Biochemical and Clinical Profiles of 52 Tunisian Patients Affected by Zellweger Syndrome Accepted Manuscript Biochemical and Clinical Profiles of 52 Tunisian Patients Affected by Zellweger Syndrome Fa[.]
Accepted Manuscript Biochemical and Clinical Profiles of 52 Tunisian Patients Affected by Zellweger Syndrome Fahmi Nasrallah, Wiem Zidi, Moncef Feki, Samia Kacem, Neji Tebib, Naziha Kaabachi PII: S1875-9572(17)30107-9 DOI: 10.1016/j.pedneo.2016.08.011 Reference: PEDN 646 To appear in: Pediatrics & Neonatology Received Date: April 2016 Revised Date: 15 August 2016 Accepted Date: 26 August 2016 Please cite this article as: Nasrallah F, Zidi W, Feki M, Kacem S, Tebib N, Kaabachi N, Biochemical and Clinical Profiles of 52 Tunisian Patients Affected by Zellweger Syndrome, Pediatrics and Neonatology (2017), doi: 10.1016/j.pedneo.2016.08.011 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain PEDN-D-16-00134_After Eng Edited_final ACCEPTED MANUSCRIPT Original article Biochemical and Clinical Profiles of 52 Tunisian Patients Affected by Zellweger RI PT Syndrome Running title SC Zellweger Syndrome in Tunisia M AN U Fahmi Nasrallaha*, Wiem Zidia, Moncef Fekia, Samia Kacemb, Neji Tebibc, Naziha Kaabachia a Laboratory of Biochemistry -Rabta Hospital, Jebbari, 1007 Tunis University Tunis El Manar, Campus University Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia b Centers of Maternity and Neonatology, 1007 Tunis, University Tunis El Manar, Campus University Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia c EP TE D Pediatric Department -Rabta Hospital, Jebbari, 1007 Tunis University Tunis El Manar, Campus University Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia AC C Corresponding author: Doctor Fahmi Nasrallah (PhD), Laboratory of Biochemistry, Rabta Hospital, 1007 Jebbari, Tunis, Tunisia Tel/fax, 216 71 561912; E-mail address: fehmi56@yahoo.fr Declaration of interest The authors state that there is no conflicts of interest regarding the publication of this article Abstract ACCEPTED MANUSCRIPT Background Zellweger syndrome (ZS) is a peroxisome biogenesis disorder due to a mutation of the PEX genes family The incidence of this disease in Africa and the Arab world remains unknown This contribution aimed at describing clinical phenotype and biochemical features in Tunisian patients with ZS in order to improve the detection and management of RI PT this severe disorder Methods A total of 52 patients diagnosed with ZS and 60 age- and gender-matched healthy controls were included Patients were recruited during the last 21 years and the diagnosis of SC ZS was based on clinical and biochemical characteristics Plasma very long chain fatty acids (VLCFA) were analyzed using capillary gas chromatography Estimated incidence of ZS was M AN U calculated using Hardy-Weinberg formula Results Estimated incidence of ZS is 1/15,898 in Tunisia Age at diagnosis varied between days and 18 months Severe neurological syndrome, polymalformative features and hepatodigestive signs were observed in 100%, 67.9% and 32% of patients, respectively Values for TE D plasma C26:0 and C26:0/C22:0 and C24:0/C22:0 ratios were noticeably higher in ZS patients than controls Distributions of values were completely different for C26:0 (0.10 to 0.37 vs 0.001 to 0.009), C26:0/C22:0 ratio (0.11 to 1.29 vs 0.003 to 0.090) and C24:0/C22:0 ratio EP (1.03 to 3.18 vs 0.46 to 0.90) in ZS patients and controls, respectively Conclusions This study highlights the high incidence of ZS in Tunisia and the possibility of AC C simple and reliable biochemical diagnosis, thus permitting early genetic counseling for families at risk Key Words: gas chromatography; hypotonia; peroxisomal disorder; very long chain fatty acids; Zellweger syndrome ACCEPTED MANUSCRIPT Introduction The peroxisome biogenesis disorders constitute a group of genetically heterogeneous diseases among which Zellweger syndrome (ZS; McKusick 21410) is the most severe.1,2 Peroxisome is a cellular organelle that exclusively performs or participates in important cellular functions RI PT such as oxidation of very long chain fatty acids (VLCFA) and synthesis of plasmalogen and bile acids.3,4 This syndrome is associated with multiple peroxisomal enzyme deficiencies that lead to an accumulation of VLCFA and a lack in plasmalogens ZS, also called the SC cerebrohepatorenal syndrome, is well known to exhibit multiple congenital anomalies Patients with ZS have profound neurologic impairment with seizures, severe retardation, and M AN U dysmorphic features.5,6 The worldwide prevalence of ZS is estimated between 1/50,000 and 1/100,000 The incidence of ZS varies widely throughout the world with the highest incidence in the Saguenay-Lac-Saint-Jean region of Quebec and the lowest incidence in Japan.7-9 The high incidence of ZS in Quebec is likely due to a founder mutation in the PEX6 gene in TE D Saguenay-Lac-Saint-Jean population The disease impairs the shortening of the chain of VLCFA, leading to tissue accumulation of these compounds Major accumulated fatty acids are hexacosanoic acid (C26:0) and EP lignoceric acid (C24:0).10 It is presumed that the increased concentrations of these metabolites cause multiple congenital abnormalities involving the brain, bone, liver, eyes, kidneys, and AC C endocrine glands ZS patients exhibit a delay in mental development, marked hypotonia, feeding difficulty and respiratory impairment and often die in early life.7 Confirmation of the diagnosis of ZS requires the demonstration of reduced peroxisomal functions by enzymatic tests or mutation within the PEX gene These sophisticated analyzes are restricted to a few specialized laboratories However, the more accessible VLCFA analysis is considered a suitable test for the screening of ZS, especially when combined with clinical features.11 ACCEPTED MANUSCRIPT Since 1994, plasma VLCFA analysis by gas chromatography has become available and has been used as a procedure for the screening of ZS and other peroxisomal disorders in Tunisia This study reports the result of screening for ZS during 21 years in Tunisia using the analysis of VLCFA gas chromatography This work highlights the epidemiological, clinical and AC C EP TE D M AN U SC ZS and permit genetic counseling for families at risk RI PT biochemical aspects of ZS in Tunisia in order to improve the diagnosis and management of ACCEPTED MANUSCRIPT Materials and methods 2.1 Samples and chemicals Plasma samples were collected from 52 patients with ZS between 1994 and 2015 in the Laboratory of Biochemistry (Rabta Hospital, Tunis, Tunisia) and 60 from healthy age-and RI PT gender-matched controls Blood was collected into heparinized tubes and maintained at 4°C The plasma was then separated by centrifugation at 1500 g for 10 minutes at 4°C and then stored at -20°C until analysis Fatty acids standards and potassium carbonate were purchased SC from Sigma (St.Louis, MO, USA) Acylchloride was purchased from Aldrich Acetonitrile, nhexane, chloroforme, methanol and other chemical reagents were of analytical grade and were M AN U of highest purity The study was performed with the approval of institutional ethics committee (Rabta University Health Center) and informed consent was obtained from parents 2.2 Methods VLCFA were determined as their methyl esters according to a modified method of Moser and TE D Moser.12 Samples, once prepared, are analyzed using a Hewlett Packard gas chromatograph (HP model 6890), equipped with a split/splitless capillary inlet system and a flame ionization detection as described by Nasrallah et al.13 Plasma cortisol and ACTH were measured using EP the respective radioimmunoassay methods Activity of dihydroxyacetone phosphate acyltransferase (DHAPAT) and genetic analysis were carried out in Laboratory of Genetic AC C and Metabolic Diseases, University of Amsterdam (Netherlands) Statistical computations were performed using SPSS 18.0 for Windows software (SPSS Inc., Ill.) Continuous variables were compared using independent-samples T test A p value < 0.05 based on two-sided calculation was considered significant The incidence of ZS was estimated using the Hardy-Weinberg formula.14 ACCEPTED MANUSCRIPT Results A total of 52 patients with ZS, belonging to 50 families, were diagnosed during the period 1994‒2015 (Table 1) Based on these data, incidence of ZS in Tunisia was estimated to be 1/15,898 Age at diagnosis varied from days to 18 months; age was below months in 40 RI PT patients and between and 18 months in the remainder All patients showed neurological impairment at birth Dysmorphia and polymalformative syndrome were noted in 65% of patients under months and in 83% of those over months Hepatodigestive manifestations SC were found in 27% of patients under months and in 50% of those above months Most common neurological signs observed were lower fetal intrauterine movements, hypotonia, M AN U poor sucking and feeding problems, seizures with or without deafness or blindness Main dysmorphic features observed in these patients were large fontanels, high forehead, epicanthus and abnormal ears Biochemical diagnosis was based on VLCFA analysis by gas chromatography Figure TE D shows chromatograms of VLCFA standards mixture, control subjects and ZS patients Reference values for C26:0 and C26:0/C22:0 and C24:0/C22:0 ratios were considered as the respective extremes values in the control group Compared to these values, plasma C26:0 and EP C26:0/C22:0 and C24:0/C22:0 ratios were obviously higher in ZS patients (Table 1) Figure illustrates that the three parameters perfectly discriminate ZS patients from controls Four AC C patients showed evidence of adrenal insufficiency with elevated ACTH and low cortisol levels For patients P45 and P46, diagnosis was confirmed by showing depleted DHAPAT activity in fibroblasts For these two patients, ultrastructural examinations showed the presence of peroxisomal ghosts in fibroblasts Diagnosis was further confirmed for patient P46, whose molecular analysis highlights a homozygous W105X mutation of the PEX26 gene This patient subsequently died at the age of months in a context of refractory seizures and apnea ACCEPTED MANUSCRIPT Discussion A total of 52 patients were diagnosed with ZS during the last 21 years in our laboratory, which is the only laboratory in Tunisia to perform of VLCFA analysis Based on these data, the incidence of ZS in Tunisia is estimated to be 1/15,898 This incidence is among the RI PT highest reported in the world However, it is lower than that reported in Saguenay-Lac-SaintJean region, where the incidence of ZS is highest worldwide (1/5,224‒1/37,544) 15 Incidence of ZS has been estimated to be 1/50,000 in the United States and 1/500,000 in Japan.16 17 A high incidence of ZS has been also noted in a small Arab community (Karaite) in Israel 18 SC Nevertheless, estimated incidence was much higher in the Okinawa Islands (1/30,000) M AN U The estimated incidence of ZS should be underestimated since screening is rather selective in the absence of a systematic neonatal screening in Tunisia Several families had at least one other child who died in similar clinical context In Tunisia, a single laboratory performs the biochemical screening for ZS, and it is likely that some ZS patients die with an irresolute TE D diagnosis before any biochemical screening request The high incidence of ZS and other inherited metabolic disorders in North Africa and the Middle East may be explained by the high rate of consanguineous marriages in these regions 19-21 In this study, consanguinity was EP considerably elevated (81.5% of cases) Clinical phenotype of patients with ZS is dominated by severe nervous system dysfunction AC C and facial dysmorphia The predominant symptoms at birth were a severe weakness and hypotonia Craniofacial abnormalities include large fontanels, high forehead, epicanthus and abnormal ears Over time, most patients developed feeding difficulties, seizures and hepatomegaly They showed a failure to thrive and usually died before the age of months (range, days to 11 months) 22 In this study, the occurrences of dysmorphic and malformative syndrome (83% vs 77%) and hepatodigestive troubles (50% vs 27%) were significantly higher in patients aged above months compared to those aged below months ACCEPTED MANUSCRIPT This difference is explained by a rapid deterioration of patients’ health with time Neurological abnormalities were present in all patients from both groups, which is in agreement with the literature.6,8 However, no obvious differences in severity of neurological troubles were observed between the two groups RI PT Although non-specific of ZS, increased C26:0 level and C24:0/C22:0 and C23:0/C22:0 ratios seem to be highly sensitive in discriminating ZS from healthy subjects The method of measurement of VLCFA developed in our laboratory is rapid and sensitive In contrast to SC other methods 10, it does not include Folch extraction or purification of samples by thin-layer chromatography The method is therefore less time-consuming and could be adopted for M AN U routine screening for peroxisomal disorders.13 In our series, analysis of patients’ samples yielded noticeably increased C26:0 and C26:0/C22:0 and C24:0/C22:0 ratios in ZS patients with almost no overlap with values in controls The reference values reported here are in good agreement with those reported in the literature.23,24 TE D Adrenocortical dysfunction is common in ZS, while the underlying mechanisms remain unclear It was suggested that high levels of VLCFA result in adrenal insufficiency Literature data indicate a correlation between plasma C26:0 levels and adrenal insufficiency25-28 Such a EP correlation was not tested in this study since ACTH and cortisol assays were achieved in few patients A recent report showed that systematic screening and early treatment of adrenal AC C insufficiency were associated with prolonged survival in ZS patients.29 In future, patients with suspicion of ZS should be screened for adrenal insufficiency in order to initiate appropriate and early therapy Confirmation of ZS diagnosis requires laboratory investigations to assess peroxisomal functions by enzymatic analysis in fibroblasts and/or PEX gene mutation studies.30,31 In these series, enzymatic test was performed in two patients, showing depleted DHAPAT activity Molecular analysis was performed in one patient who was identified a carrier of homozygous ACCEPTED MANUSCRIPT W105X mutation in the PEX26 gene PEX26 is a peroxisomal membrane protein that assists in the localization of PEX1-PEX6 complexes on the peroxisomal membrane.32,33 PEX26 mutations are less frequently observed in peroxisome biogenesis disorders-ZS spectrum patients.30 Combined with previously reported PEX26 mutations, 22 different mutations have RI PT now been identified in the PEX26 gene including the mutation (W105X) reported in this study, which has not been previously reported in North African and Arabic countries.30,31 The present study had some limitations Biochemical diagnosis was based mainly on VLCFA SC analysis and confirmatory tests (i.e enzymatic and molecular analysis) were carried out in few patients Another peroxisomal disorder could not be definitively excluded However, the M AN U clinical features observed in patients made the diagnosis of ZS very likely Another limitation was the lack of comprehensive information in some flowcharts due to limited knowledge of most neonatologists and pediatricians in the field of peroxisomal disorders The clinicians’ unawareness is also responsible for the omission of some investigations Indeed, plasma TE D ACTH and cortisol analysis were performed in only a small number of patients As a consequence, the prevalence of adrenal insufficiency could not be determined in this cohort of ZS patients Finally, due to the delay in obtaining a definitive diagnosis for this severe EP disorder and fatalities in early life, many patients were lost to follow up, so their outcomes were not known However, the study provided novel data demonstrating a high incidence of AC C ZS in Tunisia and emphasized the efficacy of VLCFA analysis in ZS diagnosis in the absence of enzymatic and molecular tests Given the high incidence of ZS in our population and the availability of a simple and reliable diagnosis test, any newborn with severe neurological syndrome dominated by hypotonia, especially when associated with dysmorphic syndrome, should be screened for ZS by analyzing plasma VLCFA The screening is especially indicated when the child is issued from a consanguineous marriage A systematic evaluation of adrenal ACCEPTED MANUSCRIPT function should be included in the screening and management of ZS patients Finally, AC C EP TE D M AN U SC RI PT enzymatic and genetic tests should be developed in Tunisia to ensure a firm diagnosis of ZS 10 ACCEPTED MANUSCRIPT Conclusions This study revealed a high incidence of ZS in Tunisia It showed a typical clinical profile in Tunisian ZS patients with severe neurological impairment and dysmorphic syndrome While not specific, VLCFA analysis was a consistent test for the screening of ZS The study also RI PT revealed several insufficiencies, such as lack diagnosis confirmation tests, rare screening for adrenal insufficiency, delay in diagnosis and poor follow up of patients These insufficiencies should be overcome to improve the diagnosis and management of ZS in Tunisia Despite the SC absence of treatment options, early and prompt diagnosis of ZS is important for providing appropriate symptomatic care, definitive genetic testing and genetic counseling for families at AC C EP TE D M AN U risk 11 ACCEPTED MANUSCRIPT Acknowledgements We gratefully acknowledge all physicians and medical specialists who referred patient material to our laboratory The authors would like to thank Aida Abidli and Saloua Skhiri for RI PT their contribution Non standards abbreviations DHAPAT, dihydroxyacetone phosphate acyltransferase; VLCFA, very long chain fatty acids; AC C EP TE D M AN U SC ZS, Zellweger syndrome 12 ACCEPTED MANUSCRIPT References Lazarow PB, Moser HW Disorders of peroxisome biogenesis In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds The metabolic and molecular basis of inherited disease 7th ed New York: McGraw Hill; 1995, p 2287‒324 RI PT Wilson GN, Holmes RG, Custer J, Lipkowitz JL, Stover J, Datta N, et al Zellweger syndrome: diagnostic assays, syndrome delineation, and potential therapy Am J Med Genet 1986; 24:69‒82 Singh I, Moser AE, Goldfischer S, Moser HW Lignoceric acid is oxidized in the SC peroxisome: implications for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy Proc Natl Acad Sci USA 1984; 81:4203‒7 M AN U Goldfischer S, Moore CL, Johnson A, Spiro AJ, Valsamis MP, Wisniewski HK, et al Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome Science 1973; 182:62‒4 Kelley RI Review: 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81:185‒8 26 Govaerts L, Sippell WG, Monnens L Further analysis of the disturbed adrenocortical function in the cerebro-hepato-renal syndrome of Zellweger J Inherit Metab Dis 1989; EP 12:423‒8 27 Van Den Branden C, Collumbien R, Roels F, Dacremont G, Roels H: Altered AC C adrenocortical response under the influence of experimentally increased serum very long chain fatty acids in rats Pathol Res Pract 1993; 189:558‒62 28 Berendse K, Engelen M, Linthorst GE, Van Trotsenburg ASP, Tien Poll B The high prevalence of primary adrenal insufficiency in Zellweger spectrum disorders Orphanet J Rare Dis 2014; 9:133 29 Berendse K, Engelen M, Ferdinandusse S, Majoie CB, Waterham HR, Vaz FM, et al Zellweger spectrum disorders: clinical manifestations in patients surviving into adulthood J Inherit Metab Dis 2016; 39: 93‒106 15 ACCEPTED MANUSCRIPT 30 Steinberg S, Chen L, Wei L, Moser A, Moser H, Cutting G, et al The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum Mol Genet Metab 2004; 83:252‒63 31 Ebberink MS, Mooijer PAW, Gootjes J, Koster J, Wanders RJA, Waterham HR Genetic syndrome spectrum disorder Hum Mutat 2011; 32:59‒69 RI PT classification and mutational spectrum of more than 600 patients with a Zellweger 32 Furuki S, Tamura S, Matsumoto N, Miyata N, Moser A, Moser HW, et al Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation Pex6p complex J Biol Chem 2006; 281:1317‒23 SC group impair its stability, peroxisomal localization, and interaction with the Pex1p x M AN U 33 Tamura S, Yasutake S, Matsumoto N, Fujiki Y Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p J Biol Chem AC C EP TE D 2006; 281:27693‒704 16 ACCEPTED MANUSCRIPT Table Clinical features and plasma C24:0/C22:0 and C26:0/C22:0 ratios and C26:0 in Zellweger syndrome patients and controls P1-P40