In the light of our research in PubMed and SCOPUS for any case presentation of WD resembling manifestations of HLH, we found eight reports (see Table 3 and 4). Perry et al described 3 infants in 2001; a brother and sister presented at 49 and 26 days with hepatosplenomegaly, anemia and elevated liver transaminases and triglycerides. With a diagnosis of familial erythrophagocytic lymphohistiocytosis, they were treated by chemotherapy and bone marrow transplant, respectively. Autopsy of the second infant revealed accumulation of cholesterol crystals and lipids in many organs, and a hypertrophied and calcified adrenal, leading to the diagnosis of WD following the finding of low acid lipase activity in liver biopsy. The third patient was a 25-day old boy with hepatosplenomegaly, elevated liver transaminases and adrenal calcification. The patients died in a median age of 67 days [11].
Santos et al in 2018 presented two cases of Wolman disease. The first patient was a 2-month-old boy admitted with hepatosplenomegaly, fever, anemia, elevated liver transaminases, hypertriglyceridemia and elevated ferritin level. Based on the criteria, the patient was treated according to HLH protocol [12]. Signs and symptoms subsided but recurred after a month. Peripheral blood smear showed lymphocyte cytoplasmic vacuolation and adrenal appeared calcified on X-ray. LAL activity in blood and fibroblasts was low. Later on, molecular PCR analysis revealed [LIPA] c.966 + 2T > G-intron 9 (in homozygosity), compatible with Lysosomal Acid Deficiency. Sebelipase alfa was administered which caused slight improvement, yet the patient died eventually with multiorgan failure. The second patient was a 4-month-old girl with hepatosplenomegaly, icterus, elevated liver transaminases, severe coagulopathy, hyperferritinemia, hypertriglyceridemia and hypercholesterolemia. Hemophagocytosis in bone marrow and elevated CD25 were found. HLH therapy made no improvement. Further investigations showed a calcified adrenal and low LAL activity. Although Sebelipase alfa was administered as a bridge therapy for HSCT, the patient died of cardiopulmonary compromise due to hepatic insufficiency. Molecular analysis of the LIPA gene identified two pathogenic mutations in compound heterozygous state: c.509C > A (p. S103R)/c.796G > T (p. G266X) [4].
Taurisano et al in 2014 discussed a a 4-month-old female case with hepatosplenomegaly, icterus, anemia, thrombocytopenia, elevated liver transaminases and hypertriglyceridemia. Liver biopsy portrayed Kupffer cells with lysosomes containing crystals of cholesteryl esters. Bone marrow aspiration revealed giant histiocytic and signs of hemophagocytosis. Reduced LAL confirmed the diagnosis of WD. The patient went through supportive therapies, and died due to respiratory failure when she was 5 months old [3].
Yavas et al reported a 2-month-old girl in 2015 with fever, diarrhea, FTT and hepatosplenomegaly. Anemia, thrombocytopenia, elevated liver transaminases and hypertriglyceridemia were also found. Hemophagocytic lymphohistiocytosis was observed in bone marrow aspiration. She was primarily diagnosed with HLH, then a diagnosis of WD was made because of low LAL activity. The patient died one month later, and molecular analysis revealed Exon 4 heterozygous variation at the LIPA gene, location c:260G>T (GGC>GTC), p. Gly87Val [13].
Elsayed et al in 2015 reported three patients with diagnosis of WD. The first patient was a 2.5-month-old boy with fever, hepatomegaly, anemia, and erythropoiesis in bone marrow aspiration. Hyperferritinemia, hypertriglyceridemia and high LDH level were also observed. Sequencing of the LIPA gene demonstrated homozygous G969A (W130X) mutation which made the diagnosis of WD. The second patient was a 3-month-old girl presented with the same presentations as the first one. Vacuolated macrophages were found in bone marrow aspiration as well as histiocytosis. Sequencing of all coding sequences of the LIPA gene revealed homozygous mutation c.438delC (p.S112X) and led to the diagnosis of WD. The third patient, a 3-month-old boy, also with the same presentation and laboratory findings, went through sequencing of LIPA gene, which revealed homozygous mutation c. G969A (p. W289X), leading to the diagnosis of WD [14].
Tinsa et al reported a 3-month-old girl in 2018 with fever and hepatosplenomegaly. Laboratory values were consistent with HLH disease. Bone marrow biopsy illustrated abnormal macrophages, but no evidence of hemophagocytosis. Three weeks later, adrenal calcification was found and bone marrow aspiration was repeated, revealing bubbles like cytoplasm in macrophages, compatible with Wolman disease. There was a novel homozygous mutation in LIPA gene exon 3: NM_000235.3: c.153 C>A (p. Tyr51*), which interrupted the reading frame by a premature STOP codon and confirmed the diagnosis of Wolman disease. The parents were heterozygous for this mutation [15].
Rabah et al reported a 2-month-old boy in 2014, primarily diagnosed with HLH due to fever, hepatosplenomegaly, icterus, anemia, thrombocytopenia, elevated liver enzymes, hyperferritinemia and hypofibrinogenemia. Soap bubbles like macrophage cytoplasm besides hemophagocytosis were observed in bone marrow biopsy. Diagnosis of WD was made by leukocytic cholesteryl esterase assay, and molecular analysis showed no mutation [16].
Alabbas et al reported a male infant in 2021, previously diagnosed with WD, but not treated due to unavailability of medication. He presented with secondary HLH at 4 months. They reported a novel mutation in Lipa gene; a deletion/duplication genetic analysis by real-time quantitative PCR (qPCR) confirmed the presence of homozygous deletion c. (428 + 1_967-1) _ (*1_?) del in the LIPA gene (NM_000235.3; chr.10): (OMIM 613,497).[17]
The discussed patients including ours consist of 14 cases. They aged from 25 to 135 days (median=90 days). Consanguinity was reported in nearly all of the cases. Hepatosplenomegaly was observed in all cases and fever was seen in most of them. Laboratory findings included anemia in all patients and thrombocytopenia, elevated liver enzymes, hypoalbuminemia, hypofibrinogenemia and elevated LDH and triglycerides in most cases. Bone marrow hemophagocytosis was observed in 9 patients and adrenal calcification was reported in 8. Except for one report which did not point to the patient’s survival, all the other patients were dead in less than 2 months from admission. A molecular analysis concordant with Wolman disease was found in 9 patients.
Witeck et al conducted a systematic review in June 2020 in which they included all published articles on WD, finally incorporating 108 articles[18]. According to Witeck’s review, the median reported ages of the patients at onset and at diagnosis were 1.5 and 3 years, respectively. The most common clinical presentations were reported as hepatomegaly (93%), splenomegaly (77%), abdominal distension (52%), failure to thrive (66%), diarrhea (51%), vomiting (36%) and jaundice (8.2%). Laboratory values revealed anemia (55%), elevated transaminases (33%), Low High-density Cholesterol (37%), and hypertriglyceridemia (22%), Hypercholesterolemia (8%). By meticulously studying features of patients presenting with HLH and keeping the important differential diagnosis of WD in mind, we can reach the correct diagnosis, which, by leading to initiation of the proper treatment, can save the patient’s life.
The Whole Exome Sequencing (WES) test is a genetic test designed to identify variations in a patient’s DNA that can be causative for or related to the patient’s medical condition. By using next-generation sequencing techniques, thousands of genes are simultaneously analyzed. The patient’s and the parents’ protein-coding genes (exomes) are sequenced and then compared to a normal reference in order to find variations, which are then matched with the medical concern. Even though WES tries to sequence all exomes of a genome, certain genes might not be completely covered in the analysis[19, 20]. In this study’s method, only the exons were selected first, and then they were sequenced using Illumina high throughput DNA sequencing technology. Sanger sequencing is indeed a technique that selectively incorporates oligonucleotide primers to detect specific variants[21].
WD and CESD are two phenotypes of the same disorder in which there is low or absent LAL activity. CESD is presented by hyperlipidemia, atherosclerosis, and hepatic fibrosis, and has less mortality than WD. CESD patients typically present missense mutations, correlating with some residual enzyme activity (5% -10%). The mutation of c.894G.A in exon 8 is the most common mutation found in CESD patients, which comprises more than 50% of all reported variants[22]. Very low or absent LAL activity can be found in WD due to several dozens of mutations in the LIPA gene; i.e. deletions, insertions, and nonsense mutations [23].
The family that we are reporting presents a novel variant, caused by a Glycine to Aspartic acid substitution within exon 4 of the LIPA gene, which is predicted as likely pathogenic. This prediction was made using variant disease and population databases in silico tools (Figure 3) and segregation analysis (Figure 2) in the nuclear family and was thus presumed to be the cause of LAL-deficiency in this infant[24]. The literature was sought in regular time intervals and still, no description of this mutation was found.
The original wild-type residue (Glycine) and newly introduced mutant residue (Aspartic acid) each have their own specific size, charge, and hydrophobicity value. The wild-type residue charge is neutral compared with the negative charge of the mutant residue and is more hydrophobic than the mutant. The mutation imposes a charge which can repulse ligands or other residues with the same charge. The mutant residue is bigger than the wild-type and might lead to the formation of bumps. The torsion angles for this residue are unusual. Only Glycine is flexible enough to make these torsion angles and mutation into another residue will force the local backbone to change into an incorrect conformation, which will disturb the local structure and impair the protein’s function. The original residue is conserved to a large extent, but a few other residue types have also been found at this position. Considering the conservation scores, this mutation can disrupt the protein’s function. The mutated residue is placed in a domain that plays a crucial role in the main activity of the protein and mutation of the residue can disrupt this function[24, 25].
In this case report, the causative point mutations result in the substitution of a Glycine (the smallest amino acid) residue by a bulkier amino acid (Aspartic acid) (Figure 3). However, a loose correlation between the location of the point mutation along the molecule and disease severity exists for LIPA1. Previous research has revealed severely pathogenic and even lethal outcomes for Glycine to Aspartic acid substitutions within fibrillar collagen genes[26]. In our case, however, we are dealing with an enzyme molecule and not a structural protein; thus, these findings cannot be extrapolated to our case. Finally, we have to bear in mind that the context of the sequence surrounding the substitution, or mutations within special helical domains, significantly influence the effect of particular mutations.