Novel NBAS mutations and fever-related recurrent acute liver failure in Chinese children: a retrospective study

Background Underlying causes in Chinese children with recurrent acute liver failure (RALF), including liver crises less than full acute liver failure, are incompletely understood. We sought to address this by searching for genes mutated in such children. Methods Five unrelated Chinese boys presenting between 2012 and 2015 with RALF of unexplained etiology were studied. Results of whole exome sequencing were screened for mutations in candidate genes. Mutations were verified in patients and their family members by Sanger sequencing. All 5 boys underwent liver biopsy. Results NBAS was the only candidate gene mutated in more than one patient (biallelic mutations, 3 of 5 patients; 5 separate mutations). All NBAS mutations were novel and predictedly pathogenic (frameshift insertion mutation c.6611_6612insCA, missense mutations c.2407G > A and c.3596G > A, nonsense mutation c.586C > T, and splicing-site mutation c.5389 + 1G > T). Of these mutations, 3 lay in distal (C-terminal) regions of NBAS, a novel distribution. Unlike the 2 patients without NBAS mutations, the 3 patients with confirmed NBAS mutations all suffered from a febrile illness before each episode of liver crisis (fever-related RALF), with markedly elevated alanine aminotransferase and aspartate aminotransferase activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy and mild to moderate jaundice. Conclusions As in other countries, so too in China; NBAS disease is a major cause of fever-related RALF in children. The mutation spectrum of NBAS in Chinese children seems different from that described in other populations. Electronic supplementary material The online version of this article (doi:10.1186/s12876-017-0636-3) contains supplementary material, which is available to authorized users.


Background
Acute liver failure (ALF) is a rare but often fatal emergency for children, especially infants. Together with non-genetic causes such as viral infections, drug or toxin exposure, and autoimmune hepatitis, identified hereditary metabolic disorders account for half the instances of ALF in children [1][2][3][4][5]. Although recent work in Europe has implicated several genes in recurrent ALF (RALF) in infancy [6][7][8][9][10], the etiology of some instances of pediatric RALF remains unexplained. Furthermore, the causes of RALF in non-European populations are largely unexplored.
Using whole-exome sequencing (WES), we evaluated 5 Chinese children with RALF. Here we describe our work and its implications.

Enrollment criteria
The probands were Chinese children evaluated for RALF from 2012 to 2015 by JSW, to whose clinic instances of pediatric liver disease from throughout China are referred (1096 new pediatric liver-disease patients seen during these 4y). Their parents and siblings also took part.
Participation required informed consent (for children, informed parental consent) under a protocol approved by Children's Hospital and Jinshan Hospital of Fudan University according to the ethical guidelines of the 1975 Declaration of Helsinki. RALF of indeterminate etiology was defined as present when a child had >1 episode of liver injury, including at least 1 episode of ALF (Pediatric Acute Liver Failure Study Group criteria) [3]. That is, no child had evidence of chronic liver disease; all children had biochemical evidence of acute liver injury; all children had hepatic-based coagulopathy, with a prothrombin time (PTT) ≥15 s or an international normalized ratio (INR) >1.5 not corrected by vitamin K in the presence of hepatic encephalopathy, or a PTT ≥20s or an INR >2.0 regardless of the presence or absence of clinical hepatic encephalopathy; and all other causes possibly responsible for liver crises were excluded through comprehensive evaluation. Liver biopsy was performed when coagulopathy permitted.

Clinical features of probands
Five unrelated boys with RALF of indeterminate etiology were enrolled in this study, including 4 of Han and 1 of Miao ancestry. The major clinical features of these probands are shown in Table 1. All parents were nonconsanguine, except those of patient 4. Patient 1, initially diagnosed with liver crisis aged 6mo 18d, is the product of a 4th pregnancy (ectopic pregnancy, surgically treated;  Clinical-biochemistry evidence of hepatobiliary injury was seen in none between liver crises or bouts of ALF 2 induced abortions) complicated by intrahepatic cholestasis manifest as pruritus that halted after delivery. The parents are otherwise well. Patient 2, initially diagnosed in liver crisis aged 7mo 21d, is the product of a 1st pregnancy. A younger brother is well. Their mother has a 3y history of hyperthyroidism. Their father is healthy. Patient 3, initially diagnosed in ALF aged 6mo 1d, is the product of a 3rd pregnancy. A sister and brother died in fever-related liver crisis aged respectively 4mo and 8mo. Material from them suitable for genetic analysis was unavailable. Patient 4, initially diagnosed in ALF aged 6y 10mo, is the product of a 5th pregnancy (2 induced abortions, 2 live births). One sister has moyamoya disease; the other is healthy. Their parents are consanguineous. Patient 5, initially diagnosed in ALF aged 2y 2mo, is the product of a first and only pregnancy. The parents are healthy. All patients were normally grown, without dysmorphism. Patients 1-3 always had febrile illnesses before liver crises or episodes of ALF. Patient 5, with one episode of liver crisis without a febrile illness, had febrile illnesses before 3 liver crises and before one episode of ALF. No febrile illness preceded liver crisis or ALF in patient 4.

Exome sequencing analysis
Detailed variant filtering strategies for each patient are outlined in Additional file 2. Variants occurring with allele frequency ≥ 1% in the Thousand Genomes Project (http://www.1000genomes.org/home) and NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/) databases or ≥4.5% in the Genesky in-house database were filtered out. Potential disease-causing mutations predicted by Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and MutationTaster [16] (http:// www.mutationtaster.org/) then were selected as suspected pathogenic variations. Suspected pathogenic variations in genes known to be associated with ALF (Additional file 3) and present in accord with inheritance modes identified candidate genes. Suspected pathogenic variations in genes not known to be associated with ALF but present in accord with recessive inheritance also identified candidate genes.

Sanger sequencing
Polymerase chain reaction (PCR) amplification was carried out using primers specific for NBAS exons 8, 22, 31, 43, and 50 (Additional file 4). PCR conditions are available on request. The amplified products were sequenced using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA) and analyzed using CodonCode Aligner software (http://www.codoncode.com). NM_015909 was used as the NBAS reference sequence.

Clinical manifestations in 3 NBAS mutant patients
Unlike the 2 patients without NBAS mutation, those harbouring NBAS mutations always had a febrile illness before a liver crisis or episode of ALF. Our 3 NBAS-disease patients clinically resembled one another: all suffered from a febrile illness, likely infective (viral / bacterial) before each episode of RALF (fever-related RALF), with markedly elevated ALT (77-9382 IU/L; normal 0-40) and AST (213-17,344 IU/L; normal 0-40) activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy (maximum INR = 6.89; normal 0.8-1.2) and mild to moderate jaundice that were ascribed to ALF. Hypoglycemia and hepatic encephalopathy were transiently observed in patients 1 and 3. Total bile acid concentrations in all 3 patients were increased (18.2-517.2 umol/ L; normal 0-10). Serum alkaline phosphatase and γglutamyltranspeptidase activities were normal or only mildly increased, as were blood ammonia values. Ages during episodes of ALF and liver crises are shown in Additional file 6. The details of episodes of RALF in patient 3 are shown in Additional file 7. Similar details for patients 1 and 2 are not available, as they received care at several hospitals other than ours. Biomarkers of hepatobiliary injury completely recovered between liver crises. Not all episodes of fever led to hepatic crisis. All patients used antipyretics and hepatoprotectives (e.g., ademetionine 1, 4-butanedisulfonate; reduced glutathione). In patient 1, however, during his 7th episode of RALF (4th of ALF) biomarkers returned to normal without hepatoprotectives. The 3 NBAS-disease patients were free from facial dysmorphism and had no broken bones. No radiogrammes were exposed in patients 1 and 2; a left lower limb radiogram in patient 3 was assessed as normal. Formal cognitive evaluation and investigations for Pelger-Huët anomaly were not conducted in any patient. Motor development was normal for all 3. Lymphocyte panels and immunoglobulin values were unremarkable in patients 1 and 3; these were not evaluated in patient 2. Ophthalmoscopy found no abnormality in patient 3 and was not conducted in patients 1 and 2.

Liver biopsy in 5 RALF patients
Light microscopy was undertaken in patients 1-5, with ultrastructural study in patients 1-4. Hepatocyte cytoplasm contained small vacuoles in patients 1 and 3, confirmed as steatosis by transmission electron microscopy. Patients 2 and 3 had centrilobular fibrosis that was worse in patient 3. In patient 4, the liver was unremarkable. Inflammation and minimal portal-tract fibrosis were seen in patient 5. Ultrastructural findings were non-specific, with questionably increased glycogen stores in patients 1-4, dilated endoplasmic reticulum (ER) and abnormal mitochondria in patient 2, dense mitochondrial matrix in patient 3, and swollen mitochondria, questionably decreased in number, in patient 4.

Discussion
Mutations in NBAS were first identified as an important cause of infantile and later-onset recurrent liver failure in 2015 [8]. NBAS is a subunit of the syntaxin 18 complex, implicated in Golgi-to-endoplasmic reticulum (ER) retrograde transport [17]. NBAS also plays an important role in nonsense-mediated mRNA decay, which regulates gene expression in response to cellular and environmental Fig. 1 Pedigrees of families carrying two mutant alleles of NBAS. Blackened symbols: affected individuals.?: Liver disease in the older sister and brother of family 3 was clinically similar to that in patient 3, but no material was available to evaluate NBAS in either deceased older sibling stress [18]. NBAS had earlier been implicated in the developmental disorder SOPH syndrome [11], in which liver disease is not a feature. And a patient with NBAS disease manifest as SOPH syndrome with fever-associated liver crises that fell short of RALF is described [19]. Correlations between NBAS mutations and clinical manifestations are incomplete.
With this report, 23 NBAS-disease children with recurrent liver crises are described: 14 from European countries [12,14,19], 3 from the United States [12,14], 3 from Lebanon (siblings; parental consanguinity known) [13], and our 3 Han Chinese. These patients' phenotypes range from isolated RALF to RALF in association with multisystemic disease. Our Han Chinese patients all had isolated RALF.
RALF patients usually exhibit recurrent vomiting, progressive lethargy and pyrexia 1 or 2d before medical assessment, which finds high serum transaminases; mild to moderate jaundice and severe coagulopathy then develop [8]. Our 3 NBAS-disease patients clinically resembled other reported children with isolated RALF [8]. All suffered from a febrile illness before each liver crisis (fever-related RALF) and at presentation had substantially elevated serum AST and ALT values, followed by mild to moderate jaundice and severe coagulopathy. However, of interest as at slight variance from published descriptions is that in our patients the frequency and severity of ALF did not lessen with increasing age (Additional file 6), and that vomiting did not usually precede rises in transaminase values.
That in NBAS disease raised body temperature itself might both mark and initiate a liver crisis has been suggested [14], with the corollary and experience that early and effective control of fever might prevent or alleviate liver crisis. However, in our patients peak body temperature and length of fever were not, episode for episode of RALF, positively correlated with the severity of ensuing liver crises (Additional file 7). Expression profiling has identified ER stress in cultured fibroblasts from patients with NBAS disease [8]. ER stress accelerates lipogenesis in the liver [20,21] and activates the unfolded protein response, which can trigger cellular destruction through apoptosis [22,23]. Small-vacuole steatosis in liver, although not specific for ER stress, is consistent with that etiology. Widespread loss of hepatocytes, however, as might be expected if apoptosis is triggered, was not identified even in liver biopsied during ALF with hypertransaminasemia (patient 1). Findings on microscopy did not contribute to diagnosis. The identified NBAS mutations in reported patients with liver crises comprise 5 nonsense, 14 missense, and 7 deletion/insertion mutations, with 4 splice-site variants. These are clustered into 3 regions in the first half of the gene, exons 2-4, exons 7-15 and exons 21-26 (Fig. 2). However, among the mutations identified in our patients, none of which has before been associated with RALF, 3 lie in the second half of the gene (c.6611_6612insCA, p.M2204Ifs*3; c.5389 + 1G > T; c.3596G > A, p. C1199Y, exons 50, 43, and 31 respectively). That patients 1 and 2, not identifiably related, share mutation c3596G > A, p.C1199Y may be of relevance to a special NBAS mutation in Han Chinese.

Conclusions
In summary, we have identified NBAS-related RALF in 3 Han Chinese children. Their disorder clinically resembled that in western European (and North American, and Lebanese) children with NBAS-related RALF, although several of the mutations in our patients lay in a region of NBAS not previously found involved. RALF in each of these patients was fever-related, suggesting that to search for NBAS lesions in Han Chinese children with fever-related RALF may be worthwhile. That 2 of the 5 patients with RALF whom we studiedone of whom was born to consanguine parentshad no demonstrable mutations in NBAS or in other genes hitherto implicated in RALF implies that heritable causes of RALF remain to be discovered.