The frequency of lysosomal acid lipase deficiency in children with unexplained liver disease

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The Frequency of Lysosomal Acid Lipase Deficiency

in Children With Unexplained Liver Disease



Zarife Kuloglu,

Aydan Kansu,

Suna Selbuz,

Ayhan G. Kalaycı,

Gu¨lseren S¸ahin,

§

Ceyda Tuna Kirsaclioglu,

Kaan Demiro¨ren,

Buket Dalgıc¸,

Erhun Kasırga,



_{Zerrin O¨nal,}

_{Ali I˙s¸lek, and National LAL-D Study Group}

ABSTRACT

Objectives: Evidence suggests that lysosomal acid lipase deficiency (LAL-D) is often underdiagnosed because symptoms may be nonspecific. We aimed to investigate the prevalence of LAL-D in children with unexplained liver disease and to identify demographic and clinical features with a prospective, multicenter, cross-sectional study.

Methods: Patients (aged 3 months–18 years) who had unexplained transaminase elevation, unexplained hepatomegaly or hepatosplenomegaly, obesity-unrelated liver steatosis, biopsy-proven cryptogenic fibrosis and cirrhosis, or liver transplantation for cryptogenic cirrhosis were enrolled. A Web-based electronic data collection system was used. LAL activity (nmol/ punch/h) was measured using the dried blood spot method and classified as LAL-D (<0.02), intermediate (0.02–0.37) or normal (> 0.37). A second dried blood spot sample was obtained from patients with intermediate LAL activity for confirmation of the result.

Results: A total of 810 children (median age 5.6 years) from 795 families were enrolled. The reasons for enrollment were unexplained transaminase elevation (62%), unexplained organomegaly (45%), obesity-unrelated liver steatosis (26%), cryptogenic fibrosis and cirrhosis (6%), and liver transplantation for cryptogenic cirrhosis (<1%). LAL activity was normal in 634 (78%) and intermediate in 174 (21%) patients. LAL-D was identified in 2 siblings aged 15 and 6 years born to unrelated parents. Dyslipidemia, liver steatosis, and mild increase in aminotransferases were common features in these patients. Moreover, the 15-year-old patient showed growth failure and microvesicular steatosis, portal inflammation, and bridging fibrosis in the liver biopsy. Based on 795 families, 2 siblings in the same family were identified as LAL-D cases, making the prevalence of LAL-D in this study population, 0.1% (0.125%– 0.606%). In the repeated measurement (76/174), LAL activity remained at the intermediate level in 38 patients.

Conclusions: Overall, the frequency of LAL-D patients in this study (0.1%) suggests that LAL-D seems to be rare even in the selected high-risk population.

Key Words:children, liver, lysosomal acid lipase deficiency

(JPGN 2019;68: 371–376)

What Is Known

 _{The precise incidence of lysosomal acid lipase}

defi-ciency is not known at this time.

 _{Lysosomal acid lipase deficiency can present with}

nonspecific manifestations and may be often under-diagnosed.

 _{The prevalence of lysosomal acid lipase deficiency has}

not been investigated in children with unexplained liver disease in a large study group.

What Is New

 _{The prevalence of lysosomal acid lipase deficiency in}

the selected high-risk population of unexplained liver disease is 0.1%.

L

inher-ited disorder that leads to progressive accumulation of cho-lesteryl esters and triglyceride (TG) in various organs. Clinical findings and age of onset depend on residual enzyme activity: a complete absence of LAL activity causes infantile-onset LAL-D, whereas in the childhood/adult-onset form, there is residual enzyme activity. Accordingly, this form of LAL-D has a slower course than the infantile-onset form (1,2).

Patients with childhood/adult-onset LAL-D typically present with hepatomegaly, liver dysfunction, liver steatosis, cirrhosis, or dyslipidemia (2–5). Liver steatosis often progresses to liver fibrosis and cirrhosis (2,4–8). Most probably, patients with LAL-D are often misdiagnosed in clinical practice, because all manifestations are nonspecific. Therefore, the possibility of LAL-D should be kept in mind in patients with liver disease of unknown origin (9). To the

Received April 25, 2018; accepted November 22, 2018.

From the Department of Pediatric Gastroenterology, Hepatology and Nutrition, Ankara University School of Medicine, Ankara, yDepartment of Pediatric Gastroenterology, Hepatology and Nutrition, Ondokuz Mayıs University, School of Medicine, Samsun, zDepartment of Pediatric Gastroenterology, Hepatology and Nutrition, Dr. Sami Ulus Children’s Hospital, Ankara, §Department of Pediatric Gastroenterology, Hepatol-ogy and Nutrition, Turkish Republic Health Ministry, Ankara Child Health Diseases, Haematology Oncology Training and Research Hospi-tal, Ankara, jjDepartment of of Pediatric Gastroenterology, Hepatology and Nutrition, Yuzuncu Yıl University, Faculty of Medicine, Van,

Nutrition, Gazi University University, Faculty of Medicine, Ankara, #Department of Pediatric Gastroenterology, Hepatology and Nutrition, Celal Bayar University Faculty of Medicine, Manisa,Department of Pediatric Gastroenterology, Hepatology and Nutrition, University of Medical Sciences, Bakırkoy Dr Sadi Konuk Research and Training Center, I˙stanbul, yyDepartment of Pediatric Gastroenterology, Hepatol-ogy and Nutrition, Atatu¨rk University, Faculty of Medicine, Erzurum, Turkey.

Address correspondence and reprint requests to Zarife Kuloglu, MD, Ankara U¨ niversitesi, Tıp Faku¨ltesi, C¸ocuk Hastanesi, 06643 Cebeci, Mamak/

best of our knowledge, apart from a study from Poland (10) that reported that none of the 89 children with elevated transaminases of unknown etiology had childhood/adult-onset LAL-D, the preva-lence of LAL-D has not been investigated in patients with a broad spectrum of hepatic manifestations including unexplained transam-inase elevation, organomegaly, liver steatosis, and chronic liver disease. We aimed to investigate the frequency of childhood/adult-onset LAL-D in children with unexplained liver disease, and to describe clinical characteristics.

METHODS

Objectives and Study Design

In this multicenter, prospective, and cross-sectional trial (Clin-icalTrials.gov Identifier NCT02372513) patients from 41 Pediatric Gastroenterology, Hepatology and Nutrition centers in Turkey were enrolled from January 10, 2015 to January 31, 2017. The Ethics Committee of Ankara University approved the study protocol. Writ-ten informed consent was obtained from all participants.

Subjects

Patients aged 3 months to 18 years of age at the time of enrollment who had unexplained transaminase elevation (serum alanine aminotransferase [ALT] levels >1.5 times the upper limit of normal) for more than 3 months, and/or unexplained hepatomegaly or hepatosplenomegaly, and/or obesity-unrelated liver steatosis, and/or biopsy-proven cryptogenic fibrosis and cirrhosis, and/or liver transplantation for cryptogenic cirrhosis were included. Major causes of liver disorders including viral hepatitis, alpha-1 antitryp-sin deficiency, Wilson disease, and autoimmune hepatitis were excluded in all patients. Patients with body mass index for-age and sex-specific percentile of >85%, drug-induced liver steatosis, transaminase elevation, or organomegaly due to infectious causes,

connective tissue disorders, hemato-oncological diseases, cardiac and vascular diseases or definitive chronic liver disease, and metabolic disorders were excluded.

Data Collection

A Web-based electronic data collection method was used for data collection (www.lalulusal.org). A file storage for all documents was provided by the Web site. Each patient’s case report form was reviewed comprehensively by the study coordinator (Z.K.). Pro-spective and retroPro-spective data were collected. Information on demographic characteristics, physical examination, laboratory find-ings, and LAL enzyme activity were recorded. In all enrolled patients, the complete blood count, serum aspartate aminotransfer-ase (AST), ALT, g-glutamyl transpeptidaminotransfer-ase, alkaline phosphataminotransfer-ase, total bilirubin, indirect bilirubin, albumin, lipid profile, prothrombin time, international normalized ratio, abdominal ultrasonography, and LAL enzyme activity results were obligatory. The abdominal ultrasonography was performed within 6 months before the start of the study. Liver histopathology findings were also recorded in terms of portal inflammation, cholestasis, macrovesicular steatosis, microvesicular steatosis, bridging fibrosis, cirrhosis, and presence of foam cells and macroscopic yellow-orange discoloration.

Anthropometry

Height (cm) and weight (kg) were measured by standardized techniques at each center. Growth and nutritional status were evaluated by WHO standardized growth references for children younger than 2 years and Center for Disease Control and Preven-tion/National Center for Health Statistics references for those older than 2 years. Z scores of weight-for-age (WAZ), height-for-age, and body mass index-for-age (BMIZ) were calculated by software programs (11,12). Height for age z score <2 was defined as

National LAL-D Study Group: The members of the Study Group are as follows: Esra Eren (Ondokuz Mayıs University, School of Medicine, Samsun), Ferda O¨ zbay Hos¸nut (Dr Sami Ulus Children’s Hospital, Ankara), Nafiye Urgancı (S¸is¸li Etfal Training and Research Hospital, I˙stanbul), Aytac¸ Yaman (Turkish Republic Health Ministry, Ankara Child Health Diseases, Haematology Oncology Training and Research Hospital, Ankara), Tanju O¨ zkan (Uludag˘ University Faculty of Medicine, Bursa), Neslihan Eks¸i Bozbulut (Gazi University, Faculty of Medicine, Ankara), Gu¨zide Dog˘an (Celal Bayar University Faculty of Medicine, Manisa), O¨ zlem Durmaz Ug˘urcan (I˙stanbul University, I˙stanbul Faculty of Medicine, I˙stanbul), Ays¸e Merve Usta (S¸is¸li Etfal Training and Research Hospital, I˙stanbul), Duran Arslan (Erciyes University, Faculty of Medicine Kayseri), Mustafa Akc¸am (Su¨leyman Demirel University, School of Medicine, Isparta), I˙shak Abdurrahman Is¸ık (Antalya Training and Research Hospital, Antalya), C¸ ig˘dem O¨ mu¨r Ecevit (Dr Behc¸et Uz Children Disease and Surgery Training and Research Hospital, I˙zmir), Yusuf Usta (Mersin University, Faculty of Medicine, Mersin), Taner O¨ zgu¨r (Uludag˘ University Faculty of Medicine, Bursa), Figen O¨ zc¸ay (Bas¸kent University, Faculty of Medicine, Ankara), Necati Balamtekin (Gu¨lhane Training and Research Hospital, Ankara), Yes¸im O¨ ztu¨rk (Dokuz Eylu¨l University, Faculty of Medicine, I˙zmir), Serdar Cantez (I˙stanbul University, I˙stanbul Faculty of Medicine, I˙stanbul), Fulya Gu¨lerman (Kırıkkale University, Faculty of Medicine, Kırıkkale), Gonca Handan U¨ stu¨ndag˘ (Bu¨lent Ecevit Uni-versity, Faculty of Medicine, Zonguldak), Halil Haldun Emirog˘lu (Selc¸uk University, Faculty of Medicine, Konya), Neslihan Karacabey (Erciyes University, Faculty of Medicine Kayseri), Atakan Comba (Hitit University, Faculty of Medicine, C¸ orum), Gu¨lin Erdemir (Dr.Behc¸et Uz Children Disease and Surgery Training and Research Hospital, I˙zmir), Ays¸en Uncuog˘lu Aydog˘an (Kocaeli University, School of Medicine, Kocaeli), Selim Go¨kc¸e (Bezmialem University, Faculty of Medicine, I˙stanbul), Pınar Kuyum (Dokuz Eylu¨l University, Faculty of Medicine,

I˙zmir), Meltem Gu¨ls¸an (Bas¸kent University, Faculty of Medicine, Ankara), Mahya Sultan Tosun (Denizli State Hospital, Denizli), Yavuz Tokgo¨z (Adnan Menderes University, Faculty of Medicine, Aydın), Burcu Gu¨ven (Kırıkkale University, Faculty of Medicine, Kırıkkale), Hasan Yu¨ksekkaya (Necmenttin Erbakan University, Faculty of Medicine, Ankara), Go¨khan Tu¨mgo¨r (C¸ ukurova University, Faculty of Medicine, Ankara), Makbule Eren (Eskis¸ehir Osmangazi University, Faculty of Medicine, Esikis¸ehir), Mas¸allah Baran (I˙zmir Tepecik Training and Research Hospital, I˙zmir), Meltem Gu¨mu¨s¸ (Konya Eg˘itim Konya Training and Research Hospital, Konya), Og˘uz Canan (Adana Bas¸kent University; Adana), Halil Kocamaz (Pamukkale University, Faculty of Medicine, Denizli), Nelgin Gerenli (U¨ mraniye Training and Research Hospital, I˙stanbul), Murat C¸ akır (Karadeniz Technical University, Faculty of Medicine, Trabzon), Mehmet Ag˘ın (C¸ ukurova University, Faculty of Medicine, Ankara), S¸amil Hızlı (Kec¸io¨ren Training and Research Hospital, Ankara), Yas¸ar Dog˘an (Fırat University, Faculty of Medicine, Elazıg˘), Cos¸kun C¸ eltik(S¸ifa University, I˙zmir), Ug˘ur Deveci (Fırat University, Faculty of Medicine Elazıg˘), Oya Balcı Sezer (Kec¸io¨ren Training and Research Hospital Ankara).

Clinical registration number: NCT02372513 https://clinicaltrials.gov/ct2/ show/NCT02372513.

This investigator sponsored research was funded by Alexion Pharmaceu-ticals Inc.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jpgn.org). Z.K. and A.K. are consultants for Alexion Pharmaceuticals Inc. The

remaining authors report no conflicts of interest.

Copyright # _{2019 by European Society for Pediatric Gastroenterology,}

Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition

stunting, WAZ <2 from the mean as undernutrition and BMIZ <2 from mean as wasting.

Laboratory Assessments

Liver tests and lipid profile were obtained and measured by standard laboratory methods at the local laboratory in each center. Liver enzymes were evaluated according to the normal reference range of each center and expressed as times the upper limit of normal. Definitions of laboratory abnormalities are shown in Supplementary Table S1 (Supplemental Digital Content, http://links.lww.com/MPG/ B530). Classification of total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein cholesterol (C) and TG levels were evaluated as shown in Supplementary Table S2 (Supplemental Digital Content, http://links.lww.com/MPG/B530) (13).

Measurement of Lysosomal Acid Lipase Activity

Peripheral blood samples for LAL activity were obtained from all participants. The dried blood spot (DBS) sample was prepared as described previously (14) and, it was sent to the reference laboratory (NHS Greater Glasgow and Clyde Biochemistry Department, Glas-gow, UK) for LAL activity measurement within 1 week.

LAL activity was measured as described previously Hamil-ton et al (14). The LAL enzyme level <0.02 nmol/punch/h was classified as LAL-D, whereas LAL enzyme level between 0.02 and 0.37 nmol/punch/h was considered as an intermediate activity and LAL enzyme level >0.37 nmol/punch/h was classified as normal. A second DBS sample was obtained from the patients with interme-diate activity for confirmation of the result.

Statistical Analysis

For statistical analysis, SPSS software (version 22, SPSS Inc, Chicago, IL) was used. Continuous variables were presented as

mean  standard deviation or median, and categorical variables

were presented as frequency (percentage). The Chi-square test for categorical variables and the Mann-Whitney test for continuous variables were used for comparisons between groups. LAL-D prevalence in the enrolled population and its corresponding 95% confidence interval (CI) was calculated. A P value <0.05 was considered statistically significant.

RESULTS

Demographic Characteristics of Study

Population

In total, 810 patients (487 boys; the median age 5.6 years [range 3 months–18 years]) were enrolled from 795 families. The median time interval between symptom onset and enrollment into the study was 7 months (range 3–210 months). The age distribution and causes of enrollment are shown in Table 1. Parental consan-guinity was noted in 27% of participants. The clinical and labora-tory findings of the study group are presented in Supplementary Table S3, S4, and S5 (Supplemental Digital Content, http:// links.lww.com/MPG/B530).

The Frequency of Lysosomal Acid Lipase

Deficiency

Based on 795 families, 2 siblings in the same family were identified as LAL-D cases, making the prevalence of LAL-D in this study population, 0.1% (95% CI: 0.125–0.606).

Features of Patients With Lysosomal Acid

Lipase Deficiency

Table 2 summarizes the features of the 2 identified LAL-D children (15-year-old boy and 6-year-old girl) born from unrelated parents. Hyperlipidemia was noted in some relatives of their parents. The duration between initial disease and diagnosis was 10 years for the boy and 3 months for the girl. When the boy was evaluated in a local hospital for short stature at 5 years of age, mild increase in liver enzymes was noticed. He underwent the liver biopsy after exclusion of several pathologies, showing microvesi-cular steatosis, portal inflammation, and bridging fibrosis. At 15 years of age, the patient was referred to a university hospital

TABLE 1. Age distribution and the causes of enrollment of study population n (%) Age groups Infants 254 (31) Preschool children 129 (16) School children 218 (27) Adolescents 209 (26)

The causes of enrollment

Unexplained transaminase elevation 466 (58) Unexplained organomegaly 369 (46) Obesity-unrelated liver steatosis 213 (26) Cryptogenic fibrosis and cirrhosis 53 (7) Liver transplantation for cryptogenic cirrhosis 7 (<1)

TABLE 2. The features of patients with lysosomal acid lipase deficiency Case 1 Case 2

Age, y 15 6

Sex Male Female

The cause of study enrollment Liver steatosis

Liver steatosisþ elevated liver enzyme

Growth failure þ 

Physical examination Normal Normal Liver biochemical findings

AST (ULN) 0.8 2.4

ALT (ULN) 1.2 4

GGT, ALP, total and direct bilirubin, albumin and INR levels Normal Normal Lipid profile, mg/dL Total cholesterol 247 240 LDL cholesterol 157 158 HDL cholesterol 54 44 Triglyceride 66 110 Abdominal USG Liver steatosis þ þ

Liver biopsy Not performed

Microvesicular steatosis þ Portal inflammation þ Bridging fibrosis þ

ALP¼ alkaline phosphatase; ALT¼ alanine aminotransferase; AST¼ aspartate aminotransferase; HDL ¼ high-density lipoprotein choles-terol; INR¼ international normalized ratio; LDL ¼ low-density lipoprotein; ULN¼ upper limit of normal; GGT g-glutamyl transpeptidase; USG¼ ultrasonography.

for further evaluation. His physical examination was normal. His

height-for-age, WAZ, and BMIZ were3, 4, and 3,

respec-tively. At this time, a slightly high transaminase level (AST 42 U/L; ALT 49 U/L), high total and LDL cholesterol levels, and liver steatosis on abdominal ultrasound were detected. After reassess-ment, he was enrolled in the LAL-D study. LAL-D was diagnosed based on reduced LAL enzyme activity in DBS.

His sister aged 6 years was also referred to the same center with a 3-month history of elevated liver enzymes and liver steatosis for which she did not have any specific diagnosis. Her physical examination was normal. She had high transaminase (AST 120 U/L; ALT 158 U/L), total and LDL cholesterol levels, borderline low high-density lipoprotein-C, and mildly high TGs. She was included to the LAL-D study with her brother simultaneously. The diagnosis of LAL-D was made by markedly low LAL enzyme activity.

Lysosomal Acid Lipase Enzyme Activity

The mean value of LAL activity was 0.63 0.33 nmol/ punch/h. Of the 810 patients, 634 (78%) had normal LAL activity, 2 (0.3%) had LAL-D, and 174 (21%) had intermediate LAL activity.

The second measurement of LAL activity was available in 76 of 174 patients (44%) with intermediate LAL activity. Significantly increased LAL enzyme activity was observed in the second

mea-surement compared with the first meamea-surement (0.38 0.18 vs

0.25 0.08 nmol/punch/h, respectively, P ¼ 0.001). Compared with the first measurement, LAL enzyme activity decreased in 14 (18%), no change was noted in 12 (16%) and an increase was observed in 50 (66%) of these patients at the second measurement. Finally, the LAL activity levels of 38 patients (50%) were found to be within normal limits, whereas intermediate activity was noted again in 38 patients (50%) in the second measurement. At the final

assessment, 136 patients had intermediate LAL enzyme activity.

Mean LAL activity in these patients was 0.26 0.09 nmol/punch/h

(range; 0.04–0.37 nmol/punch/h). LAL enzyme activity in 119 patients (87%) was in the range 0.15 to 0.4 nmol/punch/h, whereas 17 patients (13%) showed LAL enzyme activity under 0.15 nmol/ punch/h. Of 136 children with intermediate level LAL activity, 26

(19%) had low platelet count (<100 000/mm3), which is accepted as

indirect markers for cirrhosis.

The comparison of demographic and laboratory character-istics of the patients with normal and intermediate LAL activity is shown in Table 3. The 2 patients with LAL-D were excluded from the statistical analysis because of the small sample size. Intermedi-ate LAL activity was significantly more frequently noted in children older than 12 years, and in children with splenomegaly and throm-bocytopenia compared with those without. Although the frequency of intermediate LAL activity was more common in patients with fibrosis (41%) or cirrhosis (47%) compared with those without (25.8% and 30.8%, respectively), it did not reach statistical

signifi-cance (P¼ 0.54 and P ¼ 0.62). Of note, the frequency of fibrosis,

but not cirrhosis was significantly greater in the patients with thrombocytopenia (77%) compared to those without (54%)

(P¼ 0.009). On the contrary, the rate of intermediate LAL activity

was significantly lower in patients with abnormal AST and ALT concentrations and liver steatosis compared with those with normal values.

DISCUSSION

To the best of our knowledge, for the first time, LAL activity in a large population of children with unexplained liver disease was evaluated. The prevalence of LAL-D in this selected population was rare with 0.1% (95% CI: 0.125–0.606). The common features of patients with LAL-D were mildly elevated aminotransferase levels, liver steatosis, and dyslipidemia.

TABLE 3. Demographic and laboratory characteristics of participants according to different levels of lysosomal acid lipase activity LAL activity

Parameters Normal n (%) Intermediate n (%) P

Age <12 y 494 (83) 106 (17) <0.001 >12 y 140 (67) 68 (33) Sex Female 249 (77) 73 (23) n.s. Male 385 (79) 101 (21) Hepatomegaly No 138 (76) 43 (24) n.s Yes 280 (76) 89 (24) Splenomegaly No 266 (84) 49 (16) <0.001 Yes 152 (65) 83 (35) Platelet count >150,000/mm3 594 (83) 120 (17) <0.001 <150,000/mm3 40 (43) 54 (57)

ALT level Normal 190 (71) 78 (29) <0.001

Abnormal 444 (82) 96 (18)

AST level Normal 185 (73) 68 (27) 0.013

Abnormal 449 (81) 106 (19)

Total cholesterol level Normal 484 (79) 132 (21) n.s.

Abnormal 150 (78) 42 (22) Liver steatosis No 456 (77) 139 (23) 0.037 Yes 177 (84) 35 (16) Cirrhosis No 81 (69) 36 (31) n.s. Yes 20 (53) 18 (47) Fibrosis No 46 (74) 16 (26) n.s. Yes 55 (59) 38 (41)

The diagnosis of childhood/adult-onset LAL-D is commonly challenging because the clinical and histopathological findings overlap widely with other liver diseases. Previous epidemiologic studies suggested that the disease is likely to be underdiagnosed (15–17). Therefore, we focused mainly on the patients with unex-plained transaminase elevation, unexunex-plained organomegaly, obe-sity-unrelated liver steatosis, biopsy-proven cryptogenic liver fibrosis and cirrhosis, and liver transplantation for cryptogenic cirrhosis.

The precise incidence is not known at this time. Estimated prevalence in children and adults ranges from 1 in 40,000 to 1 in 300,000 (18,19). The prevalence may, however, vary depending on ethnicity and geographical region (18). In Turkey, the incidence of LAL-D is not known. Our study showed the presence of 2 siblings with childhood/adult-onset LAL-D in 810 patients from 795 fami-lies with unexplained liver disease, corresponding to a frequency of 0.1%. Recently, Wierzbicka-Rucin´ska et al (10) published their experience about screening for LAL-D in Polish children. Although detailed information is lacking, they had screened children with elevated transaminase of unknown etiology and infants with multi-organ disease. They reported that except for one infantile onset LAL-D, childhood/adult-onset LAL-D was not identified in 89 children older than 1 year (10). Our study with a large number of patients, covering all aspects of liver involvement of LAL-D and focusing on the only childhood/adult-onset form is different from this report. Indeed, our results showed that LAL-D is a rare disease even in this selected population cohort of high-risk children; however, screening of ‘‘at risk’’ patients may reveal previously unrecognized cases of LAL-D.

Among the 2 LAL-D siblings in our study, the 15-year-old boy had growth retardation with slight increase in liver enzymes, whereas his sister ages 6 years was healthy in appearance with mild liver enzyme level elevation. Hepatosplenomegaly which is one of the typical findings of LAL-D was not noticed in 2 patients. Both siblings also shared the typical findings of the disease, such as liver steatosis and dyslipidemia. These findings are consistent with previous reports (2–4,20). Although the number of our LAL-D cases was small, we would like to emphasize again that LAL-D patients may have near normal liver enzymes and liver steatosis as in the older patient in our study group (7,8).

In the measurement of LAL activity, the sample quality is important for accurate results. It is recommended that intermediate results should be confirmed by a good quality sample (21). A second measurement was available in 76 of 174 patients (44%) who had intermediate LAL enzyme activity at the first measurement. In 38 of these 76 patients, the LAL enzyme activity remained at the inter-mediate level. Finally, interinter-mediate LAL enzyme activity was observed in 136 patients in this study. Intermediate LAL enzyme activity in the range 0.15 to 0.40 nmol/punch/h has been described in heterozygous carriers of LIPA gene mutations (14), but it is not a reliable method for identifying carriers (21). Some carriers may have normal activity (21) or LAL activity may be found below the reference range a consequence of severe liver disease or nonalco-holic fatty liver disease (22–27). Moreover, some patients with homozygous carriers of LIPA gene mutations are known to show an intermediate LAL activity under 0.15 nmol/punch/h (14). Indeed, our 119 patients (87%) showed an LAL activity in the range of heterozygous carriers, whereas 17 patients (13%) presented an LAL activity in the range, which has been described in homozygous carriers of LIPA gene mutations. It is, however, not clear whether these patients are affected, carriers, or not. We think that genetic analysis is required to identify whether these patients had affected, heterozygous carriers, or not.

The relationship between DBS-determined LAL activity and transaminase levels, white blood cell and platelet count, cirrhosis

(22–24), and nonalcoholic fatty liver disease (25–27) has been reported recently. As in previous studies, intermediate LAL activity was frequently noted in our participants with splenomegaly and thrombocytopenia compared with those without. Our results, how-ever, did not support that the patients with liver steatosis have intermediate LAL activity. The relationship between transaminase levels and LAL activity is controversial. LAL activity was inversely associated with ALT concentration in the study of Baratta et al (25), whereas Shteyer et al (22) reported no relationship. In contrast to these studies, the intermediate LAL activity was significantly frequent in patients with normal transaminase concentrations com-pared with those with abnormal values. These differences may be due to the nonhomogeneity of the studies. Thus, further data are needed on this issue.

The association between age and LAL activity in childhood is unknown. In our study, intermediate LAL activity was more frequently observed in the patients older than 12 years than those of younger patients. It may be due to decreased protein production secondary to a reduction in intact hepatocytes over time. It is, however, difficult to make a definite comment due to the lack of a control group and further research is needed.

The main limitation of this study was the lack of genetic analysis of cases with intermediate LAL enzyme activity. Another milder limitation was that this study did not focus on patients with obesity-related liver steatosis or hyperlipidemia, although our study covered patients with a wide spectrum of liver manifestations of LAL-D. Recently, Himes et al (28) reported 2 children with LAL-D who have obesity-related liver steatosis. Despite these limitations, to the best of our knowledge, there are no data in the literature evaluating LAL-D in such a broad spectrum and large population. In summary; the prevalence of LAL-D in this selected population cohort of high-risk children is 0.1% (95% CI: 0.125– 0.606). Our results suggest that LAL-D seems to be rare even in this selected population; however, if the genetic analysis was available especially in the cases with intermediate LAL activity which was <0.15 nmol/punch/h, overall prevalence may have risen. We con-clude that high awareness for LAL-D is required especially in patients with liver steatosis and dyslipidemia who also may have mildly elevated transaminases. The simple DBS test is helpful to distinguish LAL-D from other diseases even if there is a gray zone of slightly decreased levels.

Acknowledgments:The authors requested a medical review of

the manuscript from Alexion Pharmaceuticals Inc. Alexion Pharmaceuticals Inc also edited the manuscript for English; the authors had final decisions of the content of this manuscript. The authors thank all the patients and their families for consenting to this study, and the investigators at each center for input of data into the CRF. The authors also thank the Turkish Society of Pediatric Gastroenterology, Hepatology and Nutrition for supporting the annual meeting. The authors also wish to acknowledge Dr John Hamilton (NHS Greater Glasgow and Clyde Biochemistry Department, Glasgow, UK) for the technical support in the measurement of LAL enzyme activity, Nazmiye Kursun (Ankara University, School of Medicine, Department of Biostatistics) for

statistical analysis, Aylin Go¨kduman (Plexus Information

Technology Inc) for the Web site support.

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