Lysosomal Acid Lipase Deficiency: Report of Five Cases across the Age Spectrum

Lysosomal acid lipase (LAL) deficiency is an autosomal recessive lysosomal storage disorder caused by mutations in the LIPA gene that leads to premature organ damage and mortality. We present retrospective data from medical records of 5 Brazilian patients, showing the broad clinical spectrum of the disease.


Introduction
Lysosomal acid lipase (LAL) de ciency (cholesteryl ester storage disease/Wolman disease; Online Mendelian Inheritance in Man database #278000) is an autosomal recessive lysosomal storage disorder caused by mutations in the LIPA gene that reduce LAL activity [1].
De cient activity of LAL leads to the accumulation of cholesteryl esters and triglycerides in most tissues, demonstrating the enzyme's role in cholesterol and triglyceride metabolism. LAL de ciency leads to a poorly de ned continuum of symptoms, historically divided into Wolman disease (early onset), rst described in 1956 [2], and cholesteryl ester storage disease (late onset) [1,3], rst described in 1963 [4].
LAL de ciency presenting in infants is a neonatal-onset, severe, and lethal form, resulting in severely a ected lysosomes, due to accumulation of cholesteryl esters and triglycerides, predominantly in the liver, spleen, gut, adrenal glands, bone marrow, lymph nodes, and macrophages. A ected infants present at an early age, with vomiting, diarrhea, and massive hepatosplenomegaly. Feeding diculties and malabsorption lead to malnutrition, growth retardation, and cachexia, which, together with the severe liver disease, contribute to the patient's early demise, generally in the rst year of life [5,6].
In other cases, LAL de ciency may present in any period of life; some patients are diagnosed in childhood, while others remain undiagnosed until adulthood [7]. e progressive accumulation of lysosomal cholesteryl esters and triglycerides leads to the characteristic liver disease, elevated serum transaminase levels, and elevated serum low-density lipoprotein cholesterol (LDL-C) concentrations, with normal to low high-density lipoprotein cholesterol (HDL-C) concentrations. Premature demise is due to liver failure and/or accelerated atherosclerotic disease secondary to the chronic hyperlipidemia [1,2,7].
Liver biopsies may not be pathognomonic for LAL de ciency, which may be misdiagnosed as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, or cryptogenic cirrhosis. Despite being an excellent exam for diagnosis and also for evaluating the degree of liver compromise, it is an invasive exam, with potential risk of complications such as hemorrhage, specially in patients with advanced liver disease [8]. Another important tool to help elucidate the diagnosis is the endoscopy, which can present speci c ndings such as disseminated lipidic accumulation or other nonspeci c signs, such as aesophagus varices [9]. Diagnostic suspicion can also be con rmed by demonstrating the de cient LAL activity via biochemical tests (i.e., dried blood spot, leukocytes, andbroblasts) and determination of mutations on the LIPA gene by performing gene sequencing, which can help identify patients with this autosomal recessive disease without potential complications [10]. To date, the genotype-phenotype correlation has not been established.
In 2013, Bernstein et al. [11] reviewed 135 cases of LAL de ciency in the literature, showing the clinical variability of the disease. Despite the variability, the majority of cases were recognized in infancy, early childhood, or adolescence (83% of cases presented with the rst clinical manifestation prior to 12 years of age), showing the important role of the pediatrician in early detection of clinical symptoms.
In this study, we report the clinical, biochemical, and histological aspects observed across the age spectrum: 1 case in infancy and 4 pediatric cases in siblings.

Patients.
is is a retrospective study reviewing data from medical records of patients from a Brazilian Reference Center for Inborn Errors of Metabolism, Universidade Federal de São Paulo (Institutional Review Board/Ethics Committee approval number CEP-UNIFESP 2007/11).

Biochemical
Diagnosis. LAL enzymatic activity assay was performed in dried blood spot (DBS) samples according to the uorimetric technique described by Hamilton et al. [12] and performed at HRG Laboratory, HIBM Research Group (Chatsworth, CA, USA), which used the reference range of 0.37-2.30 nmol/punch/h. Biochemical analysis was also performed in our laboratory-Laboratório de Erros Inatos do Metabolismo (LEIM)-UNIFESP, which used the reference range of >0.024 nmol/punch/h.

Pathology.
All patients underwent ultrasound-guided liver biopsy in order to evaluate liver damage. PAS-D stain (periodic acid-Schi + diastasis) was performed to di erentiate lipid from glycogen deposit, and Masson trichrome was performed to evaluate presence of cirrhosis.

Case 1.
e patient is a female, index case of this family, second of ve siblings from the same mother, who has a son (una ected) and a daughter (this patient) from her rst marriage, and three other sons (all a ected-cases 2, 3, and 4), from her second marriage. Both marriages were nonconsanguineous unions, and both fathers are not related. e mother of cases 1, 2, 3, and 4 also has reduced activity of LAL enzyme, but her case was not presented in this report.
Onset of symptoms occurred at 3 years of age; she was referred to a gastroenterologist for hepatomegaly, intermittent diarrhea, and failure to thrive. She underwent liver biopsy at 3 years of age, showing granulomatous hepatitis with generalized hepatocellular stu ng. Grade 3 macro-and microvesicular di use steatosis was present in 60% of the hepatocytes. e patient was rst evaluated in our service at the age of 4 years; she weighed 16 kg (z score of −0.56), her height was 93 cm (z score of −3.0), and she had hepatosplenomegaly (rounded liver with rock-hard consistency, 5 cm below the right costal margin; rock-hard consistency spleen, 5 cm below the left costal margin). Her second hepatic biopsy showed enlargement of portal spaces with enlarged microvacuolized macrophages and hepatocytes. She developed progressive increase of hepatosplenomegaly-by age 9, physical examination revealed that her liver was 20 cm below the right costal margin and her spleen was 17 cm below the left costal margin-and worsening of diarrheic episodes. Biochemical analysis showed dyslipidemia and mild elevation of liver transaminases (Table 1). Between 8 and 9 years of age, she underwent 7 hospitalizations due to respiratory distress secondary to severe hepatosplenomegaly. e patient died at age 9, due to pneumonia and septic shock; at that time, the enzymatic assay was not available in our country, and thus her diagnosis was con rmed by enzymatic assay in DBS samples postmortem showing LAL activity to be 0.0068 nmol/punch/h (reference range, 0.37-2.30 nmol/punch/h). e autopsy showed disseminated lipid and cholesterol crystal accumulation, mainly in the liver, spleen, bone marrow, and gastrointestinal tract, and severe steatosis with lipid-lled histiocytes ( Figure 1). No adrenal calci cation was noted.

Case 2.
e patient is a male, half-brother of the patient in case 1, currently 11 years old, onset of symptoms at age 3, presenting with hepatosplenomegaly. In his rst evaluation at our center at 4 years of age, clinical examination showed enlarged abdominal circumference, round liver 5 cm below the right costal margin, spleen 3 cm below the left costal margin, and growth de cit, weighing 15 kg (z score of −1) and measuring 96 cm (z score of −2.4) with a body mass index (BMI) of 16.5 (z score of 0.92). Laboratory assessment showed elevation in total cholesterol, with a low HDL-C level and high LDL-C level. His triglyceride level was elevated, with slight increase in liver enzymes (Table 1). Hepatic biopsy presented hepatocyte dilatation due to lipid accumulation, lipid-lled histiocytes, and broadening portal spaces suggesting lipid storage disease (Figure 1). Enzymatic assay in DBS samples showed LAL activity to be 0.0087 nmol/punch/h (reference range, 0.37-2.30 nmol/ punch/h), con rming the diagnosis.

Case 3.
e patient is a male, younger half-brother of the patient in case 1, currently 10 years old, diagnosed at the age of 6 years in the familial screening. In his rst evaluation, at 6 years old, no hepatosplenomegaly and no growth delay were noticed, with the patient weighing 20 kg (z score of −0.46) and measuring 114 cm (z score of −0.77), with a BMI of 15.4 kg/m 2 (z score of 0.03). Laboratory assessment showed mild elevation of total cholesterol and the LDL-C fraction, and a low HDL-C fraction. Triglycerides were also mildly elevated (Table 1), but transaminase levels were normal. Abdominal sonogram showed discrete homogeneous hepatomegaly for the patient's age. Enzymatic assay of DBS samples showed LAL activity to be 0.00 nmol/punch/h (reference range, >0.024 nmol/punch/h), con rming the diagnosis.

Case 4.
e patient is a male, younger half-brother of the patient in case 1 currently 8 years old, who was diagnosed at the age of 3 years in the familial screening. In his rst assessment in our service, at the age of 2 years and 11 months, the patient presented with sporadic episodes of  diarrhea. No hepatomegaly and no growth delay were noticed; the patient weighed 16 kg (z score of 0.9) and measured 95.5 cm (z score of −0.35), with a BMI of 17.8 kg/m 2 (z score of 1.63). Laboratory assessment showed that his total cholesterol level was within the normal range, but he had mildly low HDL-C and mildly high LDL-C levels.
Triglycerides were also elevated ( Table 1). Abdominal sonogram showed that the liver and spleen were within the upper limit of normal for the patient's age. Enzymatic assay of DBS samples showed LAL activity to be 0.00 nmol/ punch/h (reference range, >0.024 nmol/punch/h), con rming the diagnosis.

Case 5.
e patient is a male, not related to the previous patients, with neonatal cholestasis until 20 days of life. He was rst evaluated at our center in 2012 at 4 months of age with a perinatal history of cholestasis and hypoglycemia; severe jaundice (Kramer scale score of 5); severe hepatosplenomegaly (liver and spleen 5 cm below the costal margin); failure to thrive, weighing 5150 g (z score of −2.51) and measuring 60.5 cm (z score of −1.43), with a BMI of 14.1 (z score of −2.35); blood dyscrasia; impaired liver function, with severe hepatic and canalicular enzyme level elevations; and very high total cholesterol, low HDL-C, and high LDL-C levels. Triglycerides were also elevated (Table 1). e patient presented with severe pancytopenia and hemophagocytic lymphohistiocytosis syndrome. By the age of 8 months, the patient underwent liver transplantation due to hepatic failure and died soon after the procedure due to acute organ rejection. Liver biopsy showed severe macro-and microvesicular steatosis with associated severe brosis and portal ductular reaction (Figure 1). e DBS samples showed LAL activity to be 0.0324 nmol/punch/h (reference range, 0.37-2.30 nmol/punch/h). LIPA gene sequencing presented two heterozygous mutations: an exon 5 deletion (c.477delT) and an exon 7 insertion (c.822+37_38insC).

Discussion
Clinically, LAL de ciency, like other lysosomal storage disorders, can present across the age spectrum. It is often unrecognized in infants as well as in children and adults. In children and adults, once a physician notes the hepatomegaly, with or without splenomegaly and/or elevated transaminase activity, suspicion for LAL de ciency increases, but this rst requires an awareness of the disease [4]. Typically, a liver biopsy will reveal foamy, lipid-lled hepatocytes and macrophages, microvesicular steatosis, and lysosomal cholesteryl ester crystals, best observed by ultrastructural examination [13]. e hepatosplenomegaly in cases 1 and 2 was noticed by 3 years of age; this nding can be an alert to pediatricians and hepatologists, once frequent causes of hepatomegaly are discarded, with or without splenomegaly, as reported by Bernstein et al. [11] in 2013, after analyzing 135 cases of LAL de ciency found that 99,3% of the patients presented hepatomegaly and 74% presented splenomegaly, being those the most frequent rst signs of the disease. In contrast, the patient in case 3 did not have evidence of hepatomegaly at age 6, demonstrating the variety of the clinical spectrum, even between siblings, who are supposed to carry the same mutation. On the other hand, the patient in case 5 presented at an early age with severe hepatosplenomegaly and early-onset hepatic failure. When compared to the other cases, the patient in case 5 presented a much more severe and much more early-onset symptoms, showing the clinical variability of this disease.
Regarding the dyslipidemia, four out of ve patients showed abnormal levels of total cholesterol, and all patients showed abnormal cholesterol fractions and triglycerides, being higher on the patient with an early-onset "Wolman" phenotype. In children and adults with LAL de ciency, laboratory parameters of cholesterol are not always severely abnormal, as reported by Bernstein et al. in 2013, when reviewing 135 cases of patients with LAL de ciency, where 79% of the patients presented total cholesterol > 200 and 71% had HDL-C between 20 and 40 [11]. As previously reported, patients with higher levels of total cholesterol and LDL-C do not necessarily present with more severe lipid deposits and impaired liver function than patients with lower levels [7].
Regarding liver enzymes, we can observe that patients 1 to 4 had moderately increased aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and alkaline phosphatase levels, and patient 5 showed much more severe compromise of liver function, with much higher enzyme levels. e liver biopsies of the patients in cases 1 and 2 show us disseminated lipid accumulation and severe steatosis with lipid-lled histiocytes, marking the important lipid deposits in hepatocytes, leading to cellular damage and subsequent organic failure. In case 5, we can see more important tissue damage, with severe macro-and microvesicular steatosis and brosis presenting at an early stage.
at follows the pattern showed in the literature so far [14,15] and, despite the initial presenting symptoms or signs, the presence of dyslipidemia, hypertriglyceridemia, and increased liver enzyme levels must lead to the suspicion of LAL de ciency.
ese cases in addition to the published literature [1,[16][17][18] show the broad clinical spectrum of this disease and highlight the importance of health care professionals having LAL de ciency in mind as a potential diagnosis when evaluating a patient with hepatomegaly with or without splenomegaly, elevated liver enzymes, dyslipidemia, and/or growth abnormalities, because the early identi cation of patients is an important prerequisite for timely treatment with enzyme replacement therapy [19].

Conclusion
LAL de ciency should be included in the di erential diagnosis for all patients with elevated LDL-C who also may have mildly to moderately decreased HDL-C levels, elevated transaminases, and hepatomegaly. Enzyme replacement therapy with sebelipase alfa is an option in some areas of the world.
us, the early detection of patients is advised, because the earlier the patient starts the treatment, the better the outcome will probably be.
Disclosure e content is solely the responsibility of the authors. Alexion Pharmaceuticals, Inc., had no role in the preparation or approval of the manuscript.

Conflicts of Interest
Dr. Marco Antonio Curiati receives sponsorship for international conferences. Dr. Ana Maria Martins receives sponsorship for international conferences and grants for lectures.