42

Case Reports @ 1992 The Japanese Society of Pathology

An Autopsy Case of Farber‘s Lipogranulomatosis in a Japanese Boy with Gastrointestinal involvement

Mayumi Koga’, Tokuhiro lshihara2, Fumiya Uchino2, and Takehisa Fujiwaki3

A boy with Farber’s lipogranulomatosis is reported. Exces- sive ceramide was revealed by thin-layer chromatography of the extracts from the liver. Acid ceramidase activity of the liver was 31.5% of control with exogenous substrate and 33.3% without exogenous substrate. The histological appearance showed granulomatous lesions, composed of spindle or oval-shaped storage cells and proliferation of the connective tissues, in the subcutaneous tissue of the lower lip, periarticular regions and the pericardium. Histo- chemically the storage cells were revealed to contain lipid and polysaccharide. The foreign body granuloma formed by the surgical suture in the liver was surrounded by a large number of foamy cells. In gastrointestinal mucosa widespread erosion, disappearance of glands and abundant collagen fibers were noted. On electron microscopy, the spindle or oval-shaped cells i n the subcutis of the lip had intracytoplasmic inclusions containing granular or fibrillar materials and a smaller number of curvilinear structures, so called “Farber bodies”. Our case was a typical clinical and histopathological presentation of Farber’s lipogranu- lomatosis. However, ceramidase activity was higher than in previous descriptions, and severe gastrointestinal lesions and the appearance of a large number of foamy cells around the foreign body granuloma have not been described previously. Acta Pathol Jpn 42 : 42-48, 1992.

Key words : Farber‘s disease, Ceramidase deficiency, Cur- vilinea r struct ure, Gastrointestinal lesion, Ceramidase activity

Farber‘s lipogranulomatosis is a disorder of lipid metabolism due to deficiency of lysosomal acid cer-

amidase. Its clinical manifestations include growth and developmental retardation, hoarse voice, multiple subcu- taneous nodules and progressive arthropathies with periarticular swelling. I ts histological findings include granuloma formation with infiltrated lipid-laden macro- phages in the skin, periarticular regions and the larynx.

Since Farber described the first case in 1952 (l), at least 30 cases have been reported (2), including four cases(3-5) from Japan. We describe the autopsy findings of a Japanese boy with liver and gastrointestinal involvement.

CASEREPORT

A Japanese boy, the second product of healthy non- consanguineous marriage, was born at term after an uneventful pregnancy. His elder sister was healthy. A t birth he weighed 3,060 g and his length was 5 1 cm. He showed slowly progressing pes calcaneovalgus, joint contracture with painful swelling and hoarseness. At age 10 months he developed high fever and watery diarrhea. Cholecystectomy was performed at age 2 0 months to remove the gallstones. He died of broncho- pneumonia at age 2 years and 1 month. Detailed clini- cal findings will be described elsewhere (6).

His skin was sclerotic. Severe contractures with mild swell- ing were noted in the elbows, wrists, fingers, knees, ankles and toes (Fig. la) . A subcutaneous nodule with a diameter of 1 5 X 1 5 m m was noted on the lower lip, as

Autopsy was carried out three hours after death.

was a 20 m m x 2 0 m m nodule on the skin close to the anus (Fig. lb). A yellowish 10 m m x l O m m tumor was Received June 4, 1991.

Accepted for publication September 30, 1991. ‘Department of Pediatrics and 2First Department of Patho\- seen in the right lobe of the liver under the operation scar ogy, Yamaguchi University School of Medicine, Ube. of cholecystectomy. The gastrointestinal mucOSa was 3Department of Pediatrics, Kokura Memorial Hospital, Kita- hypertrophic, and the inner surface was irregular due to kyusyu. the wide-spread erosion. Other abnormal findings were Mailing address : Mayumi Koga, Department of Pediatrics, pneumonia, hydrocephalus and stones in the bile duct. Yamaguchi University School of Medicine, Ube 755, Japan. This case was presented at the 30th Annual Meeting of the Neither liver nor Spleen was enlarged. Japanese Society of Reticuloendothelial System.

-

Acta Pathologica Japonica 4 2 (1): 1992 43

MATERIALS AND METHODS

Tissues were fixed in 10% buffered formalin for his- tological and histochemical studies. For transmission electron microscopy, tissues of the skin, liver, spleen and intestine were fixed in 2.1% glutaraldehyde, postfixed in 1% osmium tetroxide and embedded in Epon 812. The ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H-800 electron microscope.

Lipid analysis of the liver was performed as follows: Crude lipid was extracted from the patient and a control liver fixed in 10% formalin and developed by thin-layer chromatography. The developing fluid was a mixture of chloroform and methanol (95/5, V/V). Commercial ceramide with non-hydroxy fatty acids and with hydroxy fatty acids (Sigma, St. Louis, Mo, USA) were used as standards. Acid ceramidase activity was measured by the method described by Mitsuo et a/. (7) with slight modifications. Liver homogenates in the sodium phos- phate buffer, pH 4.5, were incubated with and without an exogenous substrate: ceramide with non-hydroxy fatty acid. Then the amounts of sphingosine produced in this

Figure 1. A patient at autopsy. a : General appearance. Joints of extremities show severe contracture with mild swell- ing. b : A subcutaneous nodule on perianal region.

reaction were measured with the high-performance liquid chromatographic method after being labeled by o- phthalaldehyde, a fluorescent probe. In addition arylsul- fatase A, B and C and other lysosomal enzyme activity was measured using 4- met h y I um bell if ery I substrate, with the exception of arylsulfatase A, which was done using p-nitrocatechol sulfate. Substrate and liver homogenate were incubated at 37'C and after that O.D. was measured at 515 nm against water using a Hitachi spectrophotometer.

RESULTS

Microscopic findings

Subcutaneous nodules on the lip and perianal region both had a similar histological appearance including elongation of the rete ridges, proliferation of the collagen fibers and capillaries, and scattered spindle or oval- shaped cells (Fig. 2). These cells had an enlarged cyto- plasm positive for colloidal iron, alcian blue and PAS, were resistant to diastase digestion, and showed occa- sional metachromasia with toluidine blue staining. Vari-

44 Farbet's Ltpogranulornatosis (Koga et a/.)

Figure 2. Histology of subcutaneous nodule on perianal region. There are proliferation of the collagen fibers and scattered spindle (arrows) or oval-shaped (arrowheads) storage cells.

Figure3. A large number of foamy cells around the foreign body granuloma in the liver. HE.

HE.

ous lipid stainings were as follows; orange red in Sudan 111, orange in Sudan IV, black in Sudan black B and blue in Nile blue sulfate. These results indicate that these cells contain acid or neutral plysaccharides and lipid. Typi- cal foamy cells were rarely seen, and the histochemical findings were similar to those of spindle or oval-shaped cells except that foamy cells were stained more weakly for lipid stainings compared to spindle or oval-shaped ones. Such granulomas were present in the periarticular region, larynx and pericardium.

A small tumor in the right lobe of the liver showed the

foreign body granuloma formed by a surgical suture to be composed of lymphocytes and giant cells, surrounded by a large number of foamy cells (Fig. 3). The cyto- plasm of foamy cells was stained in the same way as the cytoplasm of foamy cells in the subcutis. Most Kupffer cells had enlarged eosinophilic cytoplasm stained in the same way as the spindle-shaped cells in the subcutis. Hepatocytes had clear intracytoplasmic vacuoles which tested positive for lipid staining, but negative for polysaccharide staining. Morphological and histo- chemical findings of histiocytes in the spleen were similar

Figure 4. Wide-spread erosion in the small intestine. HE.

Acta Pathologica Japonica 42 (1): 1992 45

Figure 5. On electron microscopy storage cells in the subcutaneous nodule has intracytoplasmic inclusion bodies contain- ing granular ( a ) or fibrillar (a, b) materials, or a few Farber bodies (c: arrows).

46 Farber's Lipogranulomatosis (Koga et a/.)

to those for Kupffer cells. Endothelial cells and blood cells showed no changes. Alveolar macrophages in the lungs and ganglion cells in the central and peripheral nerve system had intracytoplasmic materials which test- ed positive for lipid and polysaccharide stainings.

The gastrointestinal mucosa showed wide-spread ero- sion with disappearance of glands and proliferation of collagen fibers (Fig. 4). Lipid droplets accumulated sparsely in the lamina propria.

In subcutaneous and periarticular fat ty tissue the cytoplasm of lipocytes was atrophic, and intercellular space contained abundant polysacc ha ride. Collagen fibers and capillaries proliferated among lipocytes.

Kidney, heart and other organs showed no particular changes.

U It ra s t ruct u ra I findings

The spindle or oval-shaped storage cells in the subcu- taneous nodule, which we consider to be fibroblasts and macrophages respectively, had an enlarged cytoplasm and membrane-bound intracytoplasmic inclusions containing flocculent, granular (Fig. 5a) or fibrillar (Figs. 5a, b) materials, with a few curvilinear tubular structures, so called "Farber bodies", which consist of two electron- dense lines separated by clear space. The diameter of tubules was 1 5 nm on the average (Fig. 5c). Macro- phages and endothelial cells in the spleen also had intracytoplasmic inclusions containing granular or fibril- lar materials. The ultrastructure was similar to the storage cells in the subcutaneous nodule, but Farber bodies were never seen. Lymphocytes and other blood cells had no inclusions. Kupffer cells had membrane- bound intracytoplasmic inclusions containing fibrillar materials and a few zebra bodies. Hepatocytes contained only electron-dense fat droplets, suggesting non-specific fat degeneration. In the intestinal mucosa collagen fiber proliferation and electron-dense neutral fat droplets, which were occa sio na Ily phagocyt ized and digested by macrophages, were found along the inner lumen. No specific inclusion bodies were observed.

Lipid analysis

One-dimensional thin-layer chromatography of the patient's liver revealed two closely situated bands (Fig. 6). The upper band corresponds to the authentic stand- ard of ceramideA(CerA in the figure; ceramide with non-hydroxy fat ty acid). The lower band shows the non-specific color. These results indicate that ceramide with non-hydroxy fat ty acid accumulated in the patient's liver. Lipid fraction by DEAE-Toyopearl column in the patient's liver revealed accumulation of sulfatide and

Cer A P t Cer B Figure 6. Thin-layer chromatography of lipid. Upper band in the patient liver corresponds to ceramide with non-hydroxy fatty acids. CerA, Ceramide with non-hydroxy fat ty acid ; Pt, Crude lipid from patient's liver; CerB, Ceramide with hydroxy fat ty acid.

g a ng I io s ide.

Enzyme assay

The ceramidase activity was 0.388 nmol/mg protein per hr, which was 31.5% of control values, with the addition of exogenous substrate. Without exogenous substrate, the activity was 0.261 nmol/mg protein per hr, which was 33.3% of the control value. The activities of arylsulfatase A, 6, C and other lysosomal enzymes were within normal ranges.

DISCUSSION The specific diagnosis of Farber's lipogranulomatosis

can be made by demonstration of a deficiency of acid ceramidase, ceramide accumulation in tissue or body fluid or characteristic morphological features of subcuta- neous tissue (2). In the present case we demonstrated ceramide accumulation by thin-layer chromatography. The ceramidase activity of the liver specimen was 31.5% with exogenous substrate and 33.3% without exogenous

Acta Pathologica Japonica 42 (1): 1992 47

substrate, and these activities were higher than those of previous reports (less than 6% of control .values) (2). We speculate that the reason is that alkaline and neutral ceramidase activity, which were described to be within a normal range in a previous report (2), might be measured with acid ceramidase.

The light microscopic finding was granuloma with lipid-laden macrophages in the skin, periarticular tissue, larynx and pericardium as described previously (2).

Since van Hoof and Hers(8), several authors have stated that curvilinear tubular structures or "Farber bodies", are a characteristic ultrastructural finding in this disease (3, 9-1 l), and it is considered to consist of accumulating ceramide in lysosomes. In our case we found that a few macrophages contained curvilinear tubular structures similar in shape and size to those in the previous cases.

In the first Japanese case(3) storage cells in the subcutaneous nodule were classified into two types, spindle-shaped cells and typical foamy cells. The cyto- plasm of spindle-shaped cells was positive for PAS, colloidal iron and alcian blue, while typical foamy cells were negative. In our case, storage cells in the subcuta- neous nodule and the other organs were almost all spindle-shaped, and typical foamy cells were rarely seen except around the foreign body granuloma in the liver. Although, the histochemical differentiation of these cells was not as clear as in the first Japanese case, typical foamy cells in our case were stained more weakly for polysaccharide staining compared to spindle or oval- shaped cells. In the first Japanese case Farber bodies were found in large foamy cells, while flocculent or reticulogranular materials were in spindle-shaped cells. In our case spindle or oval-shaped cells had flocculent or granular materials as shown in the first Japanese case. However, because of the lack of typical foamy cells, Farber bodies were seen in only a small number. In neither case were Farber bodies detected in the liver or spleen.

The granulomas in Farber's lipogranulomatosis are usually present in the skin, periarticular tissue and larynx, which are subject to mechanical damage(2), and this applies to our case. Although the role of cerarnide in vivo has not been completely elucidated, it may be sug- gested that ceramide has a close relationship with mechanical damage. The infiltration of foamy cells around the foreign body granuloma in the liver may be explained as follows : Massive ceramide was synthesized during the surgical invasion and accumulated in the tissue due to the deficiency of ceramidase. Then macro- phages phagocytized it, and they developed into foamy cells.

Gastrointestinal symptoms such as vomitting, diar-

rhea or appetite loss were described in several previous cases (4, 12-1 5), but the severe gastrointestinal mucosal lesion observed in our case has not yet been documented. Since the gastrointestinal mucosa is also subject to mechanical damage, these lesions might be generated by the same process as the granulomas in the skin or periarticular tissue.

lntracytoplasmic accumulation of acid polysaccharide was described in several previous cases (3, 14-16). In our case a large amount of acid polysaccharide stained with colloidal iron and alcian blue was seen, especially in the cytoplasm of macrophages. Lipid analysis revealed sulfatide and ganglioside accumulation in the liver. As suggested previously (2), the accumulation of gan- glioside and other glycolipids may be a secondary phe- nomenon of the ceramidase deficiency, because ceramide is on the degradation pathway of these substrates. This hypothesis is supported more strongly by the normal activity of arylsulfatase and other lysosomal enzymes in our case.

Taking into account the biochemical and histopath- ological findings, the final diagnosis of our case was Farber's lipogranulomatosis. The clinical course, granuloma with lipid-laden macrophages and accumula- tion of ceramide was characteristic for this disease. However, acid ceramidase activity was higher than that in previous descriptions, and severe gastrointestinal involvement and the infiltration of foamy cells around the foreign body granuloma were found for the first time in this disease.

Acknowledgements : We thank to Dr. S. Hamanaka, Yamaguchi Rosai Hospital, for advice on lipid analysis and Dr. Y. Suzuki, The Tokyo Metropolitan Institute of Medical Science, for helpful discussion.

REFERENCES

Farber S. A lipid metabolic disorder "disseminated lipogranulomatosis": A syndrome with similarity to and important differences from Niemann-Pick and Hand-Shuller-Christian disease (Abstract). Am J Dis Child 84 : 499-500, 1952. Moser HW, Moser AB, Chen WW, and Schram AW. Cerarnide deficiency : Farber lipogranulomatosis. In Scriver DR, Beaudet AL, Sly WS, and Valle D, eds. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, 1989 : 1645-1 654. Tanaka T, Takahashi K, Hakozaki H, Kimoto H, and Suzuki Y. Farber's disease (disseminated lipogranu- lomatosis) : A pathological, histological and ultra- structural study. Acta Pathol Jpn 29: 135-155, 1979. Ohfu M, Abe K, Hirano H, et a/. Farber lipogranu- lomatosis: A report of a case with nystagmus, rnyo- clonus and convulsions. Brain Dev 9: 227, 1987.

48

5.

6.

7.

8.

9.

10.

11.

Farber's Lipogranulornatosis (Koga et a/.)

Chanoki M, lshii M, Fukai K, et a/. Farbets lip0 granulomatosis in siblings : Light and electron micro- scopic studies. Br J Dermatol 121 : 777-785, 1989. Fujiwaki T, Hamanaka S, Koga M, et a/. A case of Farber's disease. Acta Paediatr Jpn (in press). Mitsuo K, Kobayashi T, Shinooh N, and Goto I. A high-performance liquid chromatographic assay for 13. acid ceramidase activity in cultured fibroblast from patient with Farber's disease and from controls. Clin Chim Acta 173: 281-288, 1988. Van Hoof F and Hers HG. Other lysosomal storage disease. In Hers HG and van Hoof F, eds. Lyso- somes and Storage Disease. Academic Press, New York and London, 1973: 559-563. Rutsaert J, Tondeur M, Vamos-Hurwitz E, and Dustin P. The cellular lesion of Farber's disease and experi- mental reproduction in tissue culture. Lab Invest 36 : 16.

Schmoekel C and Hohlfed M. A specific ultrastructural marker for disseminated lipogranulomatosis (Farber). Arch Dermatol Res 266: 187-196, 1979. Pellissier JF, Berard-Badier M, and Pinser M. Farber's

12.

14.

15.

474-480, 1977.

disease in two siblings : Sural nerve and subcutaneous biopsies by light and electron microscopy. Acta Neu- ropathol (Berl) 72: 178-188, 1986. Zetterstorm R. Disseminated lipogranulomatosis (Far- bets disease). Acta Pediatr (Uppsala) 47: 501-510, 1958. Schultze G and Lang EK. Disseminated lipogranu- lomatosis: Report of a case. Radiology 74: 428- 431, 1960. Abul-Haj SK, Martz DG, Daglas WF, and Geppert LJ. Farber's disease: Report of a case with observation on its histogenesis and notes on the nature of the stored material. J Pediatr 61 : 221-232, 1962. Amirhakimi GH, Haghighi P, Ghalambor MA, and Honari S. Familial lipogranulomatosis (Farber's dis- ease). Clin Genet 9 : 625-630, 1967. Bierman SM, Edgingtin T, Newcomer VD, and Pearson CM. Farber's disease: A disorder of mucopolysac- charide metabolism with articular, respiratory and neurologic manifestations. Arthritis Rheum 9 : 620- 630, 1966.