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Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular alpha-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular alpha-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35 +/- 9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains of Mr = 52,000. During a 1.5-hr pulse-label with 35S-methionine, alpha-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form with Mr = 58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form with Mr = 62,000. In contrast, only 25-30% of control enzyme was processed to an extracellular form (Mr = 62,000), with the remainder retained intracellularly (Mr = 60,000). In the other two fucosidosis cell lines, alpha-L-fucosidase was synthesized as an intracellular form with Mr = 56,000 that was processed to an extracellular form with Mr = 60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of alpha-L-fucosidase as expressed by lymphoid cells.

Delayed hypersensitivity (DH) in the mouse was studied with complexes of dinitrophenyl (DHP) as hapten and bovine serum albumin (BSA), mouse immunoglobulin (MIg) and polyvinylpyrrolidone (PVP) as carrier. Priming with BSA induced strong DH against this carrier, but DN of decreasing strength against complexes with increasing DNP:carrier ratio. Priming with DNP-BSA complexes never resulted in a DH against BSA or DNP 3-minusBSA. Injections of DNP 16-minusBSA and DNP 28-minusBSA induced positive DH which increased with the hapten:carrier ratio of the eliciting antigen. The complexes with an isologous carrier DNP 48-minusMIg or DNP 90-minusMIg induced positive reactions against both complexes but not against the weakly substituted DNP 11-minusMIg. The latter only primed for itself. The importance of the DNP groups as determinant in these DH reactions is stressed by the cross-reactions between DNP-BSA and DNP-MIg complexes and by the induction by DNP 16-minusPVP of positive DH against DNP 28-minusBSA. Cyclophosphamide (Cy) treatment before priming with complexes induced enhanced DH against complexes with sufficient hapten:carrier ratio. Priming with carrier under Cy treatment induced no DH against complexes. All these results indicate that carrier determinants are not involved in the DH against complexes. After priming wtih complexes with a low hapten:carrier ratio the DH is directed against new antigenic determinants (NAD) groups. After priming with complexes with high ratios DH is directed against DNP groups. With adoptive local transfer of spleen cells of primed animals and pretreatment of these cells with anti-thymocyte serum or anti-plasma cell serum and complement it was possible to demonstrate that the T cell was responsible for the DH reactions. The involvement of different determinant groups on the hapten-carrier complexes in immune reactions is discussed.

Recently, a subset of fucosidosis patients was identified in which the single EcoRI site in the open reading frame of the human cDNA encoding alpha-L-fucosidase was obliterated. We have employed the polymerase chain reaction technique to amplify alpha-L-fucosidase DNA from the five patients known to carry the EcoRI abnormality as well as four patients and two additional fucosidosis patients who do not carry the EcoRI abnormality. Sequence analysis of the amplified DNA has determined that the EcoRI site was destroyed by a C-T transition in the last position of the EcoRI site. This single base change results in the generation of a stop codon 120 base pairs upstream of the normal stop codon. In addition, we have determined that EcoRI cleavage of amplified DNA may be a useful diagnostic tool in the diagnosis of heterozygotes and in prenatal diagnosis of fetuses at risk for this disease.

Fucosidosis is an autosomal recessive lysosomal storage disorder characterized by progressive neurological deterioration and mental retardation. The disease results from deficient activity of alpha-L-fucosidase (E.C.3.2.1.51), a lysosomal enzyme that hydrolyzes fucose from fucoglycoconjugates. In an attempt to identify the mutation(s) that result(s) in fucosidosis, we performed Southern blot analysis of the structural gene encoding alpha-L-fucosidase (FUCA 1) in 23 patients affected with fucosidosis. In five patients Southern blot analysis showed obliteration of an EcoRI restriction site in the open reading frame of FUCA 1 encoding mature alpha-L-fucosidase. This abnormality was not observed in 80 controls, and it may be the basic defect responsible for fucosidosis in these patients. Both patients with the severe type I form of fucosidosis and patients with the less severe type II were shown to be homozygous for this presumed mutation. In the remaining 18 patients the EcoRI site obliteration, major-gene deletions, or insertions were not detected. This suggests that at least two different mutations are involved in fucosidosis. The heterogeneity found at the DNA level was not present at the protein level, as all fucosidosis patients investigated had low fucosidase protein (less than 6% of normal) and negligible fucosidase activity in fibroblasts and lymphoblastoid cell lines.

We describe a girl with extreme growth retardation of prenatal onset, short limbs, and somewhat unusual facial appearance. She represents the 6th patient with osteodysplastic primordial dwarfism type II. The distant consanguinity of the parents of this patient suggests possible autosomal recessive inheritance.

Two families with five patients affected with fucosidosis are described. Within each family, both type I and type II fucosidosis are present. This suggests that environmental factors or "modifying genes" different from the fucosidase structural gene contribute to the phenotype of a fucosidosis patient.

We report on a family with X-linked hydrocephalus: progressive increase in head circumference (OFC) led to the diagnosis in 3 patients; however, in 5 with normal OFC, the initial diagnosis had been "nonspecific" mental retardation, until identification of relatedness between 3 macrocephalic boys suggested segregation of a major Mendelian gene. Moderate to severe hydrocephaly was present in all macrocephalic patients and in 3 of the 5 with normal OFC, but CT-scan of the brain did not show aqueductal stenosis in any of them. We stress the importance of a brain CT-scan in every male with "nonspecific" mental retardation, especially in cases with X-linked inheritance, because confirmation of X-linkage has important genetic counseling consequences.

The influence of hapten, carrier and their ratio in a complex on T-cell helper stimulation and antibody formation against the dinitrophenyl (DNP) hapten was studied. Complexes of DNP with bovine serum albumin (BSA), bovine gamma-globulin (BGG), isologous mouse immunoglobulin (MIG) and polyvinylpyrrolidone (PVP) as carriers were used. Optimal antibody formation against DNP was obtained with complexes with an intermediate hapten:carrier ratio (DNP 16-minusBSA, DNP 43-minusBGG and DNP 48-MINUSMIG). DNP-PVP complexes were not active either in the primary or in the secondary response. The anti-BSA titre was independent of the number of DNP groups on the complex used for immunization. Inhibition of DNP-plaque formation by spleen cells of immunized mice shows an increase of the inhibitory capacity of the complex with the increase of the hapten-carrier ratio. DNP 16-minusPVP was the only PVP complex which was inhibitory. These results suggest that helper cells involved in the antibody formation against BSA and DNP are reactive with different parts of the complex. Priming of mice with carrier or complex after cyclophosphamide (Cy) treatment followed by a secondary injection with complex 10 days later gave strong indications that there is a greater involvement in stimulation of helper T cells by determinants of the complex (new antigenic determinants (NAD) or NAP-DNP groups) or DNP, than by true BSA determinants. This holds for both the IgM and IgG responses.