To date, reports of at least 90 cases of α-mannosidosis have appeared in the medical literature.26,45– 83 This includes 67 patients reported up to 1982 and reviewed by Chester et al.9 plus an additional 23 patients described since.63,65– 83 Among other things, these reports document that, like many lysosomal diseases, this disorder is characterized by a rather wide range of clinical phenotypes (see Fig. 140-6; Tables 140-3, 140-6). The more severely affected patients, found in the infantile age group, are considered to have the “type I” form of this disorder, while a milder form of the disorder occurring in juveniles and adults is designated “type II.”9,53
Clinical features of α-mannosidosis. (A, B) Twenty-two-year-old male with α-mannosidosis type II, showing facial appearance and destructive synovitis of the knees. (C, D) Patient with α-mannosidosis at the ages of 12 and 30 years. (C and D are from Montgomery, Thomas, and Valle.66 Used by permission of The Johns Hopkins Medical Journal.)
Table 140-6: Clinical Features Specifically noted in α-Mannosidosis Patients |Favorite Table|Download (.pdf) Table 140-6: Clinical Features Specifically noted in α-Mannosidosis Patients
|Clinical Abnormality ||After 1982* ||Before 1982** |
| || n ||% || n ||% |
|Mental retardation ||23/23 ||100 ||53/55 ||96 |
|Hearing loss ||21/23 ||91 ||39/47 ||83 |
|Coarse facies ||15/23 ||65 ||60/61 ||98 |
|Recurrent infections ||14/23 ||61 ||32/48 ||66 |
|Dysostosis multiplex ||13/23 ||56 ||55/59 ||93 |
|Delayed motor development ||12/23 ||52 ||41/45 ||91 |
|Impaired speech ||11/23 ||48 ||30/33 ||91 |
|Hepatomegaly ||8/23 ||35 ||25/54 ||46 |
|Ocular changes ||5/23 ||22 ||21/61 ||34 |
|Macrocephaly ||3/23 ||13 ||7/21 ||33 |
|Hernia ||2/23 ||9 ||22/31 ||71 |
Virtually all type I and type II patients have mental retardation, facial coarsening, and some degree of dysostosis multiplex (see Fig. 140-6; Tables 140-3, 140-6). It should be noted, however, that these features have been reported to be very mild in some patients.68,77,80 Additional findings include recurrent bacterial infections, which may be related to a defect in leukocyte chemotaxis.72 Other findings include deafness, hepatomegaly, hernias, and lenticular or corneal opacities. The ocular findings are distinctive and include posterior opacities in a spokelike pattern in the lens and superficial opacities in the cornea (Fig. 140-7).54,57,73 The skeletal dysplasia55 includes thickening of the calvaria in the majority of patients. The vertebral bodies are prominently involved, with ovoid configurations, flattening, and beaking appearance, sometimes in association with gibbus deformity. Patients with the more severe infantile, or type I, phenotype suffer from rapid mental deterioration, obvious hepatosplenomegaly, more severe dysostosis multiplex than seen in type II, and often death between 3 and 10 years of age.
Spoke-like cataracts in a 3-year-old patient with α-mannosidosis. (From Wong et al.83 Reprinted by permission of Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc.)
In contrast, individuals with the milder juvenile-adult, or type II, form of the disorder are characterized by more normal early development, with mental retardation becoming apparent only during childhood or adolescence. Hearing loss is particularly prominent in type II patients. Dysostosis multiplex is milder than that found in type I individuals, and patients often survive into adulthood. Destructive synovitis,65,67 hydrocephalus,74 spastic paraplegia,69 pancytopenia,75 and hyperphagia81 have also been reported in type II patients. MRI studies of the head in three patients demonstrated bony changes, verticalization of the chiasmatic sulcus, cerebellar atrophy, and white matter signal changes.70 The clinical course of mannosidosis is often described as being gradual, sometimes with imperceptible progression.64,76,77,80 While there may be a continuum of phenotypes rather than a clear separation, it is nevertheless possible in most cases to distinguish type I from type II patients. See, for example, the comparison of the clinical, biochemical, and radiographic findings in the two forms of the disease by Bennett et al.80
One of the more useful laboratory findings is the presence of vacuolated lymphocytes in almost all α-mannosidosis patients. While there is no evidence of mucopolysacchariduria, urine from both type I and II patients, when examined by thin-layer chromatography, is characterized by abnormal oligosaccharide patterns.80 Decreased serum IgG can occur, and a decreased PR interval on EKG has been reported.78
One patient with α-mannosidosis underwent bone marrow transplantation (at 7 years of age) and died 18 weeks later from bronchopneumonia. While there was some evidence of enzymatic correction at autopsy, there was no evidence of decreased neuronal vacuolization.79 A kidney transplant has also been carried out on a 23-year-old type II mannosidosis patient with end-stage renal failure. Three years after the transplant, the graft was functioning well with no evidence of renal deposits. The clinical course of the mannosidosis at the time of the report was stated to be “silent,” while the patient's quality of life was said to be good.84
In an animal study, Walkley et al.85 examined the efficacy of bone marrow transplantation as a means of enzyme replacement. Three kittens lacking α-mannosidase received bone marrow transplants at ages 8, 10, and 12 weeks of age using normal sibs as donors. While clinical evidence of cerebellar disease was seen in two of the three treated cats, these animals showed little or no progression of neurologic signs 1–2 years after the transplants. In contrast, the untreated affected cats became severely impaired. The authors provided evidence that the bone marrow transplantation (at least in kittens) restored significant levels of the missing enzyme within neurons of the central nervous system.85
Pathologic studies of biopsy86 and autopsy45,69,74,87 material are available. Light microscopy of the liver demonstrates granular or foamy cytoplasm in the hepatocytes. Periodic acid–Schiff (PAS) staining varies with the histochemical extraction procedure. Electron microscopy (EM) studies show the presence of multiple vacuoles in hepatocytes and Kupffer cells, often with a reticulogranular pattern, although many other types of inclusions are observed. Examination of the central nervous system reveals marked and widespread ballooning of the nerve cells. The cytoplasm has an empty or vacuolated appearance. EM again demonstrates membrane-bound vacuoles with a predominantly reticulogranular pattern.
Although it is known that mannose-rich compounds accumulate in the various tissues of α-mannosidosis patients,26,88 few studies have been carried out on material from these sources. Instead, most of the analyses to date have been of mannose-rich fractions isolated from the urine of affected individuals. Early investigations established that the chemical structure of the three major urinary metabolites are as follows:89
Man(α1 → 3)Man(β1 → 4)GlcNAc
Man(α1 → 2)Man(α1 → 3)Man(β1 → 4)GlcNAc
Man(α1 → 2)Man(α1 → 2)Man(α1 → 3) Man(β1 → 4)GlcNAc
Subsequently, Strecker et al.90 and Yamashita et al.91 isolated and characterized several additional urinary mannose-containing oligosaccharides. Although the two groups found identical structures for several of the newly discovered compounds, they found different compositions for others.90,91
Since then, 17 mannose-rich oligosaccharides have been isolated and identified from pooled urine of two mannosidosis patients.92,93 Three of these compounds are identical to the major metabolites noted above, 13 are identical to those reported previously by Yamashita et al.,91 and one represented a newly discovered compound.
Each of the oligosaccharides isolated from the urine of mannosidosis patients has a single GlcNAc residue at the reducing end, not the GlcNAc(β1 → 4)GlcNAc core expected from the hydrolysis of Asn-linked glycoproteins. This finding appears to result from the enzymatic digestion of the chitobiose core found in glycoproteins and their oligosaccharide products by an endo-N-acetyl-β-glucosaminidase.14,15 In addition, although the most abundant urinary compound, Man(α1 → 3)Man(β1 → 4)GlcNAc, appears to be derived from incomplete digestion of complex glycans, it lacks the expected mannose residue linked (α1 → 6) to the core mannose residue.91 The absence of this compound appears to be due to the presence of a lysosomal (α1 → 6) mannosidase that remains active in affected patients lacking the major α-mannosidase.94– 96
Comparative studies have demonstrated that the urinary oligosaccharides differ in bovine, feline, and human mannosidoses.97 One study suggests that this is due to differences in the metabolic pathways—i.e., cattle and cats lack the endo-N-acetyl-β-glucosaminidase found in humans—and there are differences in the severity of the enzyme defects.98
The Enzyme and the Enzyme Defect
For some time, α-mannosidase activity has been known to exist in a variety of tissues.9 The enzyme became of interest to human medical geneticists following the report of a patient who lacked this enzyme and accumulated mannose-rich oligosaccharides.26
Mannosidase activity in normal tissue results from the combined actions of Golgi membrane mannosidase(s) having a neutral pH optimum99 and a lysosomal form of the enzyme (EC 18.104.22.168).100 The lysosomal form is characterized by an acidic pH optimum; broad substrate specificity, including synthetic compounds, e.g., p-nitrophenyl-α-mannoside; and sensitivity to swainsonine. While early investigations suggested that this lysosomal enzyme existed in two major forms, designated “A” and “B,”101 later studies have indicated a more complex pattern.102
Previously, the enzyme in fibroblasts was reported to be synthesized as a precursor of ∼110-kDa, with subsequent proteolytic processing to two subunits of 63 to 67 kDa and 40 to 46 kDa.103 Similarly, it was reported that mannosidase A in human liver consisted of equimolar numbers of 26- and 62-kDa subunits, whereas the B form contained the 26-kDa subunit and variable numbers of the 58- and 62-kDa subunits.104 Subsequently, Tsuji and Suzuki,105 using placental tissue, detected only 65- and 27-kDa subunits and provided evidence that differences reported in the early preparations might be due to proteolytic degradation. Although they differ in their molecular mass and subunit compositions, the A and B subunits are immunologically indistinguishable.104 It is believed, therefore, that these two forms of the enzyme are coded for by a single locus.104
In a more recent study of human placenta, Nilssen et al.102 reinvestigated the structure of this enzyme. While they agree that α-mannosidase is coded for by a single locus, their evidence indicates that the precursor is processed first into three (not two) glycoproteins, of 70, 42, and 15 kDa. It is suggested that the smallest, and previously unreported, 15-kDa peptide was missed in the early studies due to technical difficulties of the methods utilized. Evidence is also presented that the 70-kDa glycoprotein, found in both this and the earlier studies, is further processed into three more peptides joined together by disulfide bridges.
The molecular and genetic relationships of the various forms of the neutral and acidic mannosidases have not been completely elucidated; however, it is known that mannosidosis patients lack the acidic forms of the enzyme but retain the neutral enzyme(s).9,106 Genetic heterogeneity is suggested by reports of some α-mannosidosis patients who have high residual mannosidase activity.61,107– 110 In the majority of these patients, the residual enzyme appears to have increased heat lability and a marked increase in the K m against certain artificial substrates. In addition, there have been reports that the mutant enzyme is activated by cobalt and to varying degrees by zinc.53,111 Immunologic studies of this residual material have yielded conflicting results. Specifically, antibodies against human liver α-mannosidase fail to react with this material, while rabbit anti–human placenta and anti–pig kidney mannosidase antibodies yield positive reactions.53,112,113
Several investigators have found an accumulation of acid mannosidase activity in media in which mannosidosis fibroblasts have been cultured.103,114– 116 These findings were interpreted by one group as suggesting that “the defect in mannosidosis is expressed only after the enzyme has been delivered to lysosomes and presumably undergone some form of processing here.”116 Later, evidence was presented that mannosidosis fibroblasts do not synthesize acid-mannosidase and that the enzyme secreted by these cells is not related immunologically to lysosomal mannosidase.104
Inheritance and Incidence
The data provided both by reports of families containing both male and female offspring affected with mannosidosis and by the results of molecular mapping clearly indicate that this disorder is inherited in an autosomal recessive manner (MIM 248500).9,117 Therefore, for each pregnancy of a carrier couple, the risk of having an affected child is 25 percent.
Early studies of mouse-human hybrids indicated that the gene encoding acidic α-mannosidase is located on human chromosome 19.118,119 Additional studies of mouse-human hybrids containing rearranged or deleted human chromosomes provided evidence that the gene locus is located on the central region of this chromosome, i.e., 19p13–q13120,121 or, more recently, 19p13.2–q12.122 Using a panel of human/rodent somatic cell hybrids, Bachinski et al.123 showed that the DNA fragment responsible for α-mannosidase activity maps to the long arm of human chromosome 19 [proximal to D19S7 and peptidase D [PEPD]). Subsequently, Nebes and Schmidt,124 using degenerate oligonucleotide primers to generate a cDNA probe, isolated a full-length cDNA by plaque hybridization. From this they deduced the amino acid sequence of a protein believed to be human lysosomal α-mannosidase B. In agreement with the above data, this cDNA mapped to the gene symbol for α-mannosidosis locus (MANB) locus on the long arm of chromosome 19. While some of the sequence data was later shown to be incorrect (see below), expression studies provided direct evidence that this putative cDNA did in fact code for a protein with α-mannosidase activity.125
Emiliani et al.126 purified human lysosomal α-mannosidase in sufficient quantities to permit partial amino acid sequencing. Analysis of the N-terminal residue of a 30-kDa polypeptide of this material directly confirmed that the cDNA isolated by Nebes and Schmidt124 does in fact code for human α-mannosidase. Sequence analysis of cDNA isolated from several human tissues by Liao et al.,127 however, demonstrated sequence differences when compared to the data originally published by Nebes and Schmidt.124 Sequence discrepancies were also noted by Wakamatsu et al.128 and Riise et al.129 Most if not all of these differences have subsequently been reported to have been due to errors in the original published sequence, which the authors later revised;130 thus, the sequences reported by Liao et al.127 Waksmatsu et al.,128 and Riise et al.129 represent the correct data (GenBank U60885).
Liao et al127 cloned 3.0- and 3.6-kb cDNAs by RT-PCR of mRNA from human spleen. When expressed in Pichia pastoris, the shorter of the two cDNAs (containing 2964 bp) encodes a protein having all the characteristics of α-mannosidase previously isolated from human liver. Expression studies with the 3.6-kb product failed to yield enzyme activity. Northern blot analysis demonstrated the presence of a 3-kb RNA transcript in all human tissues examined: A minor amount of the 3.6-kb product was also identified in several tissues. Similar sequence results were obtained on overlapping fragments obtained by PCR of human fibroblast and human lung cDNA by Nilssen et al.102 Northern blot analysis by this group, however, revealed a single transcript of ∼3.5 kb in all tissues examined.
Wakamatsu et al.128 and Riise et al.,129 using human MANB cDNAs as probes, isolated and sequenced the entire human MANB gene. Both groups found the gene to consist of 24 exons spanning approximately 21 to 22 kb and that it appears to be controlled by a 5′ flanking sequence similar to that found previously in other lysosomal enzymes (see Table 140-4). Based on their data, Riise et al.129 concluded that the processed mRNA product of this gene should contain 3443 bp which agrees well with the 3.5-kb mRNA found earlier by Nebes and Schmidt.124
In parallel investigations, murine,131,132 feline,133 and bovine134 cDNAs have been isolated and sequenced. The deduced amino acid of the mouse α-mannosidase shows 75 percent132 to 76 percent133 identity to the human enzyme, while the feline is 81 percent identical.133 The authenticity of the isolated murine cDNA was demonstrated by expression in Pichia pastoris 131 or COS132 cells. Northern blot analysis showed a major transcript (∼3.0–3.5 kb) in all tissues examined.131,132 As was found in human tissue, the bovine gene is organized into 24 exons.134
The first detailed description of a mutation associated with α-mannosidosis was found in two children of consanguineous parents.102 These sibs, of Palestinian origin, were found to be homozygous for a 212A → T transversion resulting in a His → Leu substitution (see Table 140-4). While direct expression studies were not carried out in this family, evidence that this alteration is the cause of the disease in these sibs is provided by the fact that the mutation was not detected in a large number of controls and that the alteration occurs in a conserved position of the gene in several species.102
The results of a mutational analysis of six type II α-mannosidosis patients have also appeared in abstract form.135 With PCR followed by SSCP analysis, a total of three missense mutations were identified in four of these individuals. Three patients were found to be homozygous and one heterozygous for the mutations believed to be responsible for the presence of mannosidosis in these individuals. No mutation was identified by the PCR/SSCP analysis in the remaining two patients. While this was not directly proven, it was concluded that the missense mutations detected in the MANB gene of these patients resulted in their disease.
Patients affected with α-mannosidosis excrete increased amounts of several oligosaccharides, the major one being Man(α1 → 3) Man(β1 → 4)GlcNAc.89,136 Increased amounts of this and related compounds can most easily be demonstrated by any one of several thin-layer chromatography techniques widely utilized for the detection of inherited oligosaccharide disorders (Fig. 140-8).137– 140 For a detailed discussion of these methods of urinary screening, see the review by Sewell.141 High-performance chromatography of oligosaccharides in urine142 and fibroblasts143 has also been described. As do individuals affected with other errors of glycoprotein degradation, α-mannosidosis patients have an abnormal pattern of oligosaccharide excretion.
Thin-layer chromatogram of urine with orcinol detection. M = α-mannosidosis; G = GM1 gangliosidosis; AS = aspartylglucosaminuria; F = fucosidosis; N = normal control; S = standard mixture of fructose, lactose, and reffinose; ML1 = sialidosis. (From Sewell.137 Used by permission.)
Although its significance is currently unclear, the report that two sibs with mannosidosis, as well as 16 aspartylglucosaminuria patients, have elevated serum dolichol levels144,145 suggests that the measurement of this compound might also be a useful screening tool. Once a urinary oligosaccharide pattern suggestive of α-mannosidosis is demonstrated, the diagnosis can be confirmed or excluded by direct measurement of α-mannosidase activity in any one of several tissues, e.g., white blood cells, fibroblasts, and cultured amniotic fluid cells. Direct measurement of α-mannosidase in plasma has been less reliable than the assay of cellular enzyme levels, probably because of forms of mannosidase enzymes in plasma that are not decreased in mannosidosis patients.9 Evidence, has been presented however, that if the pH is carefully selected, e.g., pH 4.0, plasma can also be utilized for the diagnosis of α-mannosidosis.146
There are several reports of the successful prenatal diagnosis of mannosidosis. These include a report of an affected fetus, later confirmed,147 and experience with two families in which two fetuses were affected while a third was found to be free of the disease.148 Activity of α-mannosidase has been demonstrated in trophoblast biopsy material,149 and a first-trimester diagnosis using chorionic villi has been reported.150 It should be noted, however, that α-mannosidase activity in chorionic villi has been found to be less than one-third of that found in cultured amniotic fluid cells,151 emphasizing the importance of good normal control data. Although these studies were carried out with no serious difficulties, the presence of both residual activity and forms of α-mannosidase not affected in mannosidosis patients must be taken into account in interpreting the results of a prenatal study for this disorder.108
There are at least three unrelated animal models for human α-mannosidosis. The best known one, bovine mannosidosis, is an autosomal inherited disorder found in Aberdeen Angus cattle.152,153 The disorder has also been described in a number of related breeds of cattle, e.g., Red Angus, Murray Grey, Galloway, and shorthorns.154 The disease in these animals is characterized by ataxia, incoordination, tremor, and aggressive behavior. Like humans with mannosidosis, animals affected with the bovine form of this disorder have reduced levels of the acidic form of α-D-mannosidase.153 It has been estimated that in the absence of genetic testing, approximately 400 Angus calves born in Australia each year would be affected with this disorder.155 The actual prevalence of α-mannosidosis in Angus cattle in Australia, however, has been reduced as the result of an industry-sponsored disease control effort.155 Additionally, α-mannosidosis has been found in a domestic short-hair cat having similar clinical findings, i.e., multiple skeletal deformities, retarded growth, ataxia, intention tremors, and deficiency of α-mannosidase activity.156,157 More recently this disorder has been described in Persian cats.158– 162