Novel neuroblastoma amplified sequence (NBAS) mutations in a Japanese boy with fever-triggered recurrent acute liver failure

Biallelic mutations in the neuroblastoma amplified sequence (NBAS) gene have been reported to cause two different clinical spectra: short stature with optic nerve atrophy and Pelger-Huët anomaly (SOPH) syndrome and infantile liver failure syndrome 2 (ILFS2). Here, we describe a case of a 3-year-old Japanese boy who presented with fever-triggered recurrent acute liver failure (ALF). The clinical characteristics were considerable elevation of liver enzymes, severe coagulopathy, and acute renal failure. In addition to the liver phenotype, he had short stature and Pelger-Huët anomaly in the peripheral granulocytes. Whole-exome and Sanger sequencing of the patient and his parents revealed that he carried novel compound heterozygous missense mutations in NBAS, c.1018G>C (p.Gly340Arg) and c.2674 G>T (p.Val892Phe). Both mutations affect evolutionarily conserved amino acid residues and are predicted to be highly damaging. Immunoblot analysis of the patient’s skin fibroblasts showed a normal NBAS protein level but a reduced protein level of its interaction partner, p31, involved in Golgi-to-endoplasmic reticulum retrograde vesicular trafficking. We recommend NBAS gene analysis in children with unexplained fever-triggered recurrent ALF or liver dysfunction. Early antipyretic therapy may prevent further episodes of ALF.


Introduction
Acute liver failure (ALF) is a life-threatening emergency in childhood. Although~25% of ALF cases are caused by inborn errors of metabolism, the underlying cause remains unknown in ∼50% of cases [1][2][3] . Recently, biallelic mutations in the neuroblastoma amplified sequence (NBAS) gene were identified as a new cause of infantile liver failure syndrome-2 (ILFS2, OMIM 616483) 4 . ILFS2 is an autosomal recessive genetic disorder connected with recurrent episodes of ALF triggered by febrile infection. Previously, the homozygous NBAS mutation c.5741G>A (p.Arg1914His) was reported to be associated with a syndrome comprising short stature with optic nerve atrophy and Pelger-Huët anomaly (SOPH) syndrome, which occurs in the genetically isolated Yakut population (OMIM #614800) 5 . Increasing evidence has indicated a phenotypic spectrum of NBAS mutation-based disease, from isolated fever-triggered recurrent ALF to a multisystemic disease with short stature, skeletal dysplasia, immunological abnormalities, and optic nerve atrophy resembling SOPH syndrome [6][7][8][9][10][11][12][13][14][15][16][17][18] . Because early antipyretic therapy and induction of anabolism have effectively ameliorated the course of ALF with the NBAS mutation, it is important to differentially diagnose NBAS mutation-based disease. Here, we describe a 3-year-old Japanese boy who presented with recurrent episodes of ALF and carries novel compound heterozygous missense mutations in NBAS.

Subjects
After informed consent and the approval of the appropriate institutional ethics review board, peripheral blood samples were obtained from the patient, his parents, and his younger brother, and skin fibroblasts were obtained from the patient. Genomic DNA was extracted from peripheral blood leukocytes.

Whole-exome sequencing
Exomes of the patient and his parents were captured from the genomic DNA using the Agilent SureSelect Human All Exon V5 kit (Agilent Technologies, Santa Clara, CA, USA) and were sequenced (paired-end, 2 × 124 bp) using the Illumina HiSeq 2500 (Illumina, San Diego, CA, USA). Read alignment to the human reference genome hg19 was performed with Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/), and ANN-OVER was used for annotation. Germline mutations were detected through our established pipeline, as previously reported (http://genomon.hgc.jp/exome/en/index.html) 19 . Population frequencies in the Human Genetic Variation Database (HGVD), db single-nucleotide polymorphisms (SNP) 131, the Integrative Japanese Genome Variation Database (iJGVD) 20 , and an in-house SNP database were used to filter common SNPs and to confirm the novelty of each mutation. Suspected pathogenic variations in genes known to be associated with ALF or present in accordance with autosomal recessive or X-linked modes of inheritance were identified as candidate genes.

Patient clinical presentation
The patient was a Japanese boy born to healthy nonconsanguineous parents at 34 weeks gestation due to a threatened premature delivery. The pregnancy was complicated by fetal growth retardation due to the mother's pregnancy-associated hypertension. The birth weight was 1512 g (−2.3 SD), and the height was 42.5 cm (−0.8 SD). During the neonatal period, gallstones were noticed and treated with ursodeoxycholic acid. The patient's psychomotor development was normal. He showed short stature: −2.4 SDs in height at the age of 7 years (Fig. 1a). Hand radiographs revealed delayed bone maturation by 3 years (Fig. 1b). There was no family history of liver disease.
At the age of three years and four months, the patient developed an upper respiratory tract infection with 39°C fever, cough, and loss of appetite. On day 5 of his fever, he became lethargic and agitated and was then admitted to our hospital. He had jaundice with an enlarged liver palpable 4 cm below the costal margin (Fig. 2a). Laboratory results revealed greatly elevated liver enzyme levels  left, at 17,820/μL (Stabbed 26%, Segmented 21%), and the C-reactive protein (CRP) (7.15 mg/dL) and serum ferritin (2242 ng/mL [normal range: 30-200]) levels were elevated. His abdominal ultrasound showed severe hepatomegaly without splenomegaly. Electroencephalogram (EEG), magnetic resonance imaging of the brain, electrocardiogram (ECG), and echocardiography were all normal. He was diagnosed as having ALF with renal failure and was managed with intensive treatment, including plasmapheresis, hemodialysis, intravenous antibiotics, intravenous immunoglobulin, and glucose infusion. Following treatment, he made a full recovery and was discharged on the 47th day of disease.
Two months later, at the age of 3 years and 6 months, the patient was readmitted with a second episode of ALF following a sore throat for 2 days and untreated 39°C fever. Laboratory tests revealed marked elevation of liver enzymes with coagulopathy and renal failure (AST, . Serum amino acid analysis showed elevations in various amino acids, including valine, leucine, methionine, citrulline, phenylalanine, and arginine, suggestive of the secondary effects of substantial liver dysfunction. Free carnitine level was also elevated. Acylcarnitine profiles showed elevations in C0, C2, C4, C5:1, C5DC, C6, C10DC, and C12DC. Nonketotic dicarboxylic aciduria was noted in the urinalysis for organic acids, including adipate, suberate, 3-hydroxy-adipate, 3hydroxy-sebacate, dodecanedioic acid, and 3-hydroxydodecanedioic acid. Increased excretion of 2-hydroxy sebacate and tyrosine metabolite p-hydroxyphenyllactate (PHPLA) was also noted. All of these changes in metabolic biomarkers normalized between the liver crises. Very long-chain fatty acids in the plasma were normal. Ceruloplasmin, copper, and α1-antitrypsin were all normal. The activities of the mitochondrial respiratory chain complex enzymes I, II, III, and IV were all normal in both the liver tissue and skin fibroblasts, indicating that a mitochondrial disorder was not the etiology of the episodes of ALF.
The workups for the infectious agents Epstein-Barr virus, cytomegalovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus were all negative. The immunological workup, including autoantibodies, serum immunoglobulin levels, complement C3 and C4 levels, CD8 + T cell count and function, CD19 + B cell count, and natural killer cell count, were all unremarkable. The serum cytokine profile at the time of the liver crisis showed the broad elevation of IL-6, neopterin, sTNF-RI, and sTNF-RII, IL-18, but most prominently IL-6 and neopterin, suggesting a severe cytokine storm during the crisis. Bone marrow aspiration at the time of the second liver crisis revealed hypercellular marrow with occasional hemophagocytosis with Pelger-Huët anomaly in the granulocytes. A repeated peripheral blood smear showed Pelger-Huët anomaly in approximately 5% of granulocytes even in the afebrile and asymptomatic states (Fig. 2f).
A liver wedge biopsy was performed shortly after the second ALF crisis. Grossly, the liver showed a yellowish appearance with a slightly irregular surface (Fig. 2b). Histopathologically, the hepatocytes showed patchy degeneration with cytoplasmic vacuolization and moderate macrovesicular steatosis (Fig. 2c). A few microvesicular lipid droplets and bile plug formation were also seen (Fig. 2d). Infiltration of inflammatory cells, mainly CD8 + T cells, was found in the portal area. At the interface of the portal and parenchymal compartments, a ductular reaction was prominent (Fig. 2c). Reticulin staining revealed minimal bridging fibrosis (Fig. 2e). Accumulations of iron and copper were not found by Berlin blue, rhodanine, and rubeanic acid staining (not shown).

Genetic analyses
Because no etiological diagnosis was reached despite the immunological and metabolic screening, whole-exome sequencing was performed in the patient and in his parents. In total, 25,853 single-nucleotide variants were called. The candidate variations (n = 326) were selected by excluding those with (i) ambiguous (unknown) singlenucleotide variations (SNVs) (n = 572); (ii) variants only present in unidirectional reads (n = 1702); (iii) variant allele frequency <0.25 (n = 1593) and (iv) known variants listed in SNP databases (n = 21,385). From 326 candidate variations, two heterozygous sequence variants in the NBAS gene, chr2:15,629,083C>G (c.1018G>C:p.Gly340Arg) (exon 12) and chr2:15,557,740C>A (c.2674G>T:p.Val892Phe) (exon 24), were detected in the patient. Parental genotyping revealed the c.1018G>C mutation in the heterozygous state in the father and the c.2674G>T mutation in the heterozygous state in the mother, indicating compound heterozygosity of the NBAS variants in the patient. Both missense variants were validated by Sanger sequencing (Fig. 3). Neither mutation was present in >120,000 alleles from the Exome Aggregation Consortium (ExAC) Server, and they affect highly conserved amino acid residues. In iJGVD, the largest Japanese genetic variation database 20 , chr2:15,557,740C>A (c.2674G>T:p.Val892Phe) was not identified, and the allele frequency of chr2:15,629,083C>G (c.1018G>C:p.Gly340Arg) was 0.0001 (n = 1/7,102).

Western blot analysis of NBAS and p31
The NBAS protein levels in the fibroblast sample from the patient were not decreased, even after changing the culturing temperature from 37 to 40°C; however, p31 protein levels were significantly reduced (Fig. 5). As p31 forms the SNARE complex with NBAS, mutations in NBAS may impair NBAS-p31 interactions, resulting in a loss of protein stability of p31 followed by dysregulation of Golgi-to-endoplasmic reticulum (ER) retrograde vesicular trafficking in the patient.
The liver phenotype in NBAS mutation-based disease is most typically recurrent ALF triggered by febrile infections, but mild elevations of liver biochemical values occur in some cases 4, 6-14, 16, 17 . These elevations often develop during infancy or early childhood and become less frequent with age. Coagulopathy and jaundice were observed in most of the cases. Secondary metabolic abnormalities, including hypoglycemia and hyperammonemia, were often observed. Complete recovery is typically achieved with antipyretic therapy and induction of anabolism, including parenteral glucose and lipids. Our patient developed ALF twice at the age of 3 years, both episodes triggered by febrile infections. He showed severe coagulopathy with renal failure but recovered completely with conservative treatment. After the diagnosis of NBAS mutation-based disease, he suffered multiple hospitalizations with febrile illnesses associated with liver dysfunction; however, early antipyretic therapy using intravenous injection of acetaminophen and glucose infusion prevented further occurrences of ALF until the age of seven. His younger brother was found to carry the same mutations as the proband's at the age of 2 years. He shared only Pelger-Huët anomaly in peripheral granulocytes as a symptom of NBAS mutation-based disease. After the genetic diagnosis, he has received early antipyretic therapy and has never experienced fever-triggered ALF or liver dysfunction to date (he is currently 5 years old). These findings indicate the possible effectiveness of early antipyretic therapy to prevent life-threatening ALF. However, it is also possible that there is a phenotypic heterogeneity of the affected organs among the family members. Further investigation to reveal the penetration and phenotype-genotype correlation in NBAS mutationbased disease is awaited.
The molecular pathogenesis by which NBAS contributes to liver disease and to fever dependency is not fully understood. The NBAS protein is considered to be involved in Golgi-to-ER retrograde vesicular trafficking and to control nonsense-mediated mRNA decay [21][22][23] . The NBAS protein interacts directly with p31, ZW10, and RINT1 and is believed to play a primary role in SNARE assembly at the ER 21 . It is proposed that the site at amino acid positions 1-1035 is the p31 binding site. Mutations associated with ALF, including those in our patient, are mostly found at the N-terminal end of NBAS (Fig. 4). In contrast, the p.Arg1914His mutation carried by SOPHaffected Yakut individuals in the homozygous state is located near the C-terminal end and does not cause ALF 5 . Our patient's skin fibroblasts showed a decreased p31 protein level (Fig. 5). Thermal susceptibilities of NBAS and p31 proteins have been reported previously 4,8 . These findings suggest that the loss of the NBAS-p31 interaction may cause alterations to Golgi-to-ER retrograde vesicular trafficking, which leads to liver dysfunction, especially in the febrile condition 8 . Further in-depth mechanistic studies are needed to establish the genotype-phenotype correlations and to reveal the wide phenotypic spectrum of NBAS mutation-based disease. Further investigations using the patient's iPS cell-derived hepatocytes will show whether the episodes of liver disease are due to alterations in Golgi-to-ER retrograde vesicular trafficking and/or impaired nonsense-mediated mRNA decay, or a yet-tobe-discovered NBAS function.
We describe the first reported Japanese case of fevertriggered recurrent ALF carrying novel compound heterozygous missense mutations of NBAS. We recommend sequencing NBAS in any patients with unexplained Fig. 4 Gene and protein structures of NBAS and identified mutations. Schematic gene and protein structures of NBAS showing the p31 interaction site at the N-terminus and the ZW10 and RINT1 interaction site at the C-terminus. Mutations associated with acute liver failure (ALF), including those in our patient, were mapped to the N-terminal end, whereas mutations associated with SOPH syndrome or related diseases without the liver phenotype were mapped near the C-terminal end. Mutations identified in this study are boxed recurrent ALF, especially in individuals with feverassociated hepatic dysfunction.  5 Western blot of NBAS and p31 in cultured fibroblasts from the patient. a NBAS protein levels were not decreased even after a shift in the culturing temperature from 37 to 40°C; however, p31 protein levels were significantly decreased compared to the control subjects. β-actin was used as a loading control. The 15-kDa bands in the p31 western blot indicated by filled circles were isoforms of p31. A typical example of three repeated experiments is shown. b Quantification of NBAS and p31 protein levels. In each case, n = 3, and the mean ± standard deviation (SD) is shown. * denotes P < 0.01 compared to the controls