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Neuroblastoma Amplified Sequence (NBAS) mutation in recurrent acute liver failure: Confirmatory report in a sibship with very early onset, osteoporosis and developmental delay
European Journal of Medical Genetics 58 (2015) 637e641
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
Neuroblastoma Amplified Sequence (NBAS) mutation in recurrent acute liver failure: Confirmatory report in a sibship with very early onset, osteoporosis and developmental delay -Mario Capo-Chichi a, Cybel Mehawej b, 1, Valerie Delague c, d, Catherine Caillaud e, Jose Issam Khneisser b, Fadi F. Hamdan a, Jacques L. Michaud a, f, Zoha Kibar a, Me garbane b , g, * Andre a
CHU Sainte-Justine Research Center, Montreal H3T 1C5, Canada Unit e de G en etique M edicale, Facult e de M edecine, Universit e Saint-Joseph, Beirut, Lebanon c Inserm, UMR_S 910, 13385 Marseille, France d Aix Marseille Universit e, GMGF, 13385 Marseille, France e ^pital Necker Enfants Malades, Paris 75015, France Service de Biochimie M edicale, Ho f Department of Pediatrics and Department of Neurosciences, Universit e de Montreal, Montreal, Canada g ^me Lejeune, Paris, France Institut J ero b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2015 Received in revised form 6 November 2015 Accepted 7 November 2015 Available online 11 November 2015
Background: Recently, biallelic mutations in the Neuroblastoma Amplified Sequence NBAS gene have been identified in ten patients that present recurrent acute liver failure (RALF) in early infancy. In addition to severe liver dysfunction, some of these individuals also suffered from other comorbidities including cardiomyopathy, neurologic phenotypes and gastrointestinal immune defects. Here we report on a consanguineous Lebanese family with three siblings affected by RALF. Of note, neonatal spontaneous fractures, developmental delay, prominent eyes, generalized hirsutism, gum hypertrophy, and hepatosplenomegaly were also present. Methods: Whole-genome SNP genotyping in all the patients, followed by exome sequencing was performed in one of the affected siblings. Results: A homozygous c.409C > T (p.Arg137Trp) missense mutation in NBAS was identified in all patients. Conclusion: Overall, our findings confirm the involvement of NBAS in the pathogenesis of this condition characterized by severe liver dysfunction and help expand its phenotypical spectrum. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Recurrent acute liver failure Spontaneous bone fractures NBAS
1. Background Whole exome sequencing (WES) is nowadays an important tool to elucidate the molecular origin of rare disorders. We used this
de Ge n e tique Me dicale, Faculte de Me decine, * Corresponding author. Unite Saint-Joseph, 42, Rue de Grenelle, 75007 Paris, France. Universite E-mail addresses: [email protected] (J.-M. Capo-Chichi), cybel.mehawej@ usj.edu.lb (C. Mehawej), [email protected] (V. Delague), catherine. [email protected] (C. Caillaud), [email protected] (I. Khneisser), fadi. [email protected] (F.F. Hamdan), [email protected] (J.L. Michaud), [email protected] (Z. Kibar), garbane ). [email protected], [email protected] (A. Me 1 Current address: Division of Immunology, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA. http://dx.doi.org/10.1016/j.ejmg.2015.11.005 1769-7212/© 2015 Elsevier Masson SAS. All rights reserved.
approach to study three siblings born to consanguineous Lebanese parents and presenting a condition characterized by recurrent acute liver failure in early infancy, neonatal spontaneous fractures and developmental delay and hepato-splenomegaly. To our knowledge this condition has never been reported before. 2. Subjects and methods 2.1. Subjects The affected individuals (II.1e3, Fig. 1A) are three siblings born to a consanguineous Lebanese couple in good health. Blood samples were obtained from each of these individuals after informed consent and the approval of the appropriate institutional ethic
638
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
Fig. 1. Identification of a homozygous mutation in NBAS in a consanguineous family affected with developmental delay, dysmorphic features, neonatal spontaneous fractures, wrinkled skin, and hepatic failure. (A) Pedigree of the consanguineous Lebanese family studied here. (B) Sanger sequencing confirming that the c.409C > T/p.R137W (NM_015909) mutation in NBAS segregates in this family. Representative chromatograms of the three affected siblings (homozygous for c.409C > T) and their parents (heterozygous for c.409C > T) are shown. (C) Multiple alignments of NBAS showing residues of the predicted quinoprotein aminedehydrogenase beta chain like domain around the p.R137W mutation. Alignments were obtained from reference sequences of NBAS orthologues. R137 residue is identified in bold. (D) Schematic representation of NBAS showing the predicted WD40 repeats and secretory pathway sec39 motif. (Sources: Uniprot entry A2RRP1, HGNC, NCBI gene). In red, p.R137W identified herein affects the quinoprotein aminedehydrogenase beta chain €t anomaly (Maksimova et al., 2010) maps toward the like domain in N-terminal end of NBAS. In black, p.R1914H associated to the short stature, optic nerve atrophy and Pelger-Hue C-terminal end of NBAS.
review board. DNA was extracted then amplified using standard methods. 2.2. Homozygosity mapping Genomic blood DNA (2.5 mg) from individuals II.1 and II.2 was used for whole genome genotyping on the Illumina Human Omni 2.5 Chip at the McGill University and Genome Quebec Innovation Center (MUGQIC, Montreal, Canada). Genotyping reactions were performed according to the manufacturer's instructions. Homozygosity mapping was performed using PLink (v.1.06). Shared regions of homozygosity (HR) containing >30 consecutive homozygous SNPs and extending over 1 Mb were retained. 2.3. Exome capture and sequencing The exome of individual II.1 was captured from 3 mg of blood genomic DNA, using the Agilent SureSelect Human All Exon Capture 50 Mb Kit (Mississauga, ON), and sequenced (paired-end, 2x100 bp) using the Illumina Hiseq 2000 at the MUGQIC. Sequence processing, alignment, and variant calling were done using the Broad Institute Genome Analysis Tool Kit (GATK v2.2) and annotated using Annovar (Wang et al., 2010). 3. Results 3.1. Clinical findings The two elder siblings (II-1 and II-2) were previously reported garbane et al. 2008. Briefly, they had axial hypotonia, by Me developmental delay, hirsutism, large fontanels with delayed closure, dysmorphic facial features that consist of frontal bossing,
prominent eyes, slightly down-slanting palpebral fissures, hypertelorism, telecanthus, long eyelashes, and progressive gum hypertrophy, short neck, pointed chin, abnormal thoracic configuration, wrinkled skin on the hands and abdomen, progressive hepatosplenomegaly and neonatal spontaneous fractures. Liver function deteriorated during intercurrent infections and both siblings died at the age of 18 months and 3e8/12 years respectively. Liver biopsy performed in the eldest child showed steatosis without fibrosis. Laboratory exams were normal except for liver enzymes that were garbane et al., 2008). During the last years, a third elevated (Me child, a boy, was born. Pregnancy was normal and delivery was at 35 weeks of gestation. According to the parents, weight, length and OFC were in the normal limits. Like his other siblings, his major complains were overall health and liver enzymes deterioration every time he had fever. Indeed, liver dysfunction started at the age of 4 months with his first sickness episode; it was mild but got more severe leading to the death of this boy at the age of 2 years. Clinically, this patient had almost the same clinical evolution and dysmorphic features as his affected siblings. He was hypotonic, had feeding difficulties, a failure to thrive, delayed developmental milestones, as he could only sit at his own at the age of 8 months and made his first steps without help at the age of 20 months. He also had hirsutism, large fontanels with delayed closure, frontal bossing, prominent eyes, slightly down-slanting palpebral fissures, hypertelorism, long eyelashes, and gum hypertrophy, short neck, pointed chin, abnormal thoracic configuration, wrinkled skin on the hands, and hepato-splenomegaly. No fractures were identified after a total body x-rays exam. Nevertheless, as he used to cry whenever the parents hold him and because of the presence of spontaneous fractures in his other siblings, an osteodensitometry was performed and revealed a severe osteoporosis with a Z score below the 7 DS.
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
The results of cerebrospinal fluid analysis, EEG, magnetic resonance imaging of the brain, abdominal ultrasonography and echocardiography were normal. An extensive metabolic workup was performed including very long chain fatty acids, phytanic acid and pristanic acid, acylcarnitine profile, plasma amino acid concentration, plasma alkaline phosphatase, plasma cholesterol and sterol levels, organic acids, copper, ceruloplasmin, PTH, vitamin D dosages, and electrolytes were all within normal ranges. Examination of peripheral blood €t anomaly. smears from these patients has ruled out Pelger-Hue 3.2. Molecular findings We performed homozygosity mapping in two of the affected siblings (II.1 and II.2) and identified 10 shared regions of homozygosity (Additional file 1). We next performed exome sequencing in individual II.1. After mapping and removing duplicate reads, we obtained an average per target base coverage of ~90x, with 95% of the target bases covered at 10x. We identified 5277 homozygous non-synonymous coding, splicing and indels variants in the exome of this individual. Only one rare variant (<1%) was identified in all the homozygosity regions previously mapped. This variant (NBAS: NM_015909: c.409C > T: p.R137W) is absent in our in-house control exome dataset (n ¼ 657) and in 200 Lebanese control chromosomes. This p.R137W variation is reported in the Exome Aggregation Consortium (ExAC) database and has been consequently reported in the dbSNP144 database (rs368085185), but the frequency is extremely low (2/121372, 0.00001648), thereby confirming that it is indeed a rare variant. The Arginine at amino acid position 137 is well conserved among mammals and most vertebrate and the mutated Cytosine (c.409C > T) affects a CpG dinucleotide. Although p.R137W is predicted to be benign by Polyphen2 (http://genetics.bwh.harvard.edu/), several other algorithms including SIFT, Provean (http://provean.jcvi.org/), mutation-taster (http://www.mutationtaster.org/) and UMD-Predictor (umd-predictor.eu) predict a potential damaging effect for this mutation. The mutation was confirmed by Sanger sequencing to be homozygous in the three affected siblings and heterozygous in their parents (Additional file 2; Fig. 1A and B). 4. Discussion We report here a homozygous NBAS missense mutation (NM_015909: c.409C > T: p.R137W) in three siblings from a consanguineous Lebanese family, that presented with recurrent acute liver failure (RALF). Similarly to what is described in the individuals recently reported by Haack et al. (2015) and Garcia Segarra et al. (2015) our patients homozygous for p.R137W in NBAS are affected by RALF. The clinical spectrum of patients with dysfunctions in NBAS is variable. The three individuals described herein share some clinical features with the patients described by Garcia Segarra et al. (2015); particularly, signs that are evocative of abnormal bone morphogenesis including neonatal fractures (Table 1). Interestingly, patient 2 described by Garcia Segarra et al. carry a p.R137W mutation compound heterozygous with another mutation in NBAS, thus indicating that p.R137W is indeed a pathogenic allele (Table 1). NBAS, also known as neuroblastoma amplified (NAG) gene, was identified as a gene that is co-amplified with the N-myc (MYCN) gene in neuroblastoma cell lines (Scott et al., 2003). It encodes a protein with two leucine zipper domains, a ribosomal protein S14 signature domain and a Sec39 like domain, involved in Golgi-to-endoplasmic reticulum retrograde transport. NBAS is a factor of the nonsense-mediated mRNA decay (NMD), which is a highly conserved surveillance pathway that allows the organism to respond to cellular and environmental stress
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(Longman et al., 2013). NMD degrades mRNAs harboring premature termination codons (PTCs), regulates the expression of naturally occurring transcripts, and modulates the phenotypic outcome of several genetic disorders that are caused by nonsense mutations or frameshifts that generate PTCs (Palacios, 2013). Recently, Longman et al. have shown that depletion of NBAS altered the expression of 1444 genes, corresponding to nearly 10% of expressed genes. Several among them are related to embryonic development and cholesterol biosynthesis. Interestingly, the targeted gene that shows the strongest upregulation on NBAS depletion, matrix Gla protein (MGP), acts as an inhibitor of bone formation (Longman et al., 2013). With this in mind, it is tempting to postulate that the skeletal defects phenotypes observed in our patients might be due to increased MGP activity, secondary to impaired NBAS function in our patients as a result of the homozygous mutations in NBAS. Beside these skeletal features, hepatic failure occurred in the patients reported in this study and led to their early death. The implication of NBAS in the liver dysfunction can be explained by the critical role that was attributed to the NMD in the liver development, function and regeneration (Thoren et al., 2010). In addition, it was shown that morpholino-induced depletion of zebrafish Nbas protein leads to severe developmental defects and reduced embryonic viability (Anastasaki et al., 2011), which is in correlation with our clinical findings. A single point mutation in the NBAS gene was shown to cause SOPH (MIM#614800), a human syndrome with short stature, facial dysmorphism, optic nerve atrophy and leucocytes anomaly consisting of hypolobulation of granulocyte nuclei, characteristic of €t anomaly (Maksimova et al., 2010). The family reported Pelger-Hue herein share common features with the one reported by Maksimova et al. Nevertheless, the general gestalt is different and the €t anomaly, brachydactyly, optic nerve atroabsence of Pelger-Hue phy, achromatopsia in addition to hepatic failure in our patients allow a simple differentiation (Table 1). The p. R1914H mutation identified in patients with SOPH lies in the C-terminal end of NBAS, whereas all the pathogenic alleles identified in individuals with RALF are rather found in the N-terminal end of NBAS (Garcia Segarra et al., 2015). Interestingly, seven of these mutations affect the quinoprotein amine dehydrogenase beta chain like domain of NBAS, suggesting that it might play a functional role in the pathophysiology of RALF. The complexity and severity of the clinical presentations associated with NBAS mutations is mainly due to the involvement of NBAS in different physiological functions. 5. Conclusion Similar to Haack et al. (2015) and Garcia Segarra et al., 2015 we identified biallelic mutations in NBAS in patients that presented severe hepatic failure. Ranges of comorbidities are also found in individuals with dysfunction in NBAS, for example some of the patients described by Haack et al., also suffered from renal problems, cardiomyopathy, gastrointestinal autoimmune defects and neurologic phenotypes such as epilepsy. Additionally to liver disease, our patients also show other manifestations including wringarbane kled skin, spontaneous fractures and osteoporosis (Me et al., 2008). Overall, our findings confirm the involvement of NBAS in the condition described by Haack et al. and help expand its phenotypical spectrum. Authors' contribution Performed clinical investigation: A.M, C.C. Conceived and designed the experiments: A.M, Z.K J.M, V.D. Analyzed the data: J.M.C, Z.K, A.M. Contributed reagents/materials/analysis tools: V.D,
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Table 1 Clinical features of patients with pathogenic mutations in NBAS.
Capo-chichi et al, 2015 (this manuscript)
Haack et al., 2015
Maksimova et al., 2010
Origin
Sex
NBAS mutation (NM_015909.3, NP_056993.2)
AO
RALF
Dismorphic features
Other clinic findings
II.1
Lebanese
M
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp
12 m
Yes
DD, weight (0 SD), height (þ0.8 SD), OFC (0 SD) wrinkled skin, axial hypotonia, bone fractures, hepato-splenomegaly
II.2
F
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp
5m
Yes
Pointed chin gum hypertrophy, prominent forehead, large fontanels, prominent eyes, short neck slightly down-slanting palpebral fissures, hypertelorism, telecanthus, long eyelashes, Pectus excavatum, and to other features observed in II.1
II.3
M
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp c.284C > T, p.Ala95Val/c.850A > T, p.Lys284*
4m
Yes
Same as II.1
4.5 m
Yes
DD, normal stature, hypotonia, bone fractures, hepatosplenomegaly, cutis laxa Same as II.1
Patient 1
Swiss
M
Patient 2
American
F
c.409C > T, p.Arg137Trp/c.1186T > A, p.Trp396Arg
6m
Yes
Pointed chin, proptosis, triangular face, prominent eyes, narrow forehead, pectus excavatum, prominent abdomen Pointed chin, proptosis
F1:II.1
German
F
21 m
Yes
-
F2:II.1
German
F
7m
Yes
-
Acute renal failure, epilepsy
F3:II.1
German
F
10 m
Yes
-
Celiac disease
F4:II.3
German
F
8m
Yes
-
-
F5:II.2
German
M
7m
Yes
-
Cardiomyopathy
F6:II.1
German
M
18 m
Yes
-
-
F7:II.1 F7:II.2 F8:II.2
German German
F F M
c.558_560del, p.Ile187del/c.686dup, p.Ser230Glnfs*4 c.2708T > G, p.Leu903Arg/c.2708T > G, p.Leu903Arg c.603_605del, p. Leu202del/c.3164T > C, p.Leu1055Pro c.2708T > G, p.Leu903Arg/c.2827G > T, Glu943* c.3010C > T, p.Arg1004*/c.3164T > C, p.Leu1055Pro c.1533_1545del, p.Ile512Thrfs*4/ c.2951T > G, p.Ile984Ser c.1042C > T, p.Pro348Ser/c.2203_3C > G
Short stature (3 SD), skeletal dysplasia, hepatomegaly, OA, retinal dystrophy, PHA, loose skin DD, short stature (3.5 SD), skeletal dysplasia, hepatomegaly, bone fractures, PHA, loose skin -
11 m 6 8/12 y 21
Yes Yes Yes
-
Erythema nodosum, Crohn's disease -
F9:II.2 F10:II.1
German German
F F
18 4
Yes Yes
-
-
*
Yakut
N/A
N/A
No
Narrow forehead, short neck,
Short stature, PHA, OA, hypotonia, loose skin,
c.1187G > A, p.Trp396/c.2330C > A, p.Pro777His c.118_2A > G/c.2524G > T/p.Val842Phe c.686dup, p.Ser230Glnfs*4/c.3164T > C, p.Leu1055Pro c.5741G > A, p.Arg1914His/c.5741G > A, p.Arg1914His
€t anomaly, OA: Optic nerve Atrophy, OFC: Occipital Frontal Circumference, -: not reported, Male, F: Female, AO: Age at Onset, m: months, y: years, RALF: Recurrent Acute Liver Failure, DD: developmental delay, PHA: Pelger-Hue N/A: not applicable,* Yakut patients described by Maksimova et al., are summarized here. Major clinical features common to these individuals are listed.
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
Garcia Segarra et al., 2015
Patient
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
F.H, C.M, I.K. Wrote the paper: A.M, J.M.C, Z.K, C.M. Competing interests All authors have approved the submission of the manuscript and declare that there is no conflict of interest. Acknowledgments We would like to thank all participants in this study. J.M.C. holds seau de Me decine Ge ne tique Applique e a salary award from the Re bec (RMGA). We also thank the members of the RMGA du Que bioinformatics team (Alexandre Dionne-Laporte, Dan Spiegelman, Edouard Henrion Edouard Henrion, and Ousmane Diallo) for the primary analysis of the exome sequencing data. Funding this work was supported by March of Dimes (grant no. 12-FY10e236). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2015.11.005. References ceres, J.F., 2011. Dhx34 and Anastasaki, C., Longman, D., Capper, A., Patton, E.E., Ca NBAs function in the NMD pathway and are required for embryonic development in zebrafish. Nucleic Acids Res. 39, 3686e3694. http://dx.doi.org/10.1093/ nar/gkq1319. Garcia Segarra, N., Ballhausen, D., Crawford, H., Perreau, M., Campos-Xavier, B., van Spaendonck-Zwarts, K., Vermeer, C., Russo, M., Zambelli, P.-Y., Stevenson, B., Royer-Bertrand, B., Rivolta, C., Candotti, F., Unger, S., Munier, F.L., Superti, L., 2015. NBAS mutations cause a multisystem disorder Furga, A., Bonafe involving bone, connective tissue, liver, immune system, and retina. Am. J. Med.
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Genet. A. http://dx.doi.org/10.1002/ajmg.a.37338. €pke, M.G., Straub, B.K., Ko € lker, S., Thiel, C., Freisinger, P., Haack, T.B., Staufner, C., Ko Baric, I., McKiernan, P.J., Dikow, N., Harting, I., Beisse, F., Burgard, P., Kotzaeridou, U., Kühr, J., Himbert, U., Taylor, R.W., Distelmaier, F., Vockley, J., Ghaloul-Gonzalez, L., Zschocke, J., Kremer, L.S., Graf, E., Schwarzmayr, T., Bader, D.M., Gagneur, J., Wieland, T., Terrile, C., Strom, T.M., Meitinger, T., Hoffmann, G.F., Prokisch, H., 2015. Biallelic mutations in NBAS cause recurrent acute liver failure with onset in infancy. Am. J. Hum. Genet. 97, 163e169. http:// dx.doi.org/10.1016/j.ajhg.2015.05.009. ceres, J.F., Longman, D., Hug, N., Keith, M., Anastasaki, C., Patton, E.E., Grimes, G., Ca 2013. DHX34 and NBAS form part of an autoregulatory NMD circuit that regulates endogenous RNA targets in human cells, zebrafish and Caenorhabditis elegans. Nucleic Acids Res. 41, 8319e8331. http://dx.doi.org/10.1093/nar/ gkt585. Maksimova, N., Hara, K., Nikolaeva, I., Chun-Feng, T., Usui, T., Takagi, M., Nishihira, Y., Miyashita, A., Fujiwara, H., Oyama, T., Nogovicina, A., Sukhomyasova, A., Potapova, S., Kuwano, R., Takahashi, H., Nishizawa, M., Onodera, O., 2010. Neuroblastoma amplified sequence gene is associated with a novel short stature syndrome characterised by optic nerve atrophy and Pelger€t anomaly. J. Med. Genet. 47, 538e548. http://dx.doi.org/10.1136/ Hue jmg.2009.074815. garbane , A., Samaras, L., Che did, R., Chouery, E., Chre tien, D., Caillaud, C., AbouMe Ghoch, J., Jalkh, N., 2008. Developmental delay, dysmorphic features, neonatal spontaneous fractures, wrinkled skin, and hepatic failure: a new metabolic syndrome? Am. J. Med. Genet. A 146A, 3198e3201. http://dx.doi.org/10.1002/ ajmg.a.32579. Palacios, I.M., 2013. Nonsense-mediated mRNA decay: from mechanistic insights to impacts on human health. Brief. Funct. Genomics 12, 25e36. http://dx.doi.org/ 10.1093/bfgp/els051. Scott, D.K., Board, J.R., Lu, X., Pearson, A.D.J., Kenyon, R.M., Lunec, J., 2003. The neuroblastoma amplified gene, NAG: genomic structure and characterisation of the 7.3 kb transcript predominantly expressed in neuroblastoma. Gene 307, 1e11. Thoren, L.A., Nørgaard, G.A., Weischenfeldt, J., Waage, J., Jakobsen, J.S., Damgaard, I., €m, F.C., Blom, A.M., Borup, R., Bisgaard, H.C., Porse, B.T., 2010. UPF2 is a Bergstro critical regulator of liver development, function and regeneration. PloS One 5, e11650. http://dx.doi.org/10.1371/journal.pone.0011650. Wang, K., Li, M., Hakonarson, H., 2010. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164. http://dx.doi.org/10.1093/nar/gkq603.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
Neuroblastoma Amplified Sequence (NBAS) mutation in recurrent acute liver failure: Confirmatory report in a sibship with very early onset, osteoporosis and developmental delay -Mario Capo-Chichi a, Cybel Mehawej b, 1, Valerie Delague c, d, Catherine Caillaud e, Jose Issam Khneisser b, Fadi F. Hamdan a, Jacques L. Michaud a, f, Zoha Kibar a, Me garbane b , g, * Andre a
CHU Sainte-Justine Research Center, Montreal H3T 1C5, Canada Unit e de G en etique M edicale, Facult e de M edecine, Universit e Saint-Joseph, Beirut, Lebanon c Inserm, UMR_S 910, 13385 Marseille, France d Aix Marseille Universit e, GMGF, 13385 Marseille, France e ^pital Necker Enfants Malades, Paris 75015, France Service de Biochimie M edicale, Ho f Department of Pediatrics and Department of Neurosciences, Universit e de Montreal, Montreal, Canada g ^me Lejeune, Paris, France Institut J ero b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2015 Received in revised form 6 November 2015 Accepted 7 November 2015 Available online 11 November 2015
Background: Recently, biallelic mutations in the Neuroblastoma Amplified Sequence NBAS gene have been identified in ten patients that present recurrent acute liver failure (RALF) in early infancy. In addition to severe liver dysfunction, some of these individuals also suffered from other comorbidities including cardiomyopathy, neurologic phenotypes and gastrointestinal immune defects. Here we report on a consanguineous Lebanese family with three siblings affected by RALF. Of note, neonatal spontaneous fractures, developmental delay, prominent eyes, generalized hirsutism, gum hypertrophy, and hepatosplenomegaly were also present. Methods: Whole-genome SNP genotyping in all the patients, followed by exome sequencing was performed in one of the affected siblings. Results: A homozygous c.409C > T (p.Arg137Trp) missense mutation in NBAS was identified in all patients. Conclusion: Overall, our findings confirm the involvement of NBAS in the pathogenesis of this condition characterized by severe liver dysfunction and help expand its phenotypical spectrum. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Recurrent acute liver failure Spontaneous bone fractures NBAS
1. Background Whole exome sequencing (WES) is nowadays an important tool to elucidate the molecular origin of rare disorders. We used this
de Ge n e tique Me dicale, Faculte de Me decine, * Corresponding author. Unite Saint-Joseph, 42, Rue de Grenelle, 75007 Paris, France. Universite E-mail addresses: [email protected] (J.-M. Capo-Chichi), cybel.mehawej@ usj.edu.lb (C. Mehawej), [email protected] (V. Delague), catherine. [email protected] (C. Caillaud), [email protected] (I. Khneisser), fadi. [email protected] (F.F. Hamdan), [email protected] (J.L. Michaud), [email protected] (Z. Kibar), garbane ). [email protected], [email protected] (A. Me 1 Current address: Division of Immunology, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA. http://dx.doi.org/10.1016/j.ejmg.2015.11.005 1769-7212/© 2015 Elsevier Masson SAS. All rights reserved.
approach to study three siblings born to consanguineous Lebanese parents and presenting a condition characterized by recurrent acute liver failure in early infancy, neonatal spontaneous fractures and developmental delay and hepato-splenomegaly. To our knowledge this condition has never been reported before. 2. Subjects and methods 2.1. Subjects The affected individuals (II.1e3, Fig. 1A) are three siblings born to a consanguineous Lebanese couple in good health. Blood samples were obtained from each of these individuals after informed consent and the approval of the appropriate institutional ethic
638
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
Fig. 1. Identification of a homozygous mutation in NBAS in a consanguineous family affected with developmental delay, dysmorphic features, neonatal spontaneous fractures, wrinkled skin, and hepatic failure. (A) Pedigree of the consanguineous Lebanese family studied here. (B) Sanger sequencing confirming that the c.409C > T/p.R137W (NM_015909) mutation in NBAS segregates in this family. Representative chromatograms of the three affected siblings (homozygous for c.409C > T) and their parents (heterozygous for c.409C > T) are shown. (C) Multiple alignments of NBAS showing residues of the predicted quinoprotein aminedehydrogenase beta chain like domain around the p.R137W mutation. Alignments were obtained from reference sequences of NBAS orthologues. R137 residue is identified in bold. (D) Schematic representation of NBAS showing the predicted WD40 repeats and secretory pathway sec39 motif. (Sources: Uniprot entry A2RRP1, HGNC, NCBI gene). In red, p.R137W identified herein affects the quinoprotein aminedehydrogenase beta chain €t anomaly (Maksimova et al., 2010) maps toward the like domain in N-terminal end of NBAS. In black, p.R1914H associated to the short stature, optic nerve atrophy and Pelger-Hue C-terminal end of NBAS.
review board. DNA was extracted then amplified using standard methods. 2.2. Homozygosity mapping Genomic blood DNA (2.5 mg) from individuals II.1 and II.2 was used for whole genome genotyping on the Illumina Human Omni 2.5 Chip at the McGill University and Genome Quebec Innovation Center (MUGQIC, Montreal, Canada). Genotyping reactions were performed according to the manufacturer's instructions. Homozygosity mapping was performed using PLink (v.1.06). Shared regions of homozygosity (HR) containing >30 consecutive homozygous SNPs and extending over 1 Mb were retained. 2.3. Exome capture and sequencing The exome of individual II.1 was captured from 3 mg of blood genomic DNA, using the Agilent SureSelect Human All Exon Capture 50 Mb Kit (Mississauga, ON), and sequenced (paired-end, 2x100 bp) using the Illumina Hiseq 2000 at the MUGQIC. Sequence processing, alignment, and variant calling were done using the Broad Institute Genome Analysis Tool Kit (GATK v2.2) and annotated using Annovar (Wang et al., 2010). 3. Results 3.1. Clinical findings The two elder siblings (II-1 and II-2) were previously reported garbane et al. 2008. Briefly, they had axial hypotonia, by Me developmental delay, hirsutism, large fontanels with delayed closure, dysmorphic facial features that consist of frontal bossing,
prominent eyes, slightly down-slanting palpebral fissures, hypertelorism, telecanthus, long eyelashes, and progressive gum hypertrophy, short neck, pointed chin, abnormal thoracic configuration, wrinkled skin on the hands and abdomen, progressive hepatosplenomegaly and neonatal spontaneous fractures. Liver function deteriorated during intercurrent infections and both siblings died at the age of 18 months and 3e8/12 years respectively. Liver biopsy performed in the eldest child showed steatosis without fibrosis. Laboratory exams were normal except for liver enzymes that were garbane et al., 2008). During the last years, a third elevated (Me child, a boy, was born. Pregnancy was normal and delivery was at 35 weeks of gestation. According to the parents, weight, length and OFC were in the normal limits. Like his other siblings, his major complains were overall health and liver enzymes deterioration every time he had fever. Indeed, liver dysfunction started at the age of 4 months with his first sickness episode; it was mild but got more severe leading to the death of this boy at the age of 2 years. Clinically, this patient had almost the same clinical evolution and dysmorphic features as his affected siblings. He was hypotonic, had feeding difficulties, a failure to thrive, delayed developmental milestones, as he could only sit at his own at the age of 8 months and made his first steps without help at the age of 20 months. He also had hirsutism, large fontanels with delayed closure, frontal bossing, prominent eyes, slightly down-slanting palpebral fissures, hypertelorism, long eyelashes, and gum hypertrophy, short neck, pointed chin, abnormal thoracic configuration, wrinkled skin on the hands, and hepato-splenomegaly. No fractures were identified after a total body x-rays exam. Nevertheless, as he used to cry whenever the parents hold him and because of the presence of spontaneous fractures in his other siblings, an osteodensitometry was performed and revealed a severe osteoporosis with a Z score below the 7 DS.
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The results of cerebrospinal fluid analysis, EEG, magnetic resonance imaging of the brain, abdominal ultrasonography and echocardiography were normal. An extensive metabolic workup was performed including very long chain fatty acids, phytanic acid and pristanic acid, acylcarnitine profile, plasma amino acid concentration, plasma alkaline phosphatase, plasma cholesterol and sterol levels, organic acids, copper, ceruloplasmin, PTH, vitamin D dosages, and electrolytes were all within normal ranges. Examination of peripheral blood €t anomaly. smears from these patients has ruled out Pelger-Hue 3.2. Molecular findings We performed homozygosity mapping in two of the affected siblings (II.1 and II.2) and identified 10 shared regions of homozygosity (Additional file 1). We next performed exome sequencing in individual II.1. After mapping and removing duplicate reads, we obtained an average per target base coverage of ~90x, with 95% of the target bases covered at 10x. We identified 5277 homozygous non-synonymous coding, splicing and indels variants in the exome of this individual. Only one rare variant (<1%) was identified in all the homozygosity regions previously mapped. This variant (NBAS: NM_015909: c.409C > T: p.R137W) is absent in our in-house control exome dataset (n ¼ 657) and in 200 Lebanese control chromosomes. This p.R137W variation is reported in the Exome Aggregation Consortium (ExAC) database and has been consequently reported in the dbSNP144 database (rs368085185), but the frequency is extremely low (2/121372, 0.00001648), thereby confirming that it is indeed a rare variant. The Arginine at amino acid position 137 is well conserved among mammals and most vertebrate and the mutated Cytosine (c.409C > T) affects a CpG dinucleotide. Although p.R137W is predicted to be benign by Polyphen2 (http://genetics.bwh.harvard.edu/), several other algorithms including SIFT, Provean (http://provean.jcvi.org/), mutation-taster (http://www.mutationtaster.org/) and UMD-Predictor (umd-predictor.eu) predict a potential damaging effect for this mutation. The mutation was confirmed by Sanger sequencing to be homozygous in the three affected siblings and heterozygous in their parents (Additional file 2; Fig. 1A and B). 4. Discussion We report here a homozygous NBAS missense mutation (NM_015909: c.409C > T: p.R137W) in three siblings from a consanguineous Lebanese family, that presented with recurrent acute liver failure (RALF). Similarly to what is described in the individuals recently reported by Haack et al. (2015) and Garcia Segarra et al. (2015) our patients homozygous for p.R137W in NBAS are affected by RALF. The clinical spectrum of patients with dysfunctions in NBAS is variable. The three individuals described herein share some clinical features with the patients described by Garcia Segarra et al. (2015); particularly, signs that are evocative of abnormal bone morphogenesis including neonatal fractures (Table 1). Interestingly, patient 2 described by Garcia Segarra et al. carry a p.R137W mutation compound heterozygous with another mutation in NBAS, thus indicating that p.R137W is indeed a pathogenic allele (Table 1). NBAS, also known as neuroblastoma amplified (NAG) gene, was identified as a gene that is co-amplified with the N-myc (MYCN) gene in neuroblastoma cell lines (Scott et al., 2003). It encodes a protein with two leucine zipper domains, a ribosomal protein S14 signature domain and a Sec39 like domain, involved in Golgi-to-endoplasmic reticulum retrograde transport. NBAS is a factor of the nonsense-mediated mRNA decay (NMD), which is a highly conserved surveillance pathway that allows the organism to respond to cellular and environmental stress
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(Longman et al., 2013). NMD degrades mRNAs harboring premature termination codons (PTCs), regulates the expression of naturally occurring transcripts, and modulates the phenotypic outcome of several genetic disorders that are caused by nonsense mutations or frameshifts that generate PTCs (Palacios, 2013). Recently, Longman et al. have shown that depletion of NBAS altered the expression of 1444 genes, corresponding to nearly 10% of expressed genes. Several among them are related to embryonic development and cholesterol biosynthesis. Interestingly, the targeted gene that shows the strongest upregulation on NBAS depletion, matrix Gla protein (MGP), acts as an inhibitor of bone formation (Longman et al., 2013). With this in mind, it is tempting to postulate that the skeletal defects phenotypes observed in our patients might be due to increased MGP activity, secondary to impaired NBAS function in our patients as a result of the homozygous mutations in NBAS. Beside these skeletal features, hepatic failure occurred in the patients reported in this study and led to their early death. The implication of NBAS in the liver dysfunction can be explained by the critical role that was attributed to the NMD in the liver development, function and regeneration (Thoren et al., 2010). In addition, it was shown that morpholino-induced depletion of zebrafish Nbas protein leads to severe developmental defects and reduced embryonic viability (Anastasaki et al., 2011), which is in correlation with our clinical findings. A single point mutation in the NBAS gene was shown to cause SOPH (MIM#614800), a human syndrome with short stature, facial dysmorphism, optic nerve atrophy and leucocytes anomaly consisting of hypolobulation of granulocyte nuclei, characteristic of €t anomaly (Maksimova et al., 2010). The family reported Pelger-Hue herein share common features with the one reported by Maksimova et al. Nevertheless, the general gestalt is different and the €t anomaly, brachydactyly, optic nerve atroabsence of Pelger-Hue phy, achromatopsia in addition to hepatic failure in our patients allow a simple differentiation (Table 1). The p. R1914H mutation identified in patients with SOPH lies in the C-terminal end of NBAS, whereas all the pathogenic alleles identified in individuals with RALF are rather found in the N-terminal end of NBAS (Garcia Segarra et al., 2015). Interestingly, seven of these mutations affect the quinoprotein amine dehydrogenase beta chain like domain of NBAS, suggesting that it might play a functional role in the pathophysiology of RALF. The complexity and severity of the clinical presentations associated with NBAS mutations is mainly due to the involvement of NBAS in different physiological functions. 5. Conclusion Similar to Haack et al. (2015) and Garcia Segarra et al., 2015 we identified biallelic mutations in NBAS in patients that presented severe hepatic failure. Ranges of comorbidities are also found in individuals with dysfunction in NBAS, for example some of the patients described by Haack et al., also suffered from renal problems, cardiomyopathy, gastrointestinal autoimmune defects and neurologic phenotypes such as epilepsy. Additionally to liver disease, our patients also show other manifestations including wringarbane kled skin, spontaneous fractures and osteoporosis (Me et al., 2008). Overall, our findings confirm the involvement of NBAS in the condition described by Haack et al. and help expand its phenotypical spectrum. Authors' contribution Performed clinical investigation: A.M, C.C. Conceived and designed the experiments: A.M, Z.K J.M, V.D. Analyzed the data: J.M.C, Z.K, A.M. Contributed reagents/materials/analysis tools: V.D,
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Table 1 Clinical features of patients with pathogenic mutations in NBAS.
Capo-chichi et al, 2015 (this manuscript)
Haack et al., 2015
Maksimova et al., 2010
Origin
Sex
NBAS mutation (NM_015909.3, NP_056993.2)
AO
RALF
Dismorphic features
Other clinic findings
II.1
Lebanese
M
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp
12 m
Yes
DD, weight (0 SD), height (þ0.8 SD), OFC (0 SD) wrinkled skin, axial hypotonia, bone fractures, hepato-splenomegaly
II.2
F
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp
5m
Yes
Pointed chin gum hypertrophy, prominent forehead, large fontanels, prominent eyes, short neck slightly down-slanting palpebral fissures, hypertelorism, telecanthus, long eyelashes, Pectus excavatum, and to other features observed in II.1
II.3
M
c.409C > T, p.Arg137Trp/c.409C > T, p.Arg137Trp c.284C > T, p.Ala95Val/c.850A > T, p.Lys284*
4m
Yes
Same as II.1
4.5 m
Yes
DD, normal stature, hypotonia, bone fractures, hepatosplenomegaly, cutis laxa Same as II.1
Patient 1
Swiss
M
Patient 2
American
F
c.409C > T, p.Arg137Trp/c.1186T > A, p.Trp396Arg
6m
Yes
Pointed chin, proptosis, triangular face, prominent eyes, narrow forehead, pectus excavatum, prominent abdomen Pointed chin, proptosis
F1:II.1
German
F
21 m
Yes
-
F2:II.1
German
F
7m
Yes
-
Acute renal failure, epilepsy
F3:II.1
German
F
10 m
Yes
-
Celiac disease
F4:II.3
German
F
8m
Yes
-
-
F5:II.2
German
M
7m
Yes
-
Cardiomyopathy
F6:II.1
German
M
18 m
Yes
-
-
F7:II.1 F7:II.2 F8:II.2
German German
F F M
c.558_560del, p.Ile187del/c.686dup, p.Ser230Glnfs*4 c.2708T > G, p.Leu903Arg/c.2708T > G, p.Leu903Arg c.603_605del, p. Leu202del/c.3164T > C, p.Leu1055Pro c.2708T > G, p.Leu903Arg/c.2827G > T, Glu943* c.3010C > T, p.Arg1004*/c.3164T > C, p.Leu1055Pro c.1533_1545del, p.Ile512Thrfs*4/ c.2951T > G, p.Ile984Ser c.1042C > T, p.Pro348Ser/c.2203_3C > G
Short stature (3 SD), skeletal dysplasia, hepatomegaly, OA, retinal dystrophy, PHA, loose skin DD, short stature (3.5 SD), skeletal dysplasia, hepatomegaly, bone fractures, PHA, loose skin -
11 m 6 8/12 y 21
Yes Yes Yes
-
Erythema nodosum, Crohn's disease -
F9:II.2 F10:II.1
German German
F F
18 4
Yes Yes
-
-
*
Yakut
N/A
N/A
No
Narrow forehead, short neck,
Short stature, PHA, OA, hypotonia, loose skin,
c.1187G > A, p.Trp396/c.2330C > A, p.Pro777His c.118_2A > G/c.2524G > T/p.Val842Phe c.686dup, p.Ser230Glnfs*4/c.3164T > C, p.Leu1055Pro c.5741G > A, p.Arg1914His/c.5741G > A, p.Arg1914His
€t anomaly, OA: Optic nerve Atrophy, OFC: Occipital Frontal Circumference, -: not reported, Male, F: Female, AO: Age at Onset, m: months, y: years, RALF: Recurrent Acute Liver Failure, DD: developmental delay, PHA: Pelger-Hue N/A: not applicable,* Yakut patients described by Maksimova et al., are summarized here. Major clinical features common to these individuals are listed.
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Garcia Segarra et al., 2015
Patient
J.-M. Capo-Chichi et al. / European Journal of Medical Genetics 58 (2015) 637e641
F.H, C.M, I.K. Wrote the paper: A.M, J.M.C, Z.K, C.M. Competing interests All authors have approved the submission of the manuscript and declare that there is no conflict of interest. Acknowledgments We would like to thank all participants in this study. J.M.C. holds seau de Me decine Ge ne tique Applique e a salary award from the Re bec (RMGA). We also thank the members of the RMGA du Que bioinformatics team (Alexandre Dionne-Laporte, Dan Spiegelman, Edouard Henrion Edouard Henrion, and Ousmane Diallo) for the primary analysis of the exome sequencing data. Funding this work was supported by March of Dimes (grant no. 12-FY10e236). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2015.11.005. References ceres, J.F., 2011. Dhx34 and Anastasaki, C., Longman, D., Capper, A., Patton, E.E., Ca NBAs function in the NMD pathway and are required for embryonic development in zebrafish. Nucleic Acids Res. 39, 3686e3694. http://dx.doi.org/10.1093/ nar/gkq1319. Garcia Segarra, N., Ballhausen, D., Crawford, H., Perreau, M., Campos-Xavier, B., van Spaendonck-Zwarts, K., Vermeer, C., Russo, M., Zambelli, P.-Y., Stevenson, B., Royer-Bertrand, B., Rivolta, C., Candotti, F., Unger, S., Munier, F.L., Superti, L., 2015. NBAS mutations cause a multisystem disorder Furga, A., Bonafe involving bone, connective tissue, liver, immune system, and retina. Am. J. Med.
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Genet. A. http://dx.doi.org/10.1002/ajmg.a.37338. €pke, M.G., Straub, B.K., Ko € lker, S., Thiel, C., Freisinger, P., Haack, T.B., Staufner, C., Ko Baric, I., McKiernan, P.J., Dikow, N., Harting, I., Beisse, F., Burgard, P., Kotzaeridou, U., Kühr, J., Himbert, U., Taylor, R.W., Distelmaier, F., Vockley, J., Ghaloul-Gonzalez, L., Zschocke, J., Kremer, L.S., Graf, E., Schwarzmayr, T., Bader, D.M., Gagneur, J., Wieland, T., Terrile, C., Strom, T.M., Meitinger, T., Hoffmann, G.F., Prokisch, H., 2015. Biallelic mutations in NBAS cause recurrent acute liver failure with onset in infancy. Am. J. Hum. Genet. 97, 163e169. http:// dx.doi.org/10.1016/j.ajhg.2015.05.009. ceres, J.F., Longman, D., Hug, N., Keith, M., Anastasaki, C., Patton, E.E., Grimes, G., Ca 2013. DHX34 and NBAS form part of an autoregulatory NMD circuit that regulates endogenous RNA targets in human cells, zebrafish and Caenorhabditis elegans. Nucleic Acids Res. 41, 8319e8331. http://dx.doi.org/10.1093/nar/ gkt585. Maksimova, N., Hara, K., Nikolaeva, I., Chun-Feng, T., Usui, T., Takagi, M., Nishihira, Y., Miyashita, A., Fujiwara, H., Oyama, T., Nogovicina, A., Sukhomyasova, A., Potapova, S., Kuwano, R., Takahashi, H., Nishizawa, M., Onodera, O., 2010. Neuroblastoma amplified sequence gene is associated with a novel short stature syndrome characterised by optic nerve atrophy and Pelger€t anomaly. J. Med. Genet. 47, 538e548. http://dx.doi.org/10.1136/ Hue jmg.2009.074815. garbane , A., Samaras, L., Che did, R., Chouery, E., Chre tien, D., Caillaud, C., AbouMe Ghoch, J., Jalkh, N., 2008. Developmental delay, dysmorphic features, neonatal spontaneous fractures, wrinkled skin, and hepatic failure: a new metabolic syndrome? Am. J. Med. Genet. A 146A, 3198e3201. http://dx.doi.org/10.1002/ ajmg.a.32579. Palacios, I.M., 2013. Nonsense-mediated mRNA decay: from mechanistic insights to impacts on human health. Brief. Funct. Genomics 12, 25e36. http://dx.doi.org/ 10.1093/bfgp/els051. Scott, D.K., Board, J.R., Lu, X., Pearson, A.D.J., Kenyon, R.M., Lunec, J., 2003. The neuroblastoma amplified gene, NAG: genomic structure and characterisation of the 7.3 kb transcript predominantly expressed in neuroblastoma. Gene 307, 1e11. Thoren, L.A., Nørgaard, G.A., Weischenfeldt, J., Waage, J., Jakobsen, J.S., Damgaard, I., €m, F.C., Blom, A.M., Borup, R., Bisgaard, H.C., Porse, B.T., 2010. UPF2 is a Bergstro critical regulator of liver development, function and regeneration. PloS One 5, e11650. http://dx.doi.org/10.1371/journal.pone.0011650. Wang, K., Li, M., Hakonarson, H., 2010. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164. http://dx.doi.org/10.1093/nar/gkq603.