Balkan Journal of Medical Genetics

A NOVEL VARIANT IN THE LIPA GENE ASSOCIATED WITH DISTINCT PHENOTYPE
Sarajlija A.1,6, Armengol L.2, Maver A.3, Kitic I.4,6, Prokic D.4,6, Cehic M.1, Djuricic M.S.5,7, Peterlin B.3
*Corresponding Author: Adrijan Sarajlija, MD, PhD, Mother and Child Health Care Institute of Serbia “Dr. Vukan Cupic”, School of Medicine, University of Belgrade, Radoja Dakica 6-8, Belgrade, 11070, Serbia, Tel: +381113108287, E-mail: adrijans2004@yahoo.com
page: 8

CASE SERIES PRESENTATION

Family 1 Patient 1 presented with hepatosplenomegaly at the age of three years. Initial laboratory evaluation showed elevated serum transaminases with preserved synthetic liver function, mild leukopenia and thrombocytopenia. He is the second child of non-consanguineous parents, from an uneventful pregnancy and perinatal period and normal psychomotor development. His plasma lipid profile revealed very low high density lipoprotein (HDL) cholesterol, elevated TG and normal total cholesterol. Significant liver and spleen enlargement were verified via an abdominal ultrasound exam. A biomarker study was indicative of LSD with high chitotriosidase, ACE and acid phosphatase levels in the blood. On account of hepatosplenomegaly, lipid profile abnormalities and biomarker findings, LAL-D was suspected. Enzyme diagnostics for LAL-D and other LSDs (Gaucher disease, Niemann-Pick disease type A/B and C, Farber disease) were performed at the Metabolic Laboratory of the University of Mainz and results returned negative. Over the following years, hepatosplenomegaly progressed with a further decrease in platelet count. Repeated plasma measurements of chitotriosidase showed elevated values, reaching the highest level at 8083 nmol/ml/hr. With the advent of next generation sequencing, patient 1 was tested by clinical exome sequencing (Quantitative Genomic Medicine Laboratories, SL., Barcelona), and compound heterozygosity for variants in the LIPA gene was found. This consisted of a pathogenic variant NM_000235.4(LIPA):c.419G>A (p.Trp140Ter) and a novel variant of uncertain significance NM_000235.4(LIPA):c.851C>T (p.Ser284Phe). Since enzyme activity remained normal in the leukocytes over repetitive analyses, whole genome sequencing was used to discern changes in the LIPA gene, and findings from the Centogene lab confirmed the previous result from Barcelona, thus categorizing Ser284Phe as a VUS. The enzyme activity of lysosomal acid lipase in a dried blood spot was measured at the Biochemistry Department of NHS Greater Glasgow and Clyde at Glasgow, UK and was reported as normal (0.14 nmol/punch/hour). Patient 2 (the younger sibling) presented with hepatosplenomegaly upon clinical examination at the age of one and a half. He is the third child from uneventful pregnancy, with a normal perinatal period and normal psychomotor development. Elevated liver enzymes with preserved liver function and thrombocytopenia were observed during the initial laboratory evaluation. A lipidogram revealed reduced HDL cholesterol, along with hypertriglyceridemia, and normal total cholesterol (Table 1). An echographic abdominal evaluation confirmed hepatosplenomegaly, observed in clinical examination. Taking clinical findings and similarity with patient 1 into consideration, enzyme testing on a dry blood spot for LAL-D was performed and reported as normal. In the following years, hepatosplenomegaly showed progression, with persistent thrombocytopenia and consistent finding of reduced HDL cholesterol and elevated TG. Clinical exome sequencing was carried out (Quantitative Genomic Medicine Laboratories, SL., Barcelona) and patient 2 was found to be a compound heterozygote for the same variants in the LIPA gene as the older brother. Whole genome sequencing confirmed results previously obtained from Barcelona, categorizing p.Ser284Phe as a variant of uncertain significance, nevertheless vouching for the genetic diagnosis of CESD should be considered. Family 2 Patient 3 presented with hepatosplenomegaly at two months of age. Initial laboratory evaluation showed only slightly elevated serum transaminases. He is the third child of non-consanguineous parents with an uneventful perinatal period and normal psychomotor development. A lipid profile revealed normal total cholesterol, with HDL cholesterol below normal and elevated TG. An abdominal ultrasound confirmed hepatic enlargement with structural dyshomogeneity and hyperechogenicity. Chitotriosidase was extremely elevated (Table 1). The enzyme activity of LAL was tested in a dry blood spot sample in the University Medical Center Hamburg-Eppendorf lab and revealed sufficient activity. On the basis of a clinical suspicion of a diagnosis of LSD, a liver biopsy was done at the age of five. The specimen appeared bright yellow-orange in color. Standard HE and special staining were performed on paraffin and frozen sections. Microvesicular steatosis with pronounced liver fibrosis was established, and characteristic groups of uniform, foamy macrophages and Kupffer cells with PAS-positive, granular cytoplasm were found in portal spaces and focally in fibrous septa (figure 1A). The analysis of the Sudan IV stained frozen sections under polarized light revealed numerous, diffuse infiltrate of birefringent, anisotropic, needle-like crystals (figure 1B, 1C). The crystals were lost after heating the tissue sections in a thermostat to 60 degrees Celsius, which demonstrated the thermal sensitivity of the crystals. Hepatosplenomegaly showed progression over the course of time, levels of transaminases increased with development of splenomegaly, thrombocytopenia and a constant finding of low levels of HDL cholesterol (Table 1). Clinical exome sequencing (Illumina TruSight One, Illumina MiSeq) revealed that the patient 3 is homozygous for a missense variant of uncertain significance, NM_000235.4(LIPA):c.851C>T (p.Ser284Phe) in the LIPA gene. Patient 4 is the older sister of patient 3 and she also presented with hepatosplenomegaly at the age of 2.5 years. Laboratory evaluation showed elevated transaminases, dyslipidemia (elevated total cholesterol and LDL cholesterol, low HDL cholesterol and elevated TG) alongside anemia. A liver biopsy, performed at 3 years of age, revealed microvesicular steatosis with pronounced portal fibrosis and the presence of thermal-sensitive birefringent crystals in frozen tissue sections under polarized light. After the biopsy findings, a diagnosis of CESD was established. In the years that followed, hepatosplenomegaly showed regression, with persistent findings of elevated transaminases, TG, and low HDL cholesterol. Leukopenia and thrombocytopenia developed as well. Upon receiving the results of patient’s brother’s exome sequencing, genetic testing confirmed that patient 4 was homozygous for the same variant as her brother.

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