W. A. Gahl

The activity of a cystine transport system in lysosomes prepared from the leukocytes of patients with cystinosis was found to be deficient. In normal subjects, this system was resistant to N-ethylmaleimide and demonstrated saturation kinetics. Lysosomes from individuals heterozygous for cystinosis demonstrated a reduced maximum velocity for cystine egress from lysosomes. The rate of cystine escape from normal lysosomes was enhanced by adenosine triphosphate. The availability of normal and mutant lysosomes provides a means of investigating mechanisms of amino acid transport across lysosomal membranes.

BACKGROUND: Hermansky-Pudlak syndrome is characterized by oculocutaneous albinism, a storage-pool deficiency, and lysosomal accumulation of ceroid lipofuscin, which causes pulmonary fibrosis and granulomatous colitis in some cases. All identified affected patients in northwest Puerto Rico are homozygous for a 16-bp duplication in exon 15 of a recently cloned gene, HPS. We compared the clinical and laboratory characteristics of these patients with those of patients without the 16-bp duplication. METHODS: Forty-nine patients -- 27 Puerto Ricans and 22 patients from the mainland United States who were not of Puerto Rican descent -- were given a diagnosis on the basis of albinism and the absence of platelet dense bodies. We used the polymerase chain reaction to determine which patients carried the 16-bp duplication. RESULTS: Twenty-five of the Puerto Rican patients were homozygous for the 16-bp duplication, whereas none of the non-Puerto Rican patients carried this mutation. Like the patients without the duplication, the patients with the 16-bp duplication had a broad variation in pigmentation. Nine of 16 adults with the duplication, but none of the 10 without it, had a diffusing capacity for carbon monoxide that was less than 80 percent of the predicted value. High-resolution computed tomography in 12 patients with the 16-bp duplication revealed minimal fibrosis in 8, moderate fibrosis in 1, severe fibrosis in 1, and no fibrosis in 2. Computed tomography in eight patients without the duplication revealed minimal fibrosis in three and no fibrosis in the rest. Inflammatory bowel disease developed in eight patients (four in each group) between 3 and 25 years of age. CONCLUSIONS: The 16-bp duplication in exon 15 of HPS, which we found only in Puerto Rican patients, is associated with a broad range of pigmentation and an increased risk of restrictive lung disease in adults.

Exposure of intact leucocytes to 0.25 m~ ~-[~~S]cystine dimethyl ester resulted in intralysosomal ester hydrolysis and free cystine accumulation in the isolated lysosome-rich granular fractions of normal, heterozygous, and cystinotic cells. Loaded normal and cystinotic granular fractions were incubated in 0.25 m~ sucrose, 10 m~ 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.0, at 37 “C under conditions in which lysosomal integrity, assessed by latency ofhexosaminidase, was substantially preserved. Serial aliquots were washed and treated with 10 m~ N-ethylmaleimide, and [35S]cystine and [3SS]cysteine-N-ethyImaleimide were analyzed by high voltage electrophoresis. Half-times for [36S]cystine loss from loaded cystinotic granular fractions (80.8 min f 10.7 S.E., N = 12) were much slower than normal (26.1 f 1.4, N = 13), with heterozygous intermediate (43.5 f 3.1, N = 8) (all p < 0.01). In contrast, for [35S]cysteine disposal, mean cystinotic TIf2 was 18.3 min f 0.9, heterozygous 16.9 zk 0.5, and normal 14.1 * 0.7. The rate of disposal of t3H] tryptophan from cystinotic granular fractions loaded with ~-[~H]tryptophan methyl ester (mean TlIz = 28.9 min) did not differ from normal (mean TlIz = 25.6 min). Similarly, ~-[~I€Jmethionine exodus from cystinotic granular fractions (mean = 17.5 min) was indistinguishable from normal (mean Tr,z = 17.2 min). Loading with unlabeled cystine dimethyl ester and serial assay of granular fraction cystine verified that cystinotic granular fractions fail to dispose of cystine. Under appropriate conditions, loss of cystine from normal granular fractions was quantitatively accounted for by cystine recovered in the medium. We conclude that isolated cystinotic lysosomes demonstrate a pronounced, selective defect in the exodus of cystine, but not of the other amino acids examined.

Primary hypothyroidism is a known complication of nephropathic cystinosis, a lysosomal storage disorder characterized by renal failure as well as deterioration of other organs. The drug cysteamine depletes lysosomes of cystine and helps preserve renal function and enhance growth in cystinosis patients. To determine whether cysteamine also prevents hypothyroidism, we retrospectively divided 101 patients into group A (n = 28; well treated), group B (n = 26; partially treated), and group C (n = 47; poorly treated). Lifetable analysis indicated a significantly higher probability of remaining free of L-T4 replacement in group A vs. group B (P = 0.09) or group C (P = 0.004). Cysteamine therapy also improved mean height z-scores (-2.17 in group A, -3.04 in group B, and -4.07 in group C) and reduced the bone age deficit (i.e. chronological age minus bone age) by 1.5 yr for every 10 yr of previous cysteamine therapy. We conclude that in addition to its other salutary effects, oral cysteamine therapy helps prevent hypothyroidism and enhances growth in patients with nephropathic cystinosis.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.