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Infantile ceramidase deficiency (Farber disease) is an uncommon, progressive lysosomal storage disease characterized by multiple ceramide-containing nodules (lipogranulomata) in the subcutaneous tissue and upper aerodigestive tract, painful periarticular swelling, psychomotor retardation, and varying degrees of ocular, pulmonary or hepatic involvement. Management of Farber disease has been limited to symptomatic supportive care, and few affected infants survive beyond 5 years of age. We performed an allogeneic bone marrow transplant (BMT) from an HLA-identical heterozygous sister in a 9.5-month-old female with minimally symptomatic Farber disease who received a pre-transplant regimen of busulfan and cyclophosphamide. Ceramidase activity in peripheral blood leukocytes increased from 6% before transplant to 44% (donor heterozygote level) by 6 weeks after BMT. By 2 months after transplant, the patient's subcutaneous lipogranulomata, pain on joint motion, and hoarseness had resolved. Despite modest gains in cognitive and language development, hypotonia and delayed motor skills persisted. Gradual loss of circulating donor cells with autologous hematopoietic recovery occurred; VNTR analyses showed 50% donor DNA in peripheral blood cells at 8.5 months after BMT and only 1% at 21 months after transplant. Interestingly, leukocyte ceramidase activity consistently remained in the heterozygous range despite attrition of donor cells in peripheral blood. This novel observation indicates ongoing hydrolase production by non-circulating donor cells, possibly in the mononuclear phagocytic system, and uptake by recipient leukocytes. Although lipogranulomata and hoarseness did not recur, the patient's neurological and neurocognitive status progressively declined. She died 28 months after BMT (age 37.5 months) with pulmonary insufficiency caused by recurrent aspiration pneumonias. Allogeneic BMT improves the peripheral manifestations of infantile ceramidase deficiency, but may not prevent the progressive neurological deterioration, even when carried out in minimally symptomatic patients.

To date, in utero bone marrow transplantation (BMT) has had limited success, largely because of poor donor engraftment. The poor engraftment is probably the result of performing the procedure late in gestation after significant fetal immunocompetence has developed and/or transplanting insufficient numbers of donor hematopoietic stem cells for competing successfully with ongoing fetal hematopoiesis. To overcome these problems, we performed in utero BMT on a fetus with globoid cell leukodystrophy during the first trimester of gestation using selected paternal bone marrow stem (CD34+) cells. CD34 selection allowed a substantially greater number of stem cells to be transplanted. Although the fetus died 7 weeks after the procedure (during the 20th week of gestation), full donor engraftment was established. Moreover, the cause of death appeared to be overwhelming donor engraftment and leukostasis with paternal myeloid cells infiltrating most tissues. The ability of in utero BMT to produce this degree of engraftment provides great promise for the use of this approach in the treatment of a variety of inherited disorders that can be diagnosed prenatally.

In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases. We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris. Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD. Consistent with this observation, CG4 patients carry mutations in PXAAA1. The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein. Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase. Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p. We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity.

Lymphoblasts are useful cells for the diagnosis and basic studies of several human genetic disorders. Peroxisomal disorders are usually diagnosed by using fibroblasts or blood samples. Here, we report the characterization of peroxisomes in lymphoblasts. We demonstrated that lymphoblasts from a patient with Zellweger syndrome, the prototypical disorder of peroxisome biogenesis, contained peroxisomal ghosts like those described previously in Zellweger fibroblasts. We also found that lymphoblasts that carry a deletion on chromosome 7 (q11.23q22.1), a region thought to contain one Zellweger syndrome gene, contained normal peroxisomes.

The primary genetic defect of Zellweger syndrome may be related to defective synthesis or impaired import of peroxisomal proteins. We analyzed the presence and measured the abundance of the 22-kD peroxisomal integral membrane protein (PMP) in patients with Zellweger syndrome. We determined the intracellular localization of the 22-kD PMP and compared it with the localization of a peroxisomal 44-kD thiolase precursor protein. The 22-kD PMP was quantified by immunoblot analyses in liver tissue (n = 7 patients). Immunoblot signals were evaluated using transmission photometry. The intracellular localization of the 22-kD PMP and the peroxisomal 44-kD thiolase precursor protein were determined by immunoblot analyses on fibroblast subcellular fractions prepared by Nycodenz (n = 5 patients) or sucrose density gradient centrifugation (n = 2 patients). The 22-kD PMP was present and associated with membrane fractions in all patients. Its abundance varied in patients as compared with normal human liver controls. The 22-kD PMP was located in subcellular membrane fractions having a lower density than normal peroxisomes or mitochondria. Using two different gradient techniques, the 22-kD PMP and the peroxisomal 44-kD thiolase precursor protein were found in the same low-density gradient fractions. These results suggest that in Zellweger syndrome peroxisome-like elements containing both the 22-kD PMP and a 44-kD thiolase precursor protein are formed. Globally defective synthesis or import of peroxisomal proteins is therefore unlikely to be the primary genetic defect in the patients we studied.

L-Pipecolic acid, a cyclic imino acid produced during the degradation of lysine, accumulates in body fluids of infants with the generalized peroxisomal disorders, including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. Peroxisome-enriched fractions from normal human liver oxidized L-[3H]pipecolic acid to alpha-[3H]aminoadipic acid (AAA). When human liver organelles were separated on a Percoll gradient, L-[3H]pipecolic acid oxidation activity (as measured by [3H]AAA formation) most closely segregated with the peroxisomal marker, catalase, and was not associated with the mitochondria. L-Pipecolic acid oxidation was not inhibited by antimycin A and rotenone and produced H2O2, consistent with its involving a peroxisomal oxidase. We measured L-pipecolic acid oxidation in liver specimens from patients with peroxisomal disorders. While liver homogenates from adult (n = 5) and infant (n = 10) controls formed 47.1 +/- 6.6 and 48.3 +/- 10.0 pmol AAA/mg protein/h, respectively, Zellweger syndrome livers (n = 8) formed only 1.7 +/- 0.3 pmol AAA/mg protein/h. L-pipecolic acid oxidation in normal infant livers was low at birth and increased with age, but Zellweger syndrome livers showed little activity at any age. Thus, the high circulating levels of L-pipecolic acid in Zellweger syndrome probably result from defective peroxisomal oxidation of L-pipecolic acid to AAA.

We compared the formation of 14CO2 from [I-14C]fatty acids in homogenates of cultured skin fibroblasts and white blood cells from 25 patients with adrenoleukodystrophy (ALD) and from 24 controls. The ALD group included 16 boys with childhood ALD, five men with adrenomyeloneuropathy (AMN), and two boys and two girls with neonatal ALD. The substrates were unbranched saturated fatty acids ranging in chain length from 16-26 carbons. From C24:0, the radioactive CO2 production by homogenates of ALD fibroblasts and white blood cells was 17% and 37% of control, respectively, and from C26:0 it was 17% of control in ALD fibroblasts. The CO2 evolution from palmitate (C16:0) in the ALD was identical to the control group; for C18:0, the value for ALD cells was 76% of control, and fatty acids with chain lengths between C18:0 and C24:0 gave intermediate results. Results for childhood ALD patients were similar to those for the AMN patients. More limited studies with cultured amniocytes of fetuses with childhood ALD gave results similar to those obtained with cultured skin fibroblasts, and results with neonatal ALD patients appeared to be the same as for childhood ALD and AMN. Studies of three women who were carriers for childhood ALD gave values intermediate between ALD and control. The total C26:0 levels in ALD cultured skin fibroblasts and white blood cells were 4-6 times that of control; the total C24:0 levels were increased 10-30%, whereas the C22:0 levels were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniocentesis was performed in two women heterozygous for adrenoleukodystrophy (ALD). One fetus was male, and the hexacosanoic acid (C26) level in the cultured amniotic cells was 0.808 microgram per mg of protein, compared to 0.104 +/- 0.069 (S.D.) in controls. Pregnancy was interrupted at 22 wk gestation. The fetal adrenal cortex showed the ultrastructural inclusions characteristic of ALD and C26 accounted for 35% of the fatty acids in the cholesterol esters extracted from this tissue, more than one thousand times control. The second amniocentesis was performed in a woman who was also heterozygous for an electrophoretic variant of glucose 6- phosphate dehydrogenase (G6PD), and a member of a kindred showing genetic linkage of loci for ALD and G6PD. The fetus was female and the C26 level in cultured amniotic cells was 0.577. Pregnancy was interrupted at 11 wk for reasons unrelated to ALD. Study of C26 level and G6PD type in cultured fetal tissues confirmed heterozygosity for ALD.

Low arylsulfatase A levels are reported in two siblings, one with a neurologic disability not typical for metachromatic leukodystrophy, the other a healthy 18-year-old female with a normal developmental history. In both individuals, arylsulfatase A levels in white blood cells were 7-8% of control values. Cultured fibroblasts gave low values (8-10% of normal) for both cerebroside sulfatase and arylsulfatase A activities. Other family members had enzyme levels consistent with heterozygote or normal status. Cerebroside sulfate loading tests of cultured fibroblasts in 199-CO2 media were normal for all family members who were tested. In MEM-HEPES media, however, cells from the two arylsulfatase A deficient siblings showed attenuated sulfolipid catabolism. Additional clinical and laboratory studies on these individuals failed to demonstrate any features suggestive of metachromatic leukodystrophy, i.e., normal nerve conduction velocities, normal sural nerve biopsy results, and normal urinary sulfatide excretion. It is concluded that the neurologic abnormalities in the one sibling are not the result of the low enzyme activity and that both individuals represent examples of pseudo arylsulfatase A deficiency (arylsulfatase A deficiency without metachromatic leukodystrophy). These results thus call into question the ability of the high-sensitivity cerebroside sulfate loading test as carried out in MEM-HEPES media to differentiate pathologically significant defects i.e., metachromatic leukodystrophy from benign "pseudo-deficiencies."