (1977) 2969 (GM 1006 X LM/TK-), and ICR (GM 1006 X RAG) qnd human-Chinese hamster hybrids ATC (AlTr)X CHW-116i), MANA- PLA (PeLa X A3), PLC (PeLa X CHW-1102), JVA (JoVa X A3), and JVC (JoVa X CHW-1102). Additional human-mouse primary hybrid lines were REX, RAS, ATR, ALR, ALA, and JWR hybrids (17-20). a-Mannosidase Electrophoresis. Cell homogenates from |-Origin confluent monolayers were prepared in 0.05 M Tris-HCl buffer |-Origin (pH 7.5) at concentrations of 0.75 to 1.0 X 108 cells per ml (21). Supernatant fractions of cell homogenates were examined by MANB - vertical starch-gel electrophoresis (Buchler Instruments). Electrophoresis of acidic a-mannosidase (MANB) was accom- plished in 15% starch gels (Connaught Laboratories) for 17 hr 1 2 3 4 5 6 7 8 at 120 V with a bridge buffer of 0.2 M sodium phosphate (pH FIG. 1. Starch-gel electrophoretic pattern of a-mannosidase 5.0) and a gel buffer consisting of a 1/20 dilution of this. Neutral cytoplasmic (MANA) and lysosomal (MANg) components stained a-mannosidase (MANA) was examined in 12% starch gels with the fluorescent substrate 4-methyllumbelliferyl a-mannopyra- noside. (1) Human liver; (2) human kidney; (3) cultured human fi- (Electrostarch Co.) with a Tris-citrate (pH 7.0) buffer system broblasts, WI-38; (4) human mannosidosis fibroblasts, GM 654; (5) as described (22). Acidic MANB activity was observed in a 0.1 human mucolipidosis II fibroblasts, GM 1006; (6) mannosidosis cell M-phosphate/citric acid pH 5.4 buffer and neutral MANA in extracts treated with neuraminidase; (7) mucolipidosis II cell extracts 0.01 M Tris-citrate (pH 7.0) buffer with 2 mM fluorescent 4- treated with neuraminidase; and (8) cultured mouse cells, LM/ methylumbelliferyl-a-D-mannopyranoside (Koch-Light) as TK-. substrate (6, 7). Human a-mannosidase has been reported to and mouse (26) tissues. The fast and slow components of MANB show activity towards this synthetic substrate similar to that with appear to correspond to peaks A and B, respectively, demon- naturally occurring mannose-rich oligosaccharides (10, 23). strated by DEAE-cellulose chromatography of human tissue a-Mannosidase Terminology. a-Mannosidase A (MANA) extracts (9-11). Neuraminidase treatment of liver and fibroblast has previously been referred to as the neutral or cytoplasmic extracts resulted in a partial conversion of the slow MAN' band enzyme and is identical to peak C separated by DEAE-cellulose to the fast form, consistent with previous reports on the inter- column chromatography (5, 7-11). a-Mannosidase B (MANB) conversion of these forms (11). Homologous neutral and acidic is the acidic or lysosomal enzyme and is composed of peaks A forms of a-mannosidase were resolved in extracts of mouse and and B demonstrated by chromatography on DEAE-cellulose Chinese hamster tissues and cultured cells with these same (5, 7-11). The MANA and MANB terminology for these enzyme electrophoretic systems (Fig. 1, channel 8). forms has been adopted to agree with previous terminology for Extracts from skin fibroblasts derived from individuals with the mannosidosis deficiency demonstrated by cellulose-acetate mannosidosis demonstrated nearly complete absence of both electrophoresis (6) and to comply with human gene mapping forms of the lysosomal MANB enzyme, while the cytoplasmic terminology (24). MANA band was apparently unaltered (Fig. 1, channel 4). The Glucosephosphate Isomerase Electrophoresis. Human and deficiency of both MANB forms and their similiarity in bio- rodent forms of glucosephosphate isomerase were separated chemical and immunological properties demonstrate the in- by starch gel electrophoresis in a Tris-citrate (pH 7.0) buffer terrelationships of the two MANB bands. A similar mannosidase system (22) and stained as previously described (25). deficiency phenotype is observed in fibroblasts from another Neuraminidase Treatment. Supernatant fractions of cell storage disease, mucolipidosis II (Fig. 1, channel 5). This dis- homogenates were incubated with neuraminidase (acylneu- order is characterized by deficiencies of several different ly- raminyl hydrolase, EC 3.2.1.18) from Vibrio cholerae (General sosomal hydrolases and is associated with abnormal sialylation Biochemicals) as described (15). (15). The two disorders result from different genetic defects, as indicated by the fact that neuraminidase treatment of mu- RESULTS colipidosis II cell extracts showed a recovery of the MANB band while treatment of mannosidosis extracts did not alter the a-Mannosidase expression in human tissues, cultured MANB deficiency (Fig. 1, channels 6 and 7) (15). This evidence fibroblasts, and lysosomal storage diseases is consistent with reports indicating that mannosidosis is a Vertical starch-gel electrophoresis was used to separate the three mutation in the structure of a-mannosidase (13, 14). components of human a-mannosidase (Fig. 1). Two different electrophoretic systems were used to best resolve the two acidic, Expression of a-mannosidase in human-mouse and lysosomal bands (Fig. 1 lower) and the single neutral cyto- human-Chinese hamster cell hybrids plasmic enzyme (Fig. 1 upper). The cytoplasmic enzyme was Human MANg and mouse and Chinese hamster acidic a- resolved in a Tris-citrate (pH 7.0) buffer system and stained at mannosidase were separated with a sodium phosphate (pH 5.0) pH 7.0, while the lysosomal enzyme was resolved in a sodium buffer system (Fig. 2, channels 1-3). Mouse cell lines demon- phosphate (pH 5.0) buffer and stained at pH 4.5. strated a single band which migrated to the same position as the The mannosidase isozymes were investigated in human major band seen in mouse liver and kidney and showed greatest tissues and cultured fibroblasts (Fig. 1, channels 1-3). Lysoso- activity in mouse LM/TK- cells (Fig. 2, channel 1). Eight mal MANB expression consisted of a single fast, cathodally Chinese hamster cell lines examined, including cell lines A3 and migrating band common to all tissues such as kidney, liver, CHW-1 102 used in this study, showed only faint acidic a- brain, spleen, and colon and a second slower band seen in liver, mannosidase activity (Fig. 2, channel 2). This band migrated spleen, and cultured fibroblasts, yielding a double-banded to the same position as the major band in hamster tissues and pattern from these tissues. Cytoplasmic MANA showed a single comigrated with the mouse tissue culture band. The slower band in all human tissues and cultured cells. These patterns are human MANg band migrated to a similar position as the rodent similar to the electrophoretic phenotype reported in human (7) enzymes, while the faster major MANB band migrated to a Downloaded by guest on September 28, 2021 2970 Genetics: Champion and Shows Proc. Nati. Acad. Sci. USA 74 (1977)

MANB Drigin Table 1. Segregation of MANB and 30 other enzyme markers in primary cell hybrid clones Rodent MANg segregation Human- U.. - Enzyme Chromosome Concordant Discordant GPI (+) PGM1/AK2/PEPC 1 83 43 Hamster-- IDHs/MDHs 2 89 37 HEX5 5 77 43 -Origin MES 6 71 41 GUS 7 67 55 Mouse I_. GSR* 8 43 22 AK, 9 72 52 GOTS 10 87 39 Human - .- _. (-) ACP2I/ESA4/LDHA 11 81 45 1 2 3 4 5 6 7 8 LDHB/PEPB 12 74 49 FIG. 2. Starch-gel electrophoretic patterns of a-mannosidase B ESD 13 35 26 (MANB) and glucosephosphate isomerase (GPI) from parental, NP 14 74 50 human-mouse, and human-Chinese hamster hybrid cells. (1) Mouse HEXA/MPI/PKM2 15 78 45 cell line LM/TK-; (2) Chinese hamster cell line A3; (3) human fi- APRT 16 7 6 broblasts, WI-38; (4) human mannosidosis fibroblasts, GM 654; (5 TKt 17 24 24 and 7) human-mouse and human-hamster somatic cell clones (ICL-6 PEPA 18 52 28 and PLA-14, respectively) showing negative expression of the MANB GPI 19 122 4 and GPI enzymes; (6 and 8) human-mouse and human-hamster cell ADA 20 68 53 hybrids (ICL-7 and PLA-15, respectively) showing positive scoring SOD1 21 67 34 for the MANB and GPI enzymes. The intermediate GPI band in positive cell hybrids is the human-rodent GPI heterodimer. PGK/HPRT/G6PD X 60 66 The chromosome assignments, enzyme symbols, and gel electro- more cathodal position. Expression of acidic a-mannosidase in phoresis procedures for individual enzymes have been previously summarized (16, 27, 28). The concordant segregation column shows proliferating human-mouse and human-hamster hybrids the number of hybrid clones in which MANB and an enzyme mark- showed two distinct patterns (Fig. 2, channels 5-8). These er(s) were either present or absent together. Numbers of clones in consisted of hybrids with only the rodent enzyme and hybrids which only MANB or the enzyme marker(s) was expressed are indi- with a composite pattern of the rodent and human MANB ca- cated in the discordant segregation column. MANB and other enzyme thodal enzyme. The phenotype expressing both parental en- markers were determined on the same hybrid cell homogenate. zymes segregated in human-rodent cell hybrids and, when * Glutathione reductase (GSR) was best scored in sets of human- Chinese hamster hybrids. present, demonstrated the presence of the MANS gene. t Thymidine kinase (TK) has been assigned to human chromosome Since only the cathodal component of human MANB was 17 and is required in mouse and hamster (TK-)-human hybrids for separated in hybrid patterns, it was important to test in nega- growth in HAT selection medium. Expression ofMANB was scored tively scored hybrids for the presence of the slower MANB in ICL human-mouse hybrids and PLA and JVA human-hamster component, possibly masked by the rodent band. This was ac- hybrids to determine linkage relationship to chromosome 17. complished by treating hybrid cell extracts with neuraminidase to convert the slower MAN5 band to the fast position. Neu- GPI. Of the 126 hybrids examined, only 4 deviated from this raminidase treatment of MANB-positive hybrids produced pattern. The segregation characteristics of MANB and GPI for increased activity in the fast band, but treatment of negative the different hybrid sets in human-mouse and human-Chinese hybrids failed to produce activity in the fast position. These hamster hybrids is demonstrated in Table 2. Both classes of results suggest that the slower MANS component was present discordant clones were observed in the human-rodent hybrids. or absent together with the faster major MANB band and in- These discordant clones result either from chromosome dicate that the faster MANB band was a reliable marker for breakage or from differential sensitivity of the enzyme assays scoring human acidic a-mannosidase activity. in hybrids with few cells retaining the MANB-GPI linkage group. In MANB-/GPI+ clones, human GPI stained weakly. Genetic linkage of MANB and GPI This low discordancy rate (3%), suggesting relatively close linkage for MANB and GPI, has been observed for other linked Linkage of the MANB gene was investigated by examining the genes in cell hybrids (21). segregation patterns of MANB and other human enzyme The mouse a-mannosidase band demonstrated constant ex- markers previously assigned to specific chromosomes. One pression in all hybrids, while the Chinese hamster band showed hundred twenty-six independent hybrid clones established from variable expression ranging from deficient to the positive ac- fourteen sets of human-mouse and human-Chinese hamster tivity, seen in Fig. 2, channels 7 and 8. Expression of the ham- hybrids were examined. These hybrids represented fusions ster band did not correlate with the presence or absence of any using eight different human parental cells and five different human linkage groups and is considered to represent the vari- rodent cell lines. Hybrids were tested for the presence and ab- ability seen in several different Chinese hamster cell lines. sence of the catdal MANS isozyme and its cosegregation with The human CPI locus has been previously assigned to 30 other human enzymes representing 20 of the 24 different chromosome 19 by several laboratories (29, 30), and we have human chromosomes (Table 1). The MANB marker showed reconfirmed this assignment (Shows and Brown, unpublished). concordant segregation with human glucosephosphate isom- The possibility that more than one chromosome might be in- erase (GPI) (Fig. 2), but discordant segregation with all other volved in the final expression of MANB was not indicated since human enzymes tested (Table 1). Fig. 2 demonstrates cell hy- no linkage group tested, other than chromosome 19, was nec- brids either positive or negative jointly for human MANB and essary for the expression of MANB. Downloaded by guest on September 28, 2021 Genetics: Champion and Shows Proc. Nati. Acad. Sci. USA 74 (1977) 2971 Table 2. Segregation of MANB and GPI in independ94t, Table 3, Segregation of human chromosome 19, MANB, and GPI hybrid clones in human-mouse primary cell hybrids MANB/GPI Chromosome 19

Hybrid sets +/+ +1- -1+ -I- No. + _ MANn;GPI + 6 0 Human-mouse - 0 7 DUA 7 0 0 9 16 ICL 10 1 0 5 16 Chromosome identification and enzyme analyses were determined ICR 14 0 1 3 18 on the same cell passage from replicate flasks. Chromosome studies Others* 6 0 0 5 11 on human-mouse hybrids from the REX, RAS, ATR, ALR, ALA, and 37 1 1 22 61 JWR hybrid sets have been reported (17-20). Human-hamster with a secondary band formed by posttranslational modification ATC 12 0 0 7 19 of the primary product, or they share a common protein sub- PLA 9 0 0 11 20 unit. PLC 1 0 2 7 10 In human mannosidosis there is severe deficiency of both JVA 4 0 0 8 12 MANB components (Fig. 1, channel 4) (5, 6). A similar inherited JVC 2 0 0 2 4 mannosidosis has been described in Angus cattle and also results 28 0 2 35 65 in absence of both acidic a-mannosidase components (31, 32). The two purified molecular forms of human MANB show Total 65 1 3t 57 126 similar kinetic properties, molecular weights, and thermosta- Segregation columns show the numbers of primary clones of bilities, and are immunologically identical (8-12). Since the loss human-mouse and human-Chinese hamster hybrids scored for the of both forms occurs in mannosidosis and these forms appear presence (+) or absence (-) ofthe MANB and GPI enzymes. Parental to be closely interrelated, this suggests that mannosidosis results cells for hybrid sets are listed in Materials and Methods. from a structural gene defect common to both forms. Bio- * Additional human-mouse primary hybrid lines from the REX, RAS, chemical studies indicate that the residual MANB activity pu- ATR, ALR, ALA, and JWR hybrid sets were examined (17-20). rified from mannosidosis tissues is kinetically and structurally Chromosome studies were performed on these hybrids (Table 3). t GPI+/MANB- discordant hybrids were tested with neuraminidase altered (13, 14) and provides further evidence that the man- but did not generate positive activity in the faster band. nosidosis mutation exists at the MANB structural gene; Somatic MANB cell hybrids provide a means of determining the genetic and structural relationships of the mannosidase isozymes and make Chromosome assignment of MANB it possible to map the MANB structural gene and, most likely, Chromosome assignment of MANB was determined in 11 the mannosidosis defect. human-mouse primary cell hybrids whose chromosome anal- Evidence indicates that the human fast MANB component yses were previously reported (18, 29, 31). Concordant segre- is the major molecular form in human tissues (10). Data pre- gation of MANB and human chromosome 19 was observed sented here demonstrate that expression of this fast MANB form (Table 3), which confirms the assignment is linked to the human GPI locus and is encoded by a gene lo- to chromosome 19 cated on human chromosome 19. Concordant segregation of made from enzyme linkage data. All other human chromo- the MANB and GPI enzymes in 122 independent human- somes were excluded from assignment considerations because mouse and human-Chinese hamster hybrids (Tables 1 and 2) of discordant segregation. MANB and GPI segregated con- demonstrates linkage between the genes coding for GPI and cordantly in these hybrids and with chromosome 19 (Table 3), for MANB (Fig. 2). Since the human GPI locus has previously further confirming the assignment of MANB to 19. been assigned to chromosome 19 by somatic cell hybrids (29, 30), this indicates assignment of the MANB gene to chromosome DISCUSSION 19. This linkage is further supported by chromosome studies Starch-gel electrophoresis demonstrated two molecular forms in human-mouse hybrids, indicating the concordant segrega- of acidic, lysosomal a-mannosidase (MANB) from human tissues tion of the MANB enzyme with chromosome 19 (Table 3). and fibroblasts (Fig. 1, channels 1-3) in agreement with pre- Evidence suggests that the MANB structural gene has been vious evidence (5-12). Similar double bands of acidic a-man- mapped, since the MANB enzyme segregated with a single nosidase have been observed with electrophoresis in the mice linkage group and was not dependent upon the presence of any (26) and in cattle (31). The mouse pattern consists of a major other human chromosome for enzyme expression. Since only more cathodal band seen in most tissues, and a sialylated band the faster component of the MANB enzyme was separated in observed predominantly in liver and spleen. The major band hybrid enzyme patterns, cell extracts from negatively scored is observed after neuraminidase treatment of all tissues, and, as well as discordant segregating hybrids were treated with in fact, a genetic variant has been described in the mouse which neuraminidase. These hybrids failed to produce positive activity shows inability to form the sialylated band in liver (26). This in the fast MANB position, whereas treatment of positively variant has been postulated to be a defect in the sialylation of scored hybrids produced increased activity in the faster MANB the major band to the sialylated form. The MANB phenotype band. These results indicate that both components of the MANB in human tissues and fibroblasts also consists of a major band enzyme may be present or absent together incell hybrids. Re- (fast cathodal component) and a secondary band (slow com- gardless of this fact, the faster MANB band proved to be a re- ponent). This double-banded pattern can be converted to the liable marker for the MANB gene in human-rodent cell hy- single fast form by neuraminidase. This evidence and bio- brids. chemical evidence (11) suggests that human MANB consists of Preliminary scoring of the human- cytoplasmic MANA en- a major primary enzyme and a second sialylated form. It is zyme in hybrids indicates that this enzyme segregates with a likely that these forms are encoded by the same structural gene, different chromosome from chromosome 19 and is independent Downloaded by guest on September 28, 2021 2972 Genetics: Champion and Shows Proc. Natl. Acad. Sci. USA 74 (1977)

of the expression of the MANB gene (Champion and Shows, 9. Phillips, N. C., Robinson, D. & Winchester, B. G. (1974) Clin. unpublished). These results are consistent with the fact that Chim. Acta 55, 11-19. MANA shows kinetic, physical, and immunological properties 10. Phillips, N. C., Robinson, D. & Winchester, B. G. (1976) Biochem. enzyme (9-11), and in mannosi- J. 153,579-587. distinct from those of MANB 11. Chester, M. A., Lundblad, A. & Masson, P. K. (1975) Biochim. dosis the MANB enzyme is deficient while the MANA enzyme Blophys. Acta 391,341-348. is unaltered (5, 6, 31, 32). Thus, unlike molecular forms of other 12. Phillips, N., Robinson, D. & Winchester, B. (1975) Biochem. J. lysosomal enzymes which are interrelated, such as f3-hexoam- 151,469-475. inidase A and B deficient in Tay-Sachs disease and Sandhoff- 13. Hultberg, B. & Masson, P. K. (1975) Biochem. Blophys. Res. Jatzkewitz disease (16, 19), the MANA and MANB isozymes are Commun. 67, 1473-1479. encoded on separate structural genes and are expressed inde- 14. Desnick, R. J. & Ikonne, J. U. (1976) Excerpta Med. 397,29-30 pendently of one another. (Abstr). Since mannosidosis is a rare disorder, the genetic basis for this 15. Champion, M. J. & Shows, T. B. (1977) Am. J. Hum. Genet. 29, disease is poorly understood. Ockermann has suggested, from 149-163. 16. Lalley, P. A., Rattazzi, M. C. & Shows, T. B. (1974) Proc. Nati. evidence of six reported cases, that this disorder is inherited as Acad. Sci. USA 71, 1569-1573. a simple autosomal recessive trait similar to that of mannosidosis 17. Lalley, P. A., Brown, J. A., Eddy, R. L., Haley, L. L., Byers, M. in cattle (1, 5, 6). Since mannosidosis affects both forms of ly- G., Goggin, A. & Shows, T. B. (1977) Biochem. Genet. 15, sosomal MANB and appears to result from a defect in the 367-382. structural gene (13, 14), our results suggest that the mannosidosis 18. Shows, T. B. & Brown, J. A. (1975) Proc. Natl. Acad. Sci. USA mutation may be located at the MANB structural locus assigned 72,2125-2129. to chromosome 19. 19. Lalley, P. A., Brown, J. A. & Shows, T. B. (1976) Cytogenet. Cell Genet. 16, 188-191. 20. Shows, T. B. & Brown, J. A. (1975) Cytogenet. Cell Genet. 14, This work was supported by U.S. Public Health Service Grants HD 421-425. 05196 and GM 20454, and March of Dimes Grant 1-485. 21. Shows, T. B. (1972) Proc. Natl. Acad. Sci. USA 69,348-352. The costs of publication of this article were defrayed in part by the 22. Shows, T. B., Ruddle, F. H. & Roderick, T. H. (1969) Biochem. payment of page charges from funds made available to support the Genet. 3, 25-35. research which is the subject of the article. This article must therefore 23. Hultberg, B., Lundblad, A., Masson, P. K. & Ockerman, P. A. be hereby marked "advertisement" in accordance with 18 U. S. C. (1975). Biochim. Biophys. Acta 410, 156-163. §1734 solely to indicate this fact. 24. Giblett, E. R., Harris, H., Meera Khan, P., Lovrien, E. W., Mellman, W. J., Partridge, C. W. H. & Shows, T. B. (1976) Cy- 1. Ockerman, P. A. (1973) in Lysosomes and Storage Diseases, eds. togenet. Cell Genet. 16, 65-74. Hers, H. & Van Hoff, F. (Academic Press, New York), pp. 25. Carter, N. D. & Parr, C. W. (1967) Nature 216, 511. 291-304. 26. Dizik, M. & Elliott, R. W. (1977) Biochem. Genet. 15,31-46. 2. Kjellman, B., Gamstorp, I., Brun, A. Ockerman, P. A. & Palm- 27. Shows, T. B. (1975) Cytogenet. Cell Genet. 14, 199-207. gren, B. (1969) ]. Pediat. 75,366-373. 28. Baltimore Conference (1975) Third International Workshop on 3.. Norden, N. E., Ockerman, P. A. & Szabo, L. (1973) J. Pediat. 82, Human Gene Mapping (Birth Defects: Original Article Series 686688. XII:7, 1976) (The National Foundation, New York) and (1976) 4. Masson, P. K., Lundblad, A. & Autio, S. (1974) Biochem. Biophys. Cytogenet. Cell Genet. 16, 1-452. Res. Commun. 56,296-303. 29. McMorris, F. A., Chen, T. R., Ricciuti, F., Tischfield, J., Creagan, 5. Carrol, M., Dance, N., Masson, P. K., Robinson, D. & Winchester, R. & Ruddle, F. (1973) Science 179, 1129-1131. B. G. (1972) Biochem. Biophys. Res. Commun. 49,579-583. 30. Hamerton, J. L., Douglas, G. R., Gee, P. a. & Richardson, B. J. 6. Taylor, H. A., Thomas, G. H., Aylsworth, A., Stevenson, R. E. & (1973) Cytogenet. Cell Genet. 12, 128-135. Reynolds, L. W. (1975) Clin. Chim. Acta 59,93-99. 31. Hocking, J. D., Jolly, R. D. & Batt, R. D. (1972) Biochem. J. 128, 7. Poenaru, L. & Dreyfus, J. C. (1973) Biochim. Biophys. Acta 303, 69-78. 171-174. 32. Phillips, N. C., Robinson, D., Winchester, B. G. & Jolly, R. D. 8. Avila, J. L. & Convit, J. (1973) Clin. Chim. Acta 47,335-345. (1974) Biochem. J. 137,363-371. Downloaded by guest on September 28, 2021