Yerushalmi, Baruch*; Sokol, Ronald J.*; Narkewicz, Michael R.*; Smith, Debra*; Ashmead, Josephine W.†; Wenger, David A.‡
The incidence of neonatal liver disease is estimated to be as high as 1 in 2,500 live births (1). The three most common causes, based on a series of 1,086 consecutive infants referred to the King's College Hospital during a 20-year period, were biliary atresia, idiopathic neonatal hepatitis (INH), and α-1-antitrypsin deficiency (2), comprising more than 80% of infants with cholestatic liver disease. In this series, Niemann-Pick disease Type C (NPC) was the second most common genetic/metabolic disorder, with a clinical and histologic presentation similar to that of INH (2). In addition, between 45% and 65% of NPC cases present with neonatal liver disease (3,4), prompting the recommendation that the diagnosis of NPC should be considered in all cases of INH (4). However, NPC rarely has been reported in patients with neonatal cholestasis from North American centers and no published data describe its frequency in infants being evaluated for liver disease (5,6). The objectives of this study were to describe the clinical course of five children from Colorado with NPC who had perinatal or childhood liver disease and to estimate the frequency of NPC in infants who were evaluated for neonatal cholestasis at our center during a 2-year period.
We reviewed the medical records of all infants with cholestasis and all patients with NPC evaluated at our center from January 1997 through December 1998. Infants with neonatal cholestasis (defined as conjugated hyperbilirubinemia and biochemical evidence of liver injury within the first 3 months of life) were identified by a review of patients' records at the Pediatric Liver Center and by a review of all liver biopsies performed in infants during this period. Neonates with cholestasis; negative evaluations for known infectious, metabolic, genetic, toxic, or structural causes; and with liver histology consistent with “giant-cell hepatitis” were described as having INH. The diagnosis of NPC was considered in patients with INH who had persistent splenomegaly, developmental delay, suggestive liver histology, or Hispanic background. The clinical course, laboratory test results, liver histology, electron microscopy results, and the methods used for diagnosing NPC were reviewed for each patient.
Cholesterol Esterification and Sphingomyelinase Analysis
Cultured skin fibroblasts were grown in Eagle minimal essential medium until subculturing for the cholesterol esterification assay. Cholesterol esterification was measured for 6 and 24 hours, and sphingomyelinase assay was performed as described (7).
Mutation Analysis for the Rio Grande Valley Mutation
Genomic DNA was isolated from sonicated cultured skin fibroblasts, leukocytes, or homogenized liver using standard techniques. With the finding that a common mutation was responsible for NPC in individuals descending from the upper Rio Grande Valley of northern New Mexico and southern Colorado, a rapid test to identify patients and carriers was developed (8). A T→C transition at cDNA position 3182 leads to a I→T substitution at position 1061 in the NPC1 protein. A polymerase chain reaction amplification followed by restriction enzyme digestion rapidly identifies the genotype of the individual being tested. This mutation is not only restricted to Hispanics descendant from northern New Mexico and southern Colorado, but is the most frequent allele in patients of Western European descent and correlates with juvenile onset of neurologic disease (8).
Lipid Analysis of Liver
Twenty-to 100-mg wet-weight samples of liver were homogenized in 2 mL of chloroform and methanol (2:1, by volume) using a Duall homogenizer. After filtration of the homogenate through glass wool in a Pasteur pipette, one-fifth volume of distilled water was added and, after vortexing, the mixture was centrifuged at 1,500 g. The lower phase was removed and evaporated with nitrogen. The residue was dissolved in chloroform and methanol (2:1) equal to the weight of the sample in mL. Ten μL was spotted on a silica gel thin-layer chromatography plate along with lipid standards and lipid extracts from previous patients confirmed to have NPC. The plate was placed in a chamber with chloroform, methanol, and water (72:28:4, by volume), and when the solvent front reached the top of the plate it was removed, dried, and sprayed with a solution of orcinol (0.2% in 75% sulfuric acid). The plate was heated at 110°C for 10 minutes. Lipids were identified by their migration patterns and staining characteristics. Patients with NPC show a large increase in free cholesterol and sphingomyelin, and a significant increase in glucosylceramide and lactosylceramide.
The frequency of each cause of neonatal cholestasis during this period was calculated, and the 95% confidence interval was calculated for the frequency of NPC.
DESCRIPTION OF ILLUSTRATIVE CASES
The following case descriptions illustrate the difficulties in diagnosing NPC presenting as neonatal cholestasis.
A 10-day-old white infant of French descent was evaluated for jaundice and pale stools. The mother's pregnancy was normal and the infant's birth weight was 2.8 kg. He was the first child of healthy nonconsanguineous parents. He was an alert, jaundiced infant without dysmorphism. Height and weight were at the 10th percentiles. Mild hepatomegaly was present, and the tip of spleen was palpable. Stool was pale yellow. Chest and spine radiographs were normal. Abdominal ultrasonography showed normal liver echogenicity, the presence of a slitlike gallbladder, and no evidence of biliary ductal dilatation. Patency of the extra hepatic biliary tree was demonstrated on HIDA scan. Diet was changed to medium chain triglyceride–containing formula and treatment with fat-soluble vitamins, water-soluble vitamin E (d-alpha-tocopheryl polyethylene glycol succinate-1000) and ursodeoxycholic acid was begun. Jaundice gradually subsided, yet aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transpeptidase (γGT) concentrations increased to 149 IU/L, 103 IU/L, and 367 IU/L, respectively. Percutaneous liver biopsy specimen at 3 weeks of age showed portal and lobular fibrosis, marked cholestasis, multinucleated giant-cell transformation, and extramedullary hematopoiesis. Electron microscopy showed normal-appearing mitochondria and peroxisomes and no unusual storage product or infectious organisms. Liver blood test results normalized by 6 months of age; however, splenomegaly worsened. The patient had decreased muscle tone, was not sitting at 9 months of age, and would not stand at 12 months of age. Cultured skin fibroblasts at 12 months of age showed a large decrease in the esterification rate of exogenous cholesterol, confirming the diagnosis of NPC.
An 8-week-old white girl was evaluated for failure to thrive, hepatosplenomegaly, and jaundice. The mother had gestational diabetes, but pregnancy and delivery were otherwise uncomplicated. Birth weight was 2.97 kg. The infant was initially breast-fed but did not gain weight well despite supplementation with soy-based formula. Hepatosplenomegaly was noted at 7 weeks of age, and total serum bilirubin concentration was 5.4 mg/dL with conjugated fraction of 3.6 mg/dL, AST concentration was 92 IU/L, and γGT concentration was 168 IU/L. At 8 weeks of age, acholic stools developed. The family was of Irish and British origin without consanguinity, liver disease, emphysema, or metabolic or neurologic disorders.
The patient was malnourished and icteric. Weight was 3.5 kg (<5th percentile), length and head circumference were both at the 15th percentiles. The patient had no cataracts, the heart sounds were normal, the liver was 3 cm below the right costal margin with a span of 6 cm, and the spleen was palpable 3 cm below the left costal margin. Neurologic examination showed mild head lag but otherwise normal newborn reflexes. Stool was acholic with 4+ occult blood and a large amount of neutral fat. Ultrasonograph showed a very small gallbladder, and HIDA scan showed relatively good hepatic uptake of the radioisotope but the gallbladder was not visualized at 1 hour. At 16 hours, there was a minimal amount of radioisotope in the left abdomen.
Percutaneous liver biopsy tissue showed findings consistent with biliary atresia with marked cholestasis, canalicular bile plugs, bile duct proliferation, and portal and lobular fibrosis. At surgical exploration, a normal gallbladder was found, and intraoperative cholangiogram showed small but patent intrahepatic and extrahepatic bile ducts. Findings in the surgical liver biopsy specimen were similar to those of the percutaneous biopsy specimen. Electron microscopy showed a significant amount of bile within the hepatocytes, the canalicular bile had normal appearance, peroxisomes were normal, and no other metabolic storage product or infectious organisms were found. Treatment with medium chain triglyceride–containing formula and fat-soluble vitamins was started; however, weight gain was poor. Skin biopsy specimen for fibroblasts culture and repeat liver biopsy specimen were obtained at 5 months of age. Cultured fibroblast cholesterol esterification was decreased at 6 hours and within normal range at 24 hours. Lipid extraction and thin-layer chromatography of frozen liver tissue showed a pattern of lipids characteristic of that found in NPC. This finding together with the decreased cholesterol esterification rate at 6 hours confirmed the diagnosis of NPC (see Discussion).
A 1-month-old Hispanic infant was evaluated for cholestasis. Preeclampsia and induction at 37 weeks had complicated the mother's pregnancy. The infant's birth weight was 2,550 g. Cholestasis appeared at day 4 of life and was attributed to a urinary tract infection caused by Escherichia coli. However, cholestasis persisted after treatment with antibiotics and the patient was referred for further evaluation. Diet was based on soy formula, stools were pigmented, and growth was normal. Family history was negative for liver, pulmonary, or neurologic disease. Both parents were of Hispanic origin without consanguinity. Physical examination showed an alert, icteric infant with weight and length at the 10th percentiles. He had no cataracts. Heart sounds were normal. The liver was enlarged and firm at 4 cm below the right costal margin with a span of 7 cm. The spleen was palpable at 1 cm below the left costal margin. He had no ascites. Ultrasonography showed normal liver and bile ducts and good contraction of the gallbladder after a meal. Diet was changed to medium chain triglyceride–containing formula, and treatment with fat-soluble vitamins was started. By 6 weeks of age, weight gain was good and stools were pigmented; however, hepatosplenomegaly and jaundice persisted. Percutaneous liver biopsy tissue at 7 weeks of age showed cellular cholestasis, pseudoacinar transformation, and bridging fibrosis, consistent with INH. Jaundice improved by the age of 4 months, and AST and ALT concentrations become normal; however, serum γGT concentration remained increased. Spleen size increased to 5 cm below the left costal margin. Treatment with UDCA was initiated at 6 months of age, and jaundice resolved completely by 7 months of age. Persistent hepatosplenomegaly despite normal growth and development led to reevaluation for NPC at 22 months of age. A second percutaneous liver biopsy specimen showed cirrhosis with regenerative nodules, and electron microscopy showed the presence of osmiophilic, lamellar inclusions in the cytoplasm of hepatocytes, and Kupffer cells and bile duct epithelium consistent with NPC. Lipid profile of the liver specimen was consistent with the diagnosis of NPC. Analysis of DNA from the liver sample showed two copies of the Rio Grande Valley mutation.
A Hispanic infant was born after a normal pregnancy and delivery with a birth weight of 2.9 kg. At 5 days of age, the infant was evaluated for indirect hyperbilirubinemia. Total bilirubin concentration was 21.6 mg/dL, and the conjugated fraction was 1.4 mg/dL. There were no signs of hemolysis. He was treated with phototherapy for 6 days, and total bilirubin concentration decreased to 2.3 mg/dL. At 5 weeks of age, he was reevaluated for persistent jaundice that was unresolved after a change from breast milk to humanized cow milk formula. He was the first infant of a nonconsanguineous Hispanic couple. There was no family history of liver or neurologic disease. Physical examination showed an alert and icteric boy with weight at the 25th percentile, length at the 90th percentile, and head circumference at the 50th percentile. He had normal heart sounds, the liver was palpable at 5 cm below the right costal margin with a span of 8 cm, and the spleen was enlarged. Laboratory results (shown in Table 3) were consistent with neonatal hepatitis. Ultrasonography showed an enlarged liver with normal echogenicity and normal biliary tract anatomy. Percutaneous liver biopsy showed multinucleated giant cells, normal bile ducts, and interstitial fibrosis, consistent with INH. Jaundice gradually resolved during the next 5 months; however, hepatomegaly and splenomegaly persisted. A repeat liver biopsy specimen at 1.5 years of age showed micronodular cirrhosis without giant cells or inflammation. Splenomegaly with normal liver blood tests persisted, without other complications of portal hypertension. The patient was entered into an investigational trial of colchicine for childhood cirrhosis at 6 years of age. Examination showed a well-nourished boy with weight and height at the 25th to 45th percentile. The liver was 4 cm below the xiphoid with a span of 6 cm, and the spleen was firm and 7 cm below the left costal margin. Laboratory results showed the following serum concentrations: AST was 39 IU/L, ALT was 23 IU/L, alkaline phosphatase was 132 IU/L, γGT 7 was IU/L, albumin was 4.9 g/dL, total bilirubin was 0.4 mg/dL, cholesterol was 142 mg/dL, ammonia-28 was μmol/L. Complete blood count, electrolytes, BUN, and creatinine results were normal.
Prothrombin time was mildly increased at 14.3 seconds. Ceruloplasmin, urine copper, and α-1-antitrypsin concentrations were normal. The patient was stable until the development of neurologic signs at age 13 years, which included a gradual inability to ride his bicycle, mild clumsiness, dysarthria, ataxia, and impaired downward ocular movement. These findings prompted evaluation for NPC. Skin biopsy for fibroblast culture was performed, and results showed a large decrease in the esterification rate of exogenous cholesterol. Analysis of DNA from blood leukocytes showed two copies of the Rio Grande Valley mutation.
A 16-year-old girl of Hispanic origin was evaluated for hepatosplenomegaly and progressive ataxia. Indirect hyperbilirubinemia occurred at 5 days of age and was treated with phototherapy. At 2.5 years of age, the patient was hospitalized for acute myocarditis during which hepatosplenomegaly was noted. Cardiac function recovered completely 1 year later, however splenomegaly and hepatomegaly persisted. Evaluation for hematologic and collagen vascular causes was negative. Developmental milestones were normal; however, she was always noted to be clumsy. More than 3 years before her referral, changes in handwriting, dysarthria with slurred speech, dysphagia, and progressive ataxia had developed. Complete blood count, thyroid function, concentrations of electrolytes, vitamin E, serum lactate, and pyruvate, and liver blood test results were all normal. She was the only child of a healthy family of Spanish origin without consanguinity. Physical examination showed an alert well-nourished girl without jaundice. Eye examination demonstrated paresis of vertical upgaze without nystagmus. The spleen was palpable 12 cm below the left costal margin, liver span was 5 cm, and there was no ascites, palmar erythema, telangiectasia, or asterixis. Neurologic examination showed limb and truncal ataxia, wide-based gate, and slurred speech with dysarthria. Ultrasonography showed a normal liver and enlarged spleen with a normal portal vascular system and no ascites. Analysis of DNA from blood leukocytes showed two copies of the Rio Grande Valley mutation of the NPC gene. No liver or skin biopsies were performed.
Forty neonates with cholestasis were evaluated during 1997 and 1998, including the three neonates diagnosed with NPC who were originally labeled as having INH (Table 1). During this period, we had a high index of suspicion for NPC when the first case was diagnosed. Niemann-Pick disease type C accounted for 7.5% (95% confidence interval, 1.6–20.4%) of all new cases of neonatal cholestasis and 27.2% of the cases of INH during this period. Two adolescents, aged 14 and 16 years, also were diagnosed with NPC during this period, one who originally had neonatal hepatitis and cirrhosis, and the other who had hepatosplenomegaly throughout childhood. Tables 2 and 3 present demographic details and laboratory data for all patients with NPC. Diagnostic tests in the first four patients excluded α-1-antitrypsin deficiency, bile acid synthesis defects (serum and/or urine bile acid screen), tyrosinemia (urine succinylacetone), cystic fibrosis (sweat chloride test), congenital hypothyroidism (free thyroxine and thyrotropin levels), and inborn errors of organic and amino acid metabolism (serum amino acid concentration, urine organic and amino acids concentration).
Table 4 describes the specific laboratory studies used to establish the diagnosis of NPC in each patient. The diagnosis was established by using cultured skin fibroblasts to evaluate the cellular cholesterol esterification rate in three patients, by the characteristic liver lipid profile in two patients, and by identifying a specific genetic mutation (Rio Grande Valley mutation) for NPC in three patients.
In our study, NPC was the most common metabolic/genetic disorder presenting as neonatal cholestasis during a 2-year period, accounting for 7.5% of all infants evaluated for cholestasis. Eight cases of INH were identified during the same period. Because the three neonates with NPC were initially presumed to have INH, the frequency of NPC among neonates presenting as INH was 3 of 11 (27%). This frequency of NPC was higher than expected, based on the rarity of reports of NPC in North American infants with neonatal cholestasis. In contrast, Mieli-Vergani et al. (2) reported NPC to be the second most common metabolic cause of neonatal cholestasis in the United Kingdom. For unknown reasons, the possibility of NPC has not been emphasized in the differential diagnosis of neonatal cholestasis in North American centers (5,6). Although the number of NPC cases identified was relatively small during the 2-year period of our study, we found a high incidence of NPC at our center when we aggressively searched for this diagnosis. In fact, NPC was the most common metabolic disease causing neonatal cholestasis during this period, suggesting that NPC should be high on the list of diagnostic possibilities when evaluating neonatal cholestasis.
The clinical presentation of patients 1 to 3 was similar to that of neonates with INH. However, splenomegaly, with or without hepatomegaly, persisted throughout the first year of life despite resolution of cholestasis and improvement in liver blood test results. Neurologic abnormalities also developed by 12 to 24 months of life and included mild hypotonia, head lag, and delay in gross motor skills such as rolling, siting, and standing. The neurologic findings in the two adolescents were of progressive neurologic dysfunction and included clumsiness and progressive ataxia, dysarthria, and paresis of vertical gaze. Although not a specific sign, paresis of vertical gaze (either up or down) was a very helpful clinical sign, suggesting NPC in these two patients. Both adolescents had splenomegaly and no hepatomegaly at the time of diagnosis.
The biochemical markers of liver injury among the neonates with NPC were similar to those in the neonates with INH. However, the increased γGT concentration, greater than 700 IU/L, observed in two of the NPC infants is uncommon in INH. Therefore, NPC should be considered along with other diseases associated with an increased γGT concentration (e.g., biliary atresia, α-1-antitrypsin deficiency, Alagille syndrome, and MDR3 deficiency). Cholestasis completely resolved in the three neonates by age 6 to 9 months, although splenomegaly persisted. In the two adolescent patients, liver blood test results remained normal or showed slightly increased concentrations for many years.
The use of histology to diagnose NPC in infancy may not be helpful in up to 50% of patients (4). Liver histology during infancy in three patients was most consistent with “giant-cell hepatitis” and with biliary atresia in one other patient. Electron microscopy can demonstrate the presence of the characteristic lamellar inclusions and dense osmiophilic structures in the liver; however, these findings may not be apparent during the early course of this disease (present in only one of three liver biopsies performed at <1 year of age in our patients). Nevertheless, this possibility mandates electron microscopic examination of liver biopsy specimens in infants with INH. Others have used bone marrow biopsy specimens to search for foamy cells and sea-blue histiocytes when evaluating for NPC in early childhood (2,4). However these cells are nonspecific (9), and as was reported earlier, the initial bone marrow biopsy specimen may appear normal and only a few years later suggest the histologic features of NPC (10).
The NPC gene has been mapped to chromosome 18 q11–12 (11,12). Vanier et al.(13) showed that although the 18q11–12 gene is seen in most NPC patients and is associated with the whole spectrum of clinical and cellular phenotypes (NPC1), there are also small subsets of patients with a different gene defect (NPC2). This gene has been identified recently as the lysosomal protein HE1 (14). A recent study showed that the Nova Scotia form of Niemann-Pick disease (previously called Niemann-Pick disease type D) is actually an allelic variant of NPC1 (15). Three of our patients (one neonate and two adolescents) were of Hispanic descent, originating from southern Colorado or northern New Mexico. Millat et al. (8) recently described a T3182C substitution in exon 21 of the NPC1 gene that was common among Hispanic patients whose roots were in the upper Rio Grande Valley of the United States. The significant Hispanic population in Colorado, 12.9% of the state population (Colorado Department of Health), may account for the relatively high frequency of NPC among the neonates with INH in our series (16).
Cultured fibroblasts from patients with NPC, but not from patients with Niemann-Pick disease types A or B, exhibit a defect in esterification of exogenously derived low-density lipoprotein cholesterol (17). Furthermore, unesterified cholesterol is internalized and accumulates within the fibroblast lysosomes, which can be visualized by filipin histochemical staining (17). The filipin stain increases the specificity of this assay because impaired intracellular cholesterol processing also occurs in acid lipase deficiency, in familial hypercholesterolemia, and in I-cell disease (18). However, these disorders can be excluded during the evaluation of neonates with cholestasis based on clinical presentation, absence of dysmorphic features, or blood test results. Cultured skin fibroblasts from patient 2 showed a very low rate of cholesterol esterification at 6 hours (typical of classic NPC) and a normal value at 24 hours. This has been observed among patients with NPC and cross-reacting material mutations. In this type of mutation, the NPC1 protein is produced but is defective. Therefore, at 6 hours, the esterification defect is present. However, some degree of in vitro compensation occurs at 24 hours (unpublished data). The impaired esterification of cholesterol can be demonstrated in chorionic villus cells (19) or cultured amniotic fluid cells (20), offering the possibility of prenatal diagnosis.
Until the development of the cholesterol esterification assay in cultured skin fibroblasts (3), the profile of lipids extracted from liver tissue was the most specific test for diagnosing NPC. We found the lipid profile in frozen liver tissue obtained by percutaneous biopsy to be helpful in establishing the diagnosis of NPC.
There is no specific treatment for NPC. The treatment of neonates with NPC is aimed toward avoiding nutritional and fat-soluble vitamin deficiencies as long as cholestasis is present. Cholesterol-decreasing agents have been used to treat patients with NPC and have decreased both plasma and liver cholesterol concentrations (21); however, neurologic outcome was not assessed as an endpoint in this study. Transplantation of liver (22) or bone marrow (23) were shown ineffective in altering progression of neurologic deterioration.
We conclude that NPC should be considered in all neonates diagnosed with INH in North American Centers. The presence and persistence of splenomegaly with or without hepatomegaly, early soft signs of developmental motor delay, and Hispanic background make this diagnosis even more likely. Although not conclusive in all cases, electron microscopic examination of liver biopsy tissue may also assist in establishing this diagnosis. The availability of DNA mutation analysis can serve as a noninvasive and rapid method for diagnosing NPC in patients whose ancestors can be traced to the upper Rio Grande Valley of the United States. A diminishing number of infants have INH because of the discovery of other diseases that present in a similar fashion.
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