docMolecular Genetics and Metabolism
download 
Reclamo Transcript
Molecular Genetics and Metabolism
Molecular Genetics and Metabolism 96 (2009) 44–49 Contents lists available at ScienceDirect Molecular Genetics and Metabolism j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y m g m e Citrin deficiency, a perplexing global disorder David Dimmock a,1,2, Bruno Maranda b,2, Carlo Dionisi-Vici c, Jing Wang a, Soledad Kleppe d, Giuseppe Fiermonte e, Renkui Bai a, Bryan Hainline f, Ada Hamosh g, William E. O’Brien a, Fernando Scaglia a,*, Lee-Jun Wong a a Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, BCM225, Houston, TX 77030, USA Service de Génétique Médicale, Centre Hospitalier Universitaire de Québec, Université Laval, Ste-Foy, Québec, Canada c Division of Metabolism, Children’s Hospital Bambino Gesù, Rome, Italy d Division Genetica, CEMIC, Buenos Aires, Argentina e Department of Pharmaco-Biology, University of Bari, Bari, Italy f Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA g Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA b a r t i c l e i n f o Article history: Received 2 August 2008 and in revised form 14 October 2008 Accepted 14 October 2008 Available online 25 November 2008 Keywords: Citrullinemia CTLN2 NICCD Therapy Newborn screening Intrahepatic cholestasis Bipolar disorder Hepatic steatosis a b s t r a c t Citrin deficiency, caused by mutations in SLC25A13, can present with neonatal intrahepatic cholestasis or with adult onset neuropsychiatric, hepatic and pancreatic disease. Until recently, it had been thought to be found mostly in individuals of East Asian ancestry. A key diagnostic feature has been the deficient arginino succinate synthetase (ASS) activity (E.C. 6.3.4.5) in liver, with normal activity in skin fibroblasts. In this series we describe the clinical presentation of 10 patients referred to our laboratories for sequence analysis of the SCL25A13 gene, including several patients who presented with elevated citrulline on newborn screening. In addition to sequence analysis performed on all patients, ASS enzyme activity, citrulline incorporation and Western blot analysis for ASS and citrin were performed on skin fibroblasts if available. We have found 5 unre ported mutations including two apparent founder mutations in three unrelated French-Canadian patients. In marked contrast to previous cases, these patients have a markedly reduced ASS activity in skin fibroblasts. The presence of citrin protein on Western blot in three of our cases reduces the sensitivity of a screening test based on protein immunoblotting. The finding of citrin mutations in patients of Arabic, Pakistani, French Canadian and Northern European origins supports the concept that citrin deficiency is a panethnic disease. © 2008 Published by Elsevier Inc. Introduction Citrin, the mitochondrial aspartate/glutamate carrier (AGC2) plays a significant role in nitrogen metabolism by virtue of its shuttle activity. Deficiency of this protein has previously been described as the cause of citrullinemia type 2 (CTLN2) [1]. CTLN2 was initially described as an adult onset hepatic and neurological disorder in individuals of Japanese ancestry. CTLN2, was originally differentiated from classical citrullinemia by a tissue specific defi ciency of argininosuccinate synthetase (ASS) activity (E.C. 6.3.4.5) in the liver but normal enzymatic activity in renal tissue or skin fibroblast culture [2]. Citrin deficiency has subsequently been shown to be a cause of neonatal intrahepatic cholestasis through mechanisms that are not well understood [3,4]. Although initially reported among individuals of East Asian ancestry, several recent reports have described individuals with liver disease secondary to citrin deficiency from other ethnic groups [5–8]. Citrin deficiency is a dif ficult metabolic disorder to reliably distinguish from other causes of hepatic disease, particularly as the characteristic plasma amino acid profile is not consistently present [9–11]. However, in Taiwan, citrin deficiency has been shown to be the leading cause of hepatic steatosis in infants [11]. Therefore, rapid and inexpensive diagnostic testing is of significant value in evaluating children with hepatic steatosis or intrahepatic cholestasis. Consequently, more recent efforts have focused on using Western blot to diagnose patients, as all mutations reported to date have resulted in no detectable protein [12]. Methods Sequencing * Corresponding author. Fax: +1 832 825 4294. E-mail address: fsca[email protected] (F. Scaglia). 1 Current address: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA. 2 Joint first authors. Clinical samples on patients with suspected citrin deficiency were referred to the Medical Genetics laboratories at Baylor Col lege of Medicine, Houston, Texas for DNA analysis of the SCL25A13 1096-7192/$ - see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.ymgme.2008.10.007 652 D. Dimmock et al. / Molecular Genetics and Metabolism 96 (2009) 44–49 45 gene (cases 1–6 and 8–10) or in Dr Palmieri’s laboratory at the University of Bari, Italy (case 7) (see Table 1). The entire coding exons and at least 50 bases of the flanking intron regions of the SCL25A13 gene were PCR amplified, followed by automated DNA sequencing in both forward and reverse direc tions using gene specific primers (available upon request) linked to M13 universal sequence primers. The sequencing was performed on an ABI3730XL automated DNA sequencer with Sequencing Anal ysis Software v5.1 (Applied Biosystems, Foster City, CA, USA). DNA sequences were analyzed using Mutation Surveyor version 2.6.1 (SoftGenetics, State College, PA, USA) and the Genebank sequence: NM_014251.1, was used as the reference sequence. In all cases, mutations were confirmed on a second DNA extrac tion. Trans-configuration of mutations was confirmed by sequence analysis of parental DNA samples. To exclude mutations in the ASS1 gene, full sequencing was performed as noted. Research testing was performed according to protocols approved by Baylor College of Medicine Institutional Review Board and with the consent of subjects or legal guardians where appropriate and in line with the Declaration of Helsinki. Clinical details and histo pathology reports were obtained from the referring institutions. Table 1 Summary of ethnicity and mutations in SLC23A13. Western analysis Case 1, a 2 year old female, was born at term with a birth weight of 1767 g to non-consanguineous French-Canadian parents from the Bas-St-Laurent region of Quebec. She presented with an elevated citrulline on urinary newborn screening [15]. She developed jaun dice at 6 weeks with progressive cholestasis. Her aspartate ami notransferase (AST) was modestly elevated at 49 IU/L (reference range: < 34 IU/L) with an increase in gamma glutamyl transpep tidase (GGT) of 1042 IU/L (reference range: <265 IU/L). Her initial plasma amino acids at 48 days of age revealed an elevated methi onine of 90 lM (reference range 9–45 lM) and Citrulline of 235 lM (reference range <41 lM) (Table 2). Her alpha fetoprotein (AFP) was significantly elevated at 30,071 kU/L (reference range: <3395 kU/L). Given the significant elevation in citrulline, skin fibroblasts were sent for ASS assay. This revealed a complete deficiency of ASS activity (0.0 nmol/min/mg protein, normal range 0.8–3.8 nmol/ min/mg protein). Repeat analysis on re-derived cells yielded simi lar results. Clinical sequencing of the ASS1 gene revealed no muta tions. She was managed as a patient with ASS deficiency with a low protein diet and sodium benzoate. Her liver disease persisted with recurrent hypoglycemia, hypoalbuminemia, and failure to thrive. Abdominal ultrasound at one year of age showed an enlarged liver with steatosis and three echogenic foci. Due to the persistent elevation in plasma citrulline (Table 2) and clinical picture of liver disease, a liver biopsy was performed. The pathology was significant for steatosis and giant cell hepati tis. Given the predominant hepatic phenotype, sequence analysis of SCL25A13 was performed, that revealed two novel heterozygous mutations: c.127C > T (p.R43X) and c.1063C > T (p.R355X). These mutations have not been seen in over 300 control chromosomes. She was also heterozygous for a novel variant c.69 + 45 c > g, the previously reported SNPs: c.328 + 6 A > G in cis with the p.R43X mutation, and the c.1194 a > g (p.L398L) in cis with the p.R355X mutation. Western analysis for citrin using the rabbit polyclonal antibody revealed a 37 kDa protein (Fig. 1), and this was replicated when the commercial mouse monoclonal antibody was used (data not shown). The citrulline incorporation ratio was normal (26.99, reference range 3–112). Western analysis of these fibroblasts revealed an almost total absence of ASS protein with increased mRNA levels (Fig. 2). A high-protein and low carbohydrate diet was implemented as previously described [6]. The liver function returned to normal and the infant remains clinically well. Case 2, a 2 year old male, was born at term to non-consanguin eous French-Canadian parents from the Bas-St-Laurent region of Quebec. He presented with an elevated citrulline on urinary Fibroblast cultures were obtained from cases 1, 2 and 7. For cases 1 and 2 these were harvested and washed in phosphate buf fered saline and then briefly sonicated in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Protein concentration was determined by the Bio-Rad Pro tein Assay (BioRad, Hercules, CA). Samples were diluted to 1 mg/ and then 5–20 lg were loaded onto a 12% SDS PAGE gel and trans ferred to 45 lm nitrocellulose membrane. The membrane was sub sequently blocked for 1 h in 5% milk and then incubated overnight in primary antibody, washed and incubated for 1 h with a second ary antibody. Immunoreactive bands were visualized using the ECL system (Amersham Pharmacia). Membranes were then stripped and reprobed with a monoclonal anti-b-actin antibody produced in mouse (Sigma–Aldrich A2228) as a loading control. For case 7, mitochondria were isolated from patient and control fibroblasts using a commercial kit (Pierce) with the Halt protease inhibitors according to the manufacturer’s instructions. An antibody to sub unit IV of the cytochrome c oxidase was used as a loading control. Anti-citrin antibody was produced in rabbit utilizing KLH con jugation against peptide KRADPAELRTIFLK (positions p.10-23 of the precursor protein) by Open Biosystems (Huntsville, AL) according to their standard protocols. This antibody was validated on patient samples previously tested by Dr. Keiko Kobayashi [6]. A commer cial anti-citrin mouse monoclonal antibody raised against amino acid positions 2 to 81 was also evaluated (Genetex GTX95185). Anti-ASS antibody production has been previously described [13] and these results were confirmed using a commercial monoclonal antibody to the C-terminal of ASS (BD 611700). Biochemical studies Plasma amino acid analysis was performed on a Biochrom 30 amino acid analyzer according to manufacturer’s protocols. ASS enzyme activities and citrulline incorporation studies were per formed on cases 1 and 2 as previously described [13,14]. Real time PCR Total RNA was extracted from fibroblasts using TRIZOL ( Invitrogen, Carlsbad, California). Reverse transcription was per formed using iSCRIPT (Biorad, Herculaes, California). Real time PCR was then performed on cDNA using specific primers for ASS1 653 Case Ethnicity Gender Mutation 1 Mutation 2 1 2 3 4 5 6 7 8 9 10 French-Canadian French-Canadian French-Canadian French-Canadian South–East Asian Han Arabic Pakistani Pakistani Northern European Female Male Female Female Female Male Male Male Female Female p.R43X p.R43X p.R43X p.R43X c.1660_c.1661dup23 c.851delGTAT p.A25E c.172_173delGT c.172_173delGT c.848+3a>c p.R355X p.R355X p.R355X p.R43X c.1660_c.1661dup23 c.615+5G>A p.A25E c.172_173delGT c.172_173delGT p.T546R and for the beta 2 microglobulin as a reference. A patient with two missense mutations in ASS1 and a normal fibroblast line were used as controls. Results Case reports 46 D. Dimmock et al. / Molecular Genetics and Metabolism 96 (2009) 44–49 Table 2 Selected plasma amino acid profiles of selected cases in lM. Case Age/days Threonine Serine Glutamine Alanine Citrulline Valine Methionine Isoleucine Leucine Tyrosine Phe Ornithine Histidine Arginine 1 1 1 1 1 1 2 2 2 4 4 5 5 6 8 10 LLN ULN 48 110 160 178 269 563 0 94 186 54 69 14 47 34 11 14 520 167 790 95 249 252 421 138 451 1140 302 640 1208 586 787 796 20 210 84 80 523 107 147 131 110 62 218 404 192 183 444 280 353 145 56 188 94 64 175 113 487 220 135 46 212 118 156 400 300 684 — 326 238 842 102 68 336 154 552 302 319 129 299 408 230 268 398 412 — 183 148 420 235 47 157 2 25 38 492 28 101 948 599 354 1128 530 547 325 2 41 90 79 32 11 221 44 136 22 17 598 634 99 500 210 134 578 9 45 33 50 35 14 48 100 48 — 36 78 39 71 104 76 — 43 10 86 53 28 73 27 88 203 89 11 79 131 57 133 190 156 — 63 30 142 36 31 83 38 56 80 134 32 48 103 59 160 124 71 — 24 23 79 108 55 380 44 80 56 126 36 191 346 163 237 368 91 304 97 5 129 63 45 176 26 88 198 88 48 126 147 103 155 174 77 — 118 37 97 64 7 162 51 179 311 142 47 183 219 119 212 292 298 — 123 50 242 51 55 118 11 47 72 64 5 29 337 187 351 620 185 112 100 20 96 76 45 42 15 41 285 60 30 48 252 283 186 701 250 207 192 42 132 Key: LLN, lower limit of normal; ULN, upper limit of normal; Phe Phenylalanine; —, Data not available. Fig. 1. (a) Western blot analysis in skin fibroblasts demonstrates a normal immu noreactive band in a control cell line and an approximately 37 kDa band in cases 1 (“1”) and 2 (“2”), compound heterozygous for a p.R355X and p.R43X mutation; “A” is an unpublished case. (b) Western blot analysis of mitochondria from fibroblasts of a control (lane 1) and case 7 (lane 2); with anti-citrin in the top panel, and antisubunit IV of the cytochrome c oxidase in the bottom panel. Fig. 2. (a) Western blot analysis demonstrates that reduced ASS enzyme activity is as a result of a reduction in the ASS protein in case 1 (“1”) less markedly in case 2 (“2”) compared with controls C1 and C2. (b) Quantitative RT-PCR of ASS1 normal ized to Beta-2 Microglobulin in patient fibroblasts demonstrates increased tran scription when compared to a normal control (C) or a patient with classical ASS deficiency (ASS). ewborn screening. He subsequently developed jaundice with n progressive cholestasis. However, his liver function tests were all within the normal range. His alpha fetoprotein (AFP) and ferritin were not significantly elevated. His initial plasma amino acids at birth were significant for an elevated citrulline and methionine with a modest elevation of threonine (Table 2). Given the signif icant elevation in citrulline (492 lM), an ASS enzyme assay was performed on cultured fibroblasts. This assay revealed reduced ASS activity (0.5 nmol/min/mg protein normal range 0.8–3.8 nmol/ min/mg protein). A low protein diet was implemented but failure to thrive and liver dysfunction appeared. As a result of the concurrent experience with case 1, sequencing of the SCL25A13 gene was performed, which revealed the same novel heterozygous mutations: p.R43X and p.R355X with the same novel variant and SNPs as case 1. Consistent with case 1, Western analysis for citrin detected a 37 kDa protein (Fig. 1). The citrulline incor poration ratio was normal (27.98 reference range 3–112). Western analysis of fibroblasts for ASS revealed a significant reduction in ASS protein with increased mRNA (Fig. 2). A high protein, low car bohydrate diet was prescribed as previously described [6]. How ever this dietary approach has proved dif ficult for the parents to implement and he still has persistent failure to thrive. Case 3 is the 24 year old mother of case 2. She was diagnosed as a result of mutation testing to clarify the configuration of her son’s mutations. She was born at term and, by history, had no sig nificant neonatal cholestasis. She had an uneventful childhood and performed appropriately at school. She has a lifelong dislike of simple carbohydrates and a preference for protein rich foods. In adulthood she has exhibited episodic confusion and a mood dis order. Although, at this point undiagnosed, she had no significant complications during pregnancy and no postpartum decompensa tion. More recently she has presented with significant epigastric pain secondary to pancreatitis with a lipase of 1500 U/L (reference range <190 U/L). Her ALT, AST, bilirubin, ammonia and triglycerides have always remained within normal range. Sequence analysis of the SCL25A13 gene revealed the same novel heterozygous muta tions: p.R43X and p.R355X. Testing of her parents established the trans configuration of the mutations with the same novel variant and SNPs as case 1. Case 4 is a 6 month old French-Canadian female who presented with elevated citrulline on urinary newborn screening at three weeks of age. Reflex PAA revealed a pattern consistent with NICDD with a citrulline of 948 (see Table 2). Given previous experience, sequencing analysis of the citrin gene was performed and revealed 654 D. Dimmock et al. / Molecular Genetics and Metabolism 96 (2009) 44–49 a homozygous p.R43X mutation and the homozygosity for the same c.328 + 6 a > g SNP as seen in cases 1, 2, and 3. A skin biopsy was not performed for clinical or research studies. She has subse quently been managed on a high-protein, low carbohydrate diet as previously described [6]. She is growing well and has not devel oped clinically significant liver disease. Case 5, a 3 year old South–East Asian child, presented with elevated citrulline (350 lM) with borderline elevation in tyrosine on expanded newborn screening. Plasma amino acids at 3 weeks of age showed a citrulline of 850 lmol with elevations in tyrosine, methionine, threonine to serine ratio and arginine (Table 2). She exhibited significant cholestasis and deranged coagulation for over 8 weeks. The liver failure did not initially respond to the high protein, low carbohydrate diet we have previously used [6]. Con sistent with another reported case, significant improvement was seen after galactose was excluded from the diet [16]. Sequencing revealed a previously reported homozygous insertion mutation, c.1660_c.1661ins23 (c.1638_1660dup23) [1]. She was subsequently lost to follow up. Case 6, a 2 year old child, was born at 38 weeks, to non-con sanguous ethnically Han parents in Buenos Aires, Argentina. He presented with a phenylalanine seven times the upper limit of normal on newborn screening. On the second screen he had a nor mal phenylalanine level but elevated total galactose of 12.5 mg/dL, (cutoff <11 mg/dL) rising to 40 mg/dl in a third sample with positive reducing substances in the urine. On initial evaluation he had a sig nificant coagulopathy, which responded to IM vitamin K. He also had significant intrahepatic cholestasis with a total bilirubin of 15 mmol/L (reference range 5.1–17.0 mmol/L) and a direct bilirubin of 13 mmol/L (reference range 1.0–5.1 mmol/L). AST and ALT were three times the upper limit of normal with a GGT of 559 (reference range: <265 IU/L). He was also noted to have anemia, hypoprotein emia (4.4 gm/dL; reference range 6.0–8.3 gm/dL) and hypoalbumi nemia (2.9 gm/dL; reference range 3.4 - 5.4 g/dL), ammonia (71 lM; reference range <50 lM) and lactate (5.0 mM; reference range <2.2 mM) were elevated with an increased anion gap metabolic acidosis. Reflex plasma amino acids revealed a profile character istic for NICCD (Table 2). Sequencing revealed heterozygosity for two previously described mutations: c.851delGTAT and c.615 + 5 G > A [1,17]. The cholestasis responded to a high protein, low car bohydrate diet as previously described [6] with the exclusion of galactose at about 1 month of age. He is growing well, with normal laboratory values and has normal neurocognitive development at follow up. Case 7 is the third child born to consanguineous Tunisian parents. He was breast-fed and, starting at one month of age, he developed progressive scleral jaundice with hepatomegaly while maintaining normal growth. At 6 months of age laboratory inves tigations revealed raised transaminases, increased conjugated bil irubin, absent antibody titres against common viral infections, a normal serum alpha-1-antitrypsin and normal sweat test. Liver ultrasound showed increased echogenicity. The child underwent a liver biopsy that revealed cirrhosis with macrovesicular steato sis. He was discharged with vitamin K and ursodeoxycholic acid therapy. His jaundice resolved spontaneously at 8 months of age. By 3 years of age he was growing well but had mild coarse facial features, and modest hepatomegaly. Routine laboratory and meta bolic investigations including ammonia, lactate, alpha-1-antitryp sin, alpha-fetoprotein, vitamin A and E and plasma amino acids (citrulline 29 lM, methionine 23 lM, tyrosine 53 lM) were normal. Liver biopsy showed incomplete septal cirrhosis with microvesicu lar steatosis. Based on the previous history of transient cholestasis, citrin deficiency was suspected and molecular studies of SLC25A13 revealed a previously undescribed homozygous missense muta tion c. 74C>A (p. A25E). This mutation was not found in 108 unre lated control chromosomes. The substitution of the non-polar to 655 47 polar amino acid is predicted to be pathogenic by SIFT and Polphen [18,19]. Western blotting showed no difference in the amount or size of the citrin protein (Fig. 1). Case 8 was the 6 lb 1 oz product of an uncomplicated gestation and vaginal delivery born to consanguineous (first cousin) Pakistani parents. He has two healthy older full siblings. The initial Maryland state newborn screen was performed prior to suf ficient milk feeds and was not reported. On day 11, the results of the second screen revealed that the citrulline was elevated at 276 lM (cutoff 100 lM). A repeat on day 19 was 298 lM. At initial clinical evaluation on day 26, he was mildly jaundiced and somewhat thin although he had regained birth weight. Plasma amino acids showed a characteris tic pattern for citrin deficiency (Table 2). He also had a modestly increased ammonia of 86 lM with significant elevations of total bilirubin (7.6 g/dL, NR <2.2) and alkaline phosphatase 1347 (ULN 390) but with normal transaminases (AST 54, ALT 17). Given the clinical suspicion of citrin deficiency, breast milk was discontin ued and replaced with a soy based formula. Subsequently his liver status improved and by 4 months of age, his cholestasis and GGT had normalized. At 5 months of age, he is growing and developing normally. Molecular studies of SLC25A13 revealed a homozygous frameshift mutation, c.172_173delGT (p.V58GfsX24). This is pre dicted to result in a premature termination codon. Both parents are carriers for this mutation. Case 9 is the clinically asymptomatic sibling of case 8. She was the full term product of an uncomplicated gestation. Her citrulline levels were 146, 514 and 126 lM on three blood spot specimens at 2 days, 2 weeks, and 4 weeks of age respectively. However, she was not referred for metabolic evaluation at that time. She did not have any clinically detected jaundice or liver disease in the new born period and she was breast fed for 18 months. She craves pro tein at every meal and has a dietary avoidance of fruits and rice. At 4 1/2 years of age, she is growing and developing normally and has normal ammonia, liver function tests and plasma amino acids except for a modestly elevated citrulline of 53 lM (Table 2). Molec ular testing found her to be homozygous for the c.172_173delGT (p.V58GfsX24) frameshift mutation. Case 10 is a 5 year old Caucasian female who was born with a birth weight of 2.2 kg. She was fed on cow milk based formula, and passed her Indiana state newborn screen at less than 48 h but failed the second screen at 8 days of age with an elevated citrulline and citrulline to tyrosine ratio. Reflex plasma amino acids showed a significantly elevated threonine (769 lM), citrulline (325 lM), and methionine (578 lM) with an essentially normal tyrosine (100 lM, reference range <96). With the suspicion of citrin deficiency, a gal actose free diet was instituted. She has not been on a high protein diet but growth has remained at the 50th percentile. She has not had any liver dysfunction and neurocognitive development remains on target. Sequencing revealed a previously reported splice site muta tion, c.848+3a > c [6,20], and a heterozygous unclassified variant, c.1637C > G (p.T546R). Threonine at this position is conserved from yeast to human. SIFT and PolyPhen [18,19] predict this variant to be deleterious. A different substitution, p.T546M, at the same amino acid position, has previously been reported in patients with citrin deficiency [21]. Discussion A total of 14 patients, that we are aware of, have been diagnosed in North America in the past 4 years with the addition of 2 cases from Texas (one Caucasian American reported elsewhere [6] and one of Vietnamese ancestry) and 2 cases in California of Taiwanese and Korean descent. The majority of these patients are not of Asian ancestry. It remains unclear to what extent this finding reflects a different prevalence of this disease among different ethnic groups. Nevertheless, from our data it is clear that citrin deficiency 48 D. Dimmock et al. / Molecular Genetics and Metabolism 96 (2009) 44–49 should be considered in any infant presenting with intrahepatic cholestasis, steatosis or cirrhosis regardless of ethnicity. The diagnosis of three newborns in Quebec with citrin deficiency in the past 2 years would suggest, if these years are representative, a minimum birth incidence of 1 case per 50,000 live births. How ever, since all of these patients come from the same region, it is likely that the incidence within the Bas-St-Laurent region may be higher. Although we did not identify common ancestors, the com mon haplotype and the relatively small geographically defined region of origin suggests that these are founder mutations. Citrin deficiency may be a significant cause of neonatal liver disease in this region and further investigation will be required to determine if specific screening for this condition in this population is war ranted. In addition, the diagnosis of this condition in a patient of Arabic descent and another patient of Pakistani origin suggests that this disorder is truly a pan-ethnic condition and should be considered when indicated in all ethnic groups (see Table 1). To date 110 patients have been referred to the Medical Genetics Laboratories at Baylor College of Medicine Medical Genetics Labora tories for citrin sequencing, and among them twelve cases (11%) have confirmed bi-allelic mutations. Although not systematically evaluated in all patients, 7 of the remaining cases (7%) had confirmed muta tions in ASS1. Five of these seven patients had an elevated citrulline in the range of 100–1000 lM on newborn screening. As only two other genetic diseases are currently associated with an elevated citrulline (pyruvate carboxylase deficiency type B, OMIM #266150 and argi ninosuccinate lyase deficiency, OMIM #207900) it is likely that the biochemical phenotype seen in citrin deficiency may also be caused by other, as yet undefined, pathological processes. It is noteworthy that one patient, with a similar amino acid profile and cholestasis, had mutations in the DGUOK gene and is published elsewhere [22]. The original reports of CTLN2, described a tissue-specific deficiency of ASS activity in the liver but not in skin fibroblasts [2] while subsequent studies have shown that this activity may be nor mal in the liver of some patients [23], the activity has always been normal in skin fibroblasts [1,2,6]. We considered that this may be useful diagnostic distinction between this disorder and primary ASS deficiency. However, in this paper we present two French-Canadian patients with reduced ASS activity in fibroblasts harboring the same novel mutations in the SLC25A13 gene. In addition, the normal citrul line incorporation is consistent with results seen in patients with less severe mutations in ASS1 [24]. It is unclear why citrin deficiency alters ASS activity in the fibroblasts of our patients and why the reduction is restricted to the liver of the other patients previously described. Although metabolite concentrations, particularly arginine and citrul line in the culture media may alter ASS activity, this change is med iated by altered transcription [25]. As the ASS1 mRNA levels are not reduced in either of our patients fibroblasts, or in the liver of Japa nese patients with liver specific ASS deficiency [2], it seems unlikely that this is the mechanism. Subsequently, regulation of endothelial, but not hepatic ASS through alternative translation initiation sites of the 59 UTR has been described. These transcripts can suppress ASS transcription [26]. However, as they are not expressed in the liver, it seems unlikely that this is the mechanism of reduction of ASS activ ity. More recently, it has been shown that the NADPH to NADP+ ratio directly alters the ASS activity and changes the association with the regulatory protein HSCARG [27]. Since these ratios are expected to be perturbed in our patients, this is the most plausible explanation to date. Further studies will be required to evaluate the role of this pathway in citrin deficiency. Additional cases are necessary to deter mine if the reduced ASS activity in fibroblasts in the French-Cana dians is a result of the specific mutations in the SLC25A13 gene or a reflection of modifier genes. The ASS enzyme assay has been the standard diagnostic test for primary ASS deficiency for over 25 years. These cases have significant implications for the diagnosis of primary ASS deficiency. Our results demonstrate that primary ASS deficiency cannot always be distinguished from citrin deficiency by ASS assay. These findings also suggest the need for careful re-evaluation of all patients with apparent ASS deficiency on skin fibroblast testing and only modest elevations in citrulline levels, especially those not responding to, or worsening on, a low protein diet. Given that the ASS deficiency is not restricted to the liver, we would suggest that the term CTLN2 is no longer used, as it is clear that our patients have citrin (AGC2) deficiency but not CTLN2. Instead we would propose that the dis ease classification reflect the underlying metabolic cause that is the deficiency of the citrin protein. The dif ficulty in distinguishing hypo morphic ASS deficiency [28] from citrin deficiency is further com plicated by the almost isolated, markedly elevated citrulline seen in case 2’s presenting chromatogram and the significant elevation in case 4 (Table 2), suggesting that, at presentation, amino acid profiles may not help distinguish one disorder from another. The availability of dietary therapy for this condition, and the significant worsening seen in cases 1 and 2 when protein was restricted, make it vital to distinguish these conditions when a patient is detected with mod est elevations of citrulline on newborn screening. This suggests that molecular diagnosis by sequencing of SLC25A13 and ASS1 is essential in the prompt diagnosis and management of such patients ascer tained through the newborn screening program. Consistent with previous experience using blood spots where approximately half of all patients were not detected by expanded newborn screening on blood [9], it is clear from case 7 that not all patients will be detected with an elevated citrulline. Additionally, even at times of acute crises the plasma amino acid profile might be non-diagnostic. The lack of symptomatic disease in childhood of cases 3 and 9 must be considered when deciding if this disease would be an appropriate primary target for newborn screening. Consequently, further mutation-based studies will be required to evaluate the true prevalence and natural history of this disorder outside of Asia. The psychiatric features of case 3 and the natu ral history of CTLN2 patients in Japan emphasize the etiologic role citrin deficiency plays in organic psychiatric illness. However, fur ther studies are needed to evaluate how common this disorder is amongst given populations before recommending routine evalua tion for this disease in patients with psychiatric illness. The mutations observed in cases 7 and 10 emphasize the emerging role of missense mutations in this disorder [7,8,29]. Since the mutation in case 7 is in the N-terminal domain, this mutation would not affect the transport activity of citrin in our expression system [30] However, this disruptive change in an amino acid, highly conserved in the calcium-binding domain of both aralar (AGC1) and citrin (AGC2), is expected to impair calcium regulation. Noticeably, since cases 1, 2 and 7 and a recently reported patient of Pakistani origin [7] (data not shown) have protein detected on Western blotting, protein based screening [12] may not be as sen sitive as one would hope. In summary, we present five novel mutations in SLC25A13, including apparent founder mutations in a French Canadian pop ulation. Additional novel mutations in patients of Tunisian, Paki stani and Northern European descent support the notion that this is truly a pan-ethnic disorder. Further mutation-based studies will be required to evaluate the true prevalence and natural history of this disorder outside of East Asia. Citrin deficiency may present with elevated citrulline on newborn screening. Moreover, in con trast to previously described patients, ASS enzyme deficiency may be detected on skin fibroblast culture suggesting a pivotal role for DNA sequencing in these patients. Acknowledgments The authors wish to thank Jacqueline Heidorn for assistance with the ASS enzyme assay and citrulline incorporation, Robert 656 D. Dimmock et al. / Molecular Genetics and Metabolism 96 (2009) 44–49 Trieu and Yewei Ma for assistance with the Western blotting. The authors also wish to acknowledge Stephanie B. Gurnon, MS, CGC, Rebecca Roberts, MS, RD, V. Reid Sutton, MD and the late Rebecca Wappner, MD, for assistance in obtaining clinical information and providing clinical care to our patients. This study was funding in part by an NIH fellowship award to DD (K12 RR17665) and by grants from Ministero dell’Università e della Ricerca, Ministero della Salute to GF. References [1] K. Kobayashi, D.S. Sinasac, M. Iijima, A.P. Boright, L. Begum, J.R. Lee, T. Yasuda, S. Ikeda, R. Hirano, H. Terazono, M.A. Crackower, I. Kondo, L.C. Tsui, S.W. Scherer, T. Saheki, The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein, Nat. Genet. 22 (2) (1999) 159–163. [2] K. Kobayashi, N. Shaheen, R. Kumashiro, K. Tanikawa, W.E. O’Brien, A.L. Beau det, T. Saheki, A search for the primary abnormality in adult-onset type II cit rullinemia, Am. J. Hum. Genet. 53 (5) (1993) 1024–1030. [3] T. Saheki, K. Kobayashi, Mitochondrial aspartate glutamate carrier (citrin) defi ciency as the cause of adult-onset type II citrullinemia (CTLN2) and idiopathic neonatal hepatitis (NICCD), J. Hum. Genet. 47 (7) (2002) 333–341. [4] Y. Tazawa, D. Abukawa, O. Sakamoto, I. Nagata, J. Murakami, T. Iizuka, M. Okamoto, A. Kimura, T. Kurosawa, K. Iinuma, K. Kobayashi, T. Saheki, T. Ohura, A possible mechanism of neonatal intrahepatic cholestasis caused by citrin deficiency, Hepatol. Res. 31 (3) (2005) 168–171. [5] E. Ben-Shalom, K. Kobayashi, A. Shaag, T. Yasuda, H.Z. Gao, T. Saheki, C. Bach mann, O. Elpeleg, Infantile citrullinemia caused by citrin deficiency with increased dibasic amino acids, Mol. Genet. Metab. 77 (3) (2002) 202–208. [6] D. Dimmock, K. Kobayashi, M. Iijima, A. Tabata, L.J. Wong, T. Saheki, B. Lee, F. Scaglia, Citrin deficiency: a novel cause of failure to thrive that responds to a high-protein, low-carbohydrate diet, Pediatrics 119 (3) (2007) e773–e777. [7] G. Fiermonte, D. Soon, A. Chaudhuri, E. Paradies, P.J. Lee, S. Krywawych, F. Palmieri, R.H. Lachmann, An adult with type 2 citrullinemia presenting in Europe, N. Engl. J. Med. 358 (13) (2008) 1408–1409. [8] A. Tabata, J.S. Sheng, M. Ushikai, Y.Z. Song, H.Z. Gao, Y.B. Lu, F. Okumura, M. Iijima, K. Mutoh, S. Kishida, T. Saheki, K. Kobayashi, Identification of 13 novel mutations including a retrotransposal insertion in SLC25A13 gene and fre quency of 30 mutations found in patients with citrin deficiency, J. Hum. Genet. 53 (6) (2008) 534–545. [9] A. Tamamori, A. Fujimoto, Y. Okano, K. Kobayashi, T. Saheki, Y. Tagami, H. Takei, Y. Shigematsu, I. Hata, H. Ozaki, D. Tokuhara, Y. Nishimura, T. Yorifuji, N. Igarashi, T. Ohura, T. Shimizu, K. Inui, N. Sakai, D. Abukawa, T. Miyakawa, M. Matsumori, K. Ban, H. Kaneko, T. Yamano, Effects of citrin deficiency in the perinatal period: feasibility of newborn mass screening for citrin deficiency, Pediatr. Res. 56 (4) (2004) 608–614. [10] Y. Tazawa, K. Kobayashi, D. Abukawa, I. Nagata, S. Maisawa, R. Sumazaki, T. Iizuka, Y. Hosoda, M. Okamoto, J. Murakami, S. Kaji, A. Tabata, Y.B. Lu, O. Sakam oto, A. Matsui, S. Kanzaki, G. Takada, T. Saheki, K. Iinuma, T. Ohura, Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients, Mol. Genet. Metab. 83 (3) (2004) 213–219. [11] J.N. Yeh, Y.M. Jeng, H.L. Chen, Y.H. Ni, W.L. Hwu, M.H. Chang, Hepatic steatosis and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) in Taiwanese infants, J. Pediatr. 148 (5) (2006) 642–646. [12] D. Tokuhara, M. Iijima, A. Tamamori, T. Ohura, J. Takaya, S. Maisawa, K. Kobay ashi, T. Saheki, T. Yamano, Y. Okano, Novel diagnostic approach to citrin defi ciency: analysis of citrin protein in lymphocytes, Mol. Genet. Metab. 90 (1) (2007) 30–36. [13] T.S. Su, H.G. Bock, A.L. Beaudet, W.E. O’Brien, Molecular analysis of arginino succinate synthetase deficiency in human fibroblasts, J. Clin. Invest. 70 (6) (1982) 1334–1339. 657 49 [14] H. Northrup, A.L. Beaudet, W.E. O’Brien, Prenatal diagnosis of citrullinaemia: review of a 10-year experience including recent use of DNA analysis, Prenat. Diagn. 10 (12) (1990) 771–779. [15] C. Auray-Blais, D. Cyr, R. Drouin, Quebec neonatal mass urinary screening programme: from micromolecules to macromolecules, J. Inherit. Metab. Dis. 30 (4) (2007) 515–521. [16] E. Naito, M. Ito, S. Matsuura, Yokota, T. Saijo, Y. Ogawa, S. Kitamura, K. Kobay ashi, T. Saheki, Y. Nishimura, N. Sakura, Y. Kuroda, Type II citrullinaemia (citrin deficiency) in a neonate with hypergalactosaemia detected by mass screening, J. Inherit. Metab. Dis. 25 (1) (2002) 71–76. [17] Y.B. Lu, K. Kobayashi, M. Ushikai, A. Tabata, M. Iijima, M.X. Li, L. Lei, K. Kawabe, S. Taura, Y. Yang, T.T. Liu, S.H. Chiang, K.J. Hsiao, Y.L. Lau, L.C. Tsui, D.H. Lee, T. Saheki, Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13 gene of Japanese patients with citrin deficiency, J. Hum. Genet. 50 (7) (2005) 338–346. [18] P.C. Ng, S. Henikoff, Predicting the effects of amino acid substitutions on pro tein function, Ann. Rev. Genomics Hum. Genet. 7 (2006) 61–80. [19] V. Ramensky, P. Bork, S. Sunyaev, Human non-synonymous SNPs: server and survey, Nucleic Acids Res. 30 (17) (2002) 3894–3900. [20] L.J. Wong, D. Dimmock, M.T. Geraghty, R. Quan, U. Lichter-Konecki, J. Wang, E.K. Brundage, F. Scaglia, A.C. Chinault, Utility of oligonucleotide array-based comparative genomic hybridization for detection of target gene deletions, Clin. Chem. 54 (7) (2008) 1141–1148. [21] K. Kobayashi, Y. Bang Lu, M. Xian Li, I. Nishi, K.J. Hsiao, K. Choeh, Y. Yang, W.L. Hwu, J.K. Reichardt, F. Palmieri, Y. Okano, T. Saheki, Screening of nine SLC25A13 mutations: their frequency in patients with citrin deficiency and high carrier rates in Asian populations, Mol. Genet. Metab. 80 (3) (2003) 356–359. [22] D.P. Dimmock, Q. Zhang, C. Dionisi-Vici, R. Carrozzo, J. Shieh, L.Y. Tang, C. Tru ong, E. Schmitt, M. Sifry-Platt, S. Lucioli, F.M. Santorelli, C.H. Ficicioglu, M. Rodriguez, K. Wierenga, G.M. Enns, N. Longo, M.H. Lipson, H. Vallance, W.J. Craigen, F. Scaglia, L.J. Wong, Clinical and molecular features of mitochondrial DNA depletion due to mutations in deoxyguanosine kinase, Hum. Mutat. 29 (2) (2008) 330–331. [23] T. Yasuda, N. Yamaguchi, K. Kobayashi, I. Nishi, H. Horinouchi, M.A. Jalil, M.X. Li, M. Ushikai, M. Iijima, I. Kondo, T. Saheki, Identification of two novel muta tions in the SLC25A13 gene and detection of seven mutations in 102 patients with adult-onset type II citrullinemia, Hum. Genet. 107 (6) (2000) 537–545. [24] D. Dimmock, P. Trapane, A. Feigenbaum, C. Keegan, S. Cederbaum, J. Gibson, M. Gambello, K. Vaux, P. Ward, G. Rice, J. Wolff, W. O’Brien, P. Fang, The Role of Molecular Testing and Enzyme Analysis in the Management of Hypomorphic Citrullinemia American Journal of Medical Genetics, in press. [25] M.J. Jackson, S.J. Allen, A.L. Beaudet, W.E. O’Brien, Metabolite regulation of argininosuccinate synthetase in cultured human cells, J. Biol. Chem. 263 (31) (1988) 16388–16394. [26] L.C. Pendleton, B.L. Goodwin, L.P. Solomonson, D.C. Eichler, Regulation of endothelial argininosuccinate synthase expression and NO production by an upstream open reading frame, J. Biol. Chem. 280 (25) (2005) 24252–24260. [27] Y. Zhao, J. Zhang, H. Li, Y. Li, J. Ren, M. Luo, X. Zheng, An NADPH sensor protein (HSCARG) downregulates nitric oxide synthesis by association with arginino succinate synthetase and is essential for epithelial cell viability, J. Biol. Chem. 283 (16) (2008) 11004–11013. [28] J. Haberle, S. Pauli, E. Schmidt, B. Schulze-Eilfing, C. Berning, H.G. Koch, Mild citrullinemia in Caucasians is an allelic variant of argininosuccinate synthetase deficiency (citrullinemia type 1), Mol. Genet. Metab. 80 (3) (2003) 302–306. [29] N. Yamaguchi, K. Kobayashi, T. Yasuda, I. Nishi, M. Iijima, M. Nakagawa, M. Osame, I. Kondo, T. Saheki, Screening of SLC25A13 mutations in early and late onset patients with citrin deficiency and in the Japanese population: Identifi cation of two novel mutations and establishment of multiple DNA diagnosis methods for nine mutations, Hum. Mutat. 19 (2) (2002) 122–130. [30] L. Palmieri, B. Pardo, F.M. Lasorsa, A. del Arco, K. Kobayashi, M. Iijima, M.J. Runswick, J.E. Walker, T. Saheki, J. Satrustegui, F. Palmieri, Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria, EMBO J. 20 (18) (2001) 5060–5069.
