NNT Mutations: a Cause of Primary Adrenal Insufficiency, Oxidative Stress and Extra- Adrenal Defects

175:1

F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 73–84 Clinical Study defects

NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra- adrenal defects

Florence Roucher-Boulez1,2, Delphine Mallet-Motak1, Dinane Samara-Boustani3, Houweyda Jilani1, Asmahane Ladjouze4, Pierre-François Souchon5, Dominique Simon6, Sylvie Nivot7, Claudine Heinrichs8, Maryline Ronze9, Xavier Bertagna10, Laure Groisne11, Bruno Leheup12, Catherine Naud-Saudreau13, Gilles Blondin13, Christine Lefevre14, Laetitia Lemarchand15 and Yves Morel1,2

1Molecular Endocrinology and Rare Diseases, Lyon University Hospital, Bron, France, 2Claude Bernard Lyon 1 University, Lyon, France, 3Pediatric Endocrinology, Gynecology and Diabetology, Necker University Hospital, Paris, France, 4Pediatric Department, Bab El Oued University Hospital, Alger, Algeria, 5Pediatric Endocrinology and Diabetology, American Memorial Hospital, Reims, France, 6Pediatric Endocrinology, Robert Debré Hospital, Paris, France, 7Department of Pediatrics, Rennes Teaching Hospital, Rennes, France, 8Pediatric Endocrinology, Queen Fabiola Children’s University Hospital, Brussels, Belgium, 9Endocrinology Department, L.-Hussel Hospital, Vienne, France, 10Endocrinology Department, Cochin University Hospital, Paris, France, 11Endocrinology Department, Lyon University Hospital, Bron-Lyon, France, 12Paediatric and Clinical Genetic Department, Correspondence Nancy University Hospital, Vandoeuvre les Nancy, France, 13Pediatric Endocrinology and Diabetology, should be addressed Bretagne Sud Hospital Center, Lorient, France, 14Pediatric Endocrinology, Jeanne de Flandre Hospital, to F Roucher-Boulez Lille, France, and 15Pediatric Department, La Rochelle-Ré-Aunis Hospital Group, La Rochelle, France Email [email protected]

Abstract

Objective: Nicotinamide nucleotide transhydrogenase (NNT), one of the several genes recently discovered in familial glucocorticoid deficiencies (FGD), is involved in reactive oxygen species detoxification, suggesting that extra-adrenal

European Journal European of Endocrinology manifestations may occur, due to the sensitivity to oxidative stress of other organs rich in mitochondria. Here, we sought to identify NNT mutations in a large cohort of patients with primary congenital adrenal insufficiency without molecular etiology and evaluate the degree of adrenal insufficiency and onset of extra-adrenal damages. Methods: Sanger or massive parallel sequencing of NNT and patient monitoring. Results: Homozygous or compound heterozygous NNT mutations occurred frequently (26%, 13 unrelated families, 18 patients) in our cohort. Seven new mutations were identified: p.Met337Val, p.Ala863Glu, c.3G>A (p.Met1?), p.Arg129*, p.Arg379*, p.Val665Profs*29 and p.Ala704Serfs*19. The most frequent mutation, p.Arg129*, was found recurrently in patients from Algeria. Most patients were diagnosed belatedly (8–18 months) after presenting severe hypoglycemia; others experiencing stress conditions were diagnosed earlier. Five patients also had mineralocorticoid deficiency at onset. One patient had congenital hypothyroidism and two cryptorchidism. In follow-up, we noticed gonadotropic and genitalia impairments (precocious puberty, testicular inclusions, interstitial Leydig cell adenoma, azoospermia), hypothyroidism and hypertrophic cardiomyopathy. Intrafamilial phenotype heterogeneity was also observed. Conclusions: NNT should be sequenced, not only in FGD, but also in all primary adrenal insufficiencies for which the most frequent etiologies have been ruled out. As NNT is involved in oxidative stress, careful follow-up is needed to evaluate mineralocorticoid biosynthesis extent, and gonadal, heart and thyroid function. European Journal of Endocrinology (2016) 175, 73–84

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10.1530/EJE-16-0056 Clinical Study F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 74 defects

Introduction systems involving enzymes such as GPX1 (glutathione peroxidase 1), TXNRD2 and PRDX3 (peroxiredoxin Primary adrenal insufficiency (PAI) is a life-threatening 3). NADPH is a cofactor of P450 enzymes, notably in disorder. Three types of PAI can occur: isolated steroidogenesis (19, 20, 21, 22) (Fig. 1). mineralocorticoid deficiency, isolated glucocorticoid Meimaridou et al. (9) showed reduced, basal and deficiency or combined mineralocorticoid and ACTH-stimulated corticosterone, revealing impaired glucocorticoid deficiency (global adrenal insufficiency). steroidogenesis in C57BL/6J mice with a spontaneous NNT Glucocorticoid deficiencies, also called adrenocorticotropic mutation (an in-frame 5 exon deletion). Furthermore, hormone (ACTH) resistance syndromes, are autosomal they showed increased ROS levels in an NNT knockdown recessive disorders. They include familial glucocorticoid human adrenocortical cell line. Oxidative stress impedes deficiency (FGD) (OMIM#202200) and triple A syndrome steroidogenesis, which in turn induces more oxidative (AAAS) (OMIM#231550), also known as Allgrove stress resulting from electron leaks throughout the syndrome (1, 2). Patients present episodes of hypoglycemia steroidogenic pathway. Why it affects adrenal hormone in the neonatal period or early childhood with low or production preferentially remains unknown. All tissues unquantifiable cortisol, elevated ACTH levels, and normal rich in mitochondria may be affected, resulting in a wide aldosterone and plasma renin measurements. Until 2012, spectrum of diseases. Phenotypically, C57BL/6J mice do only a half of FGD cases could be explained by homozygous not have adrenal defects but show glucose intolerance and or compound heterozygous mutations in genes involved impaired insulin secretion (23). At present, in humans, in the steroidogenic pathway: MC2R (25%), MRAP (20%), NNT mutations are known to be associated with adrenal STAR (5%), and more rarely CYP11A1 (3, 4, 5, 6, 7). Over insufficiency. Additionally, relationships between decreased the last 3 years, thanks to whole exome sequencing, NNT activity, modified mitochondrial redox regulation three more causative genes have been discovered: mini and cardiac failure have been recently reported (24, 25, 26). chromosome maintenance deficient 4 homolog (MCM4), The aim of our study was to screen for NNT mutations nicotinamide nucleotide transhydrogenase (NNT) and in 50 families with PAI with no identified molecular thioredoxin reductase 2 (TXNRD2) (8, 9, 10). etiologies and to perform a careful follow-up so as to As these genes encode proteins that work together identify any extra-adrenal defects. We found 13 families for reactive oxygen species (ROS) detoxification or DNA replication, the spectrum of pathogenic mechanisms H+ causing PAI is not limited to genes involved in adrenal NNT

European Journal European of Endocrinology ATP development and steroidogenesis. Innermitochondial ETC membrane synthase The incidence of gene mutations in the FGD NNT Matrix NADH NADP+ ADP ATP H+ cohort reported by Clark et al. (3) was around 10% (15 NAD+ families) and no predominant mutation was reported H+ NADPH GLUTATHIONE SYSTEM THIOREDOXIN SYSTEM (21 private mutations) (9). Twelve more families have NADPH NADPH been reported (12 additional mutations), some with mineralocorticoid defects (11, 12, 13, 14, 15, 16). GR TXNRD2

NNT encodes an integral protein of the inner GSSG GSH Trx(SH)2 TrxS2 mitochondrial membrane that acts as a proton pumping GPX PRDX3 transhydrogenase (17). In prokaryotic cells, the enzyme is H O H O H O H O composed of two or three different subunits, whereas in 2 2 2 2 2 2 O +e- O MnSOD eukaryotic cells, it is usually composed of a single subunit. 2 2- H2O2 The active form of the enzyme is always a homodimer Free radical&steroidogenesis of ~220 kDa. All NNTs show a similar structure with three major domains. Domain I contains the hydrophilic Figure 1 NAD(H) binding site and domain III contains the Role of NNT in free radical metabolism in the mitochondria. hydrophilic NADP(H) binding sites. Domain II constitutes ETC, electron transport chain; GSSG, glutathione disulfide; the hydrophobic transmembrane part of the enzyme that GSH, glutathione; GPX, glutathione peroxidase; GR, connects domains I and III and forms the proton channel glutathione reductase; TXNRD2, thioredoxin reductase; (18). NNT supplies the high concentrations of NADPH PRDX3, peroxiredoxin 3. A full colour version of this figure is needed for glutathione and thioredoxin antioxidant available at http://dx.doi.org/10.1530/EJE-16-0056.

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Downloaded from Bioscientifica.com at 10/01/2021 04:43:07AM via free access European Journal of Endocrinology preparation. Acustompanel targeting57genes,involved DNAs weretestedusing an amplicon-basedlibrary sequencing Massive parallelsequencing ornext-generation NP_036475 builtontheGRCh37/hg19. reference sequencesNC_000005.9,NM_012343.3and National CenterforBiotechnologyInformation(NCBI) recommendations (www.hgvs.org/rec.html) usingthe designated accordingtotheHumanGenomeSociety software v2.5(LifeTechnologies). Sequence variantswere loaded on an ABI-3730XL and analyzed using SeqScape using Big-Dye Terminators. Sequencing products were sequencing ofexonsandexon-intronboundarieswasdone primers (availableonrequest).Conventionaldideoxy gene wasperformedin20fragmentsbyPCRusingspecific Selective amplificationofthe21codingexonsNNT Sanger sequencing 13 inTable 1). parallel sequencing(MPS)forthree(patients11,12and Sanger sequencingwasdonefor47patientsandmassive blood using the Nucleon BACC3 kit (GE Healthcare). Genomic DNAwasextracted from EDTA-preserved whole Molecular geneticanalysisoftheNNTgene sequencing, aswerethoseinNR0B1forboys. STAR, negative, excludinganautoimmunedisorder. Mutationsin 21-hydroxylase deficiency, andadrenalautoantibodieswere normal limitsorlow, excludingadrenoleukodystrophyand 17-hydroxyprogesterone in all patients were either within of Helsinki.Very long-chainfattyacidsinboysand in accordance with the principlesof the Declaration by allenrolledpatientsandthestudywasconducted no moleculardiagnosis.Informedconsentwasprovided The Patients Patients andmethods thyroid andgrowth,weresubjectedtolong-termmonitoring. mineralocorticoid, puberty, fertility, heart, pancreatic, insufficiency atonset.Arangeoffunctions,suchasadrenal/ diagnosed withFGDand5globaladrenal (18 patients)withNNTmutations:13patientswere Clinical Study NNT genewasanalyzedin50patientswithPAI with were excluded by Sanger CYP11A1, MC2RandMRAPwereexcludedbySanger F Roucher-Boulez andothers request). in thesection‘Sangersequencing’(primersavailableon sequencing ofthePCRproductwasdoneasdescribed the supplier’s recommendations.Conventionaldideoxy the QiagenLongRangePCRKit(Qiagen)accordingto Technologies). Thiswasfollowedbylong-rangePCRusing CGH Microarray4x180KAMADID022060(Agilent instructions, usingtheAgilentSurePrintG3Human (CMA) was performed according to the manufacturer’s hybridization (aCGH) or chromosomal microarray To confirmadeletion,arraycomparativegenomic long-range PCR Array comparativegenomichybridizationand mutations werevalidatedbySangersequencing. with thesequenceranddefaultparameters.NNT pipeline usedwastheTorrent Suitesoftwareimplemented with thePGMSequencing200Kit.Thebioinformatics on theIonTorrent PersonalGenomeMachine(PGM) were loadedonanIon316Chip.Sequencingwasdone Technologies). Theresultingliveionsphereparticles(ISPs) emulsion PCRwiththeIonOneTouch 2System(Life template-positive Ion PGM spheres were prepared by (> 100×) at sequencing. For the sequencing step, enriched groups ofeighttogetasufficientdepthcoverage MA, USA).Thepatientswerebarcoded andpooledby high-sensitivity assaykit(CaliperLifeSciencesWaltham, DNA werevalidatedontheCaliperLabChip-GXusing Technologies). Enrichmentandquantificationoftarget Kitv2.0(Life instructions withtheIonAmpliSeqLibrary preparation wasdoneaccordingtothemanufacturer’s (article underway, listavailableonrequest).Thelibrary 50 bp) software (LifeTechnologies) (codingregions ± including in adrenalinsufficiencyanddisordersofsexdevelopment, software/genedoc). Genedoc (http://www.psc.edu/index.php/user-resources/ using defaultparameters,displayed andtheneditedusing ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) Uniprot database(http://www.uniprot.org/), alignedwith missense mutations.Thesequenceswerefoundinthe regionsandtopredictputative effectsof conserved from differentspecieswasusedtoanalyzestructurally Multiple sequencealignmentofNNTproteinsequences Multiple sequencealignment Pathogenicity prediction defects NNT, adrenalandextra-adrenal NNT, wasdesignedusingIonAmpliSeqdesigner Downloaded fromBioscientifica.com at10/01/202104:43:07AM 175:1 www.eje-online.org 75 via freeaccess Clinical Study F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 76 defects ( 15 ) Reference ( 9, 14 ) ( 9 ) ( 16 ) 2, 3, 4, 11 1, 4, 12 Family number 9 13 6 5 10 12 7 ExAC 0/121286 0/121286 0/121286 0/121286 0/121286 0/121286 0/121286 1/120146 0/121286 0/121286 Allele count ESP 0/13002 0/13006 0/13006 0/13006 0/13006 0/13006 0/13006 0/13006 0/13006 dbSNP ID NA NA NA NA NA NA NA NA NA causing causing causing Disease Mutation Taster Disease Disease damaging damaging damaging Probably Polyphen-2 Probably Probably Predictive software Predictive Deleterious SIFT Deleterious Deleterious Less likely GVGD Less likely Most likely European Journal European of Endocrinology dI Domain dI dI dI dI Pre-sequence dII TMH14 dII TMH9 dII TMH7 at amino acid 337 in the DH binding domain protein truncation at amino acid 129. NMD? truncation at amino acid 71. NMD? at amino acid 437 Role in dI-dII/dIII communication truncation at amino acid 379. NMD? at amino acid 863 in the transmem - brane domain (helix 14) premature truncation at amino acid 723 premature truncation at amino acid 694 of protein Missense mutation Protein Protein consequence Premature Premature Missense mutation Premature Start loss Missense mutation Frameshift: Frameshift: Start loss. Absence p.M337V p.R129* Protein change Protein (NP_036475) p.R71* p.P437L p.R379* p.M1? p.A863E p.A704Sf s *19 p.V665Pf s *29 p.0? 8 4 Exon 3 10 9 2 17 15 14 2–3 NNT mutations in 13 families with PAI. (381 + 1_382-1)del c.1009A>G c.385 C>T Table 1 Table Nucleotide change (NM_012343.3) c.211 C>T c.1310C>T c.1135C>T c.3G>A c.2588C>A c.2106_2109dup c.1992_2005del d, domain; TMH, transmembrane helix; NA, not applicable; NMD, nonsense-mediated decay. c.(-51 + 1_-53-1)_

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Downloaded from Bioscientifica.com at 10/01/2021 04:43:07AM via free access European Journal of Endocrinology splicing mutation.Afullcolour versionofthisfigureisavailableathttp://dx.doi.org/10.1530/EJE-16-0056. frameshift mutations;below:missense mutations.Newmutationsindicatedbyrectangles.Underlined mutationisprobablya NNT mutations.Comparisonbetween thedomainstructureofNNTproteinandexonsinhumans.Above:nonsense or Figure 2 found in 11 families. No consanguinity was found in in 8ofthe13familiesandhomozygousmutationswere 2).Consanguinitywaspresent of Algerianorigin(Table The p.Arg129*mutationwasfoundinfourfamilies,all p.Ala704Serfs*19), andonestartloss(c.3G>A(p.Met1?). and p.Ala863Glu),twoframeshift(p.Val665Profs*29 and (p.Arg129* andp.Arg379*),twomissense(p.Met337Val Table 1). Sevenofthemwerenewmutations:twononsense and gene, werefoundin13families(18patients)(Fig. 2 Ten different NNT genesequencing Results if variantshadalreadybeenreported. and ExACbrowserdatabasesweresearched todetermine effect onproteinstructureandfunction.ThedbSNP, EVS molecular volume)andmayindicateastronger(negative) amino acids in the chemical properties (i.e. polarity and Higher scoresindicategreaterdifferencesbetweentwo properties todefineascorerangebetween0and215. between aminoacids.Thisscorelooksatchemical score wascalculatedtopredicttheeffectofsubstitutions Polyphen-2, SIFTandMutationTaster. TheGrantham predicted For eachnewmissensemutation,pathogenicitywas Software anddatabases Clinical Study p.M1? p.S22Pfs*6 p.0? sequence p.W4 Pre 2 (del ex

in silicousingseveralprograms:Align-GVGD,

3Vfs*2

p.R71* NNT mutations, scattered throughout the 2-3) 3 p.D178G p.S193N

p.G200S

p.R129* p.Y2 4 Domain I,NA p.F215S 01Kf 5 s*1 NA D(H) 6 D(H) F Roucher-Boulez andothers 7 site p.M337V 9 p.T binding 357A p.H370*

8 p.H365P

p.H370R p.H421Sfs*4 p.Q383* p.R379* 9 p.Y3 p.Q452Rfs*42 p.P437L 88S 10

p.A533V 11 p.Q557X

p.D590Efs*29 12 Domain II,membranespanning by MPS,allvariantsfoundinothergeneswerebenign. PCR sequencedstepbystep.Forthethreepatientsstudied thereafter confirmed by aCGH analysis and long-range the mutation and thus a deletion was suspected and carry for themutationp.Arg71*.However, themotherdidnot heterozygotes. Patient12wasfirstthoughthomozygous France. Thepatientsoffamilies4and12werecompound parents infamily10werefromthesamesmallvillage the otherhomozygousfamilies(1,5and10),but highly conserved betweenspecies(Fig. 3).H14appearsto highly conserved transmembrane helix 14 (H14) of domain II and is betweenspecies(18,27)Fig. 3). identify conservation to locatethechangedresidueinproteinstructureand multiple alignmentsofNNTproteinsweredoneinorder part, theresultingNNTshouldbenon-functional. methionine atcodon192.IntheabsenceofitsN-terminal site, should switch this latter to an in-frame downstream c.3G>A (p.Met1?),affectingthetranslationinitiation nonsense-mediated decaysystem.The new mutation, ofthe or anabsenceofproteinduetointervention the consequencesshouldbeprematuretruncatedproteins described (9,15).Fortheframeshiftornonsensemutations, The p.Pro437Leuandp.Arg71*mutationshavealreadybeen Pathogenicity prediction defects NNT, adrenalandextra-adrenal

13 p.I622Dfs*1 p.G664R The p.Ala863Glumutationislocatedinthe To predictthepathogenicityofmissensemutations,

p.V665Pfs*2 p.T6 14 p.G678R

89Lf

s*32 p.A704Sfs*19 15

p.P799Qfs*22 16 p.G862D Downloaded fromBioscientifica.com at10/01/202104:43:07AM

p.A863E

17 p.M880*

18 p.Asp999Gl NA

175:1 DP(H) Domain III, 19

NA www.eje-online.org

p.L977P 100 DP(H) 20 p.A1008P binding

yf am s*2 p.N1009K 21 ino acid

site

22 77

s via freeaccess Clinical Study F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 78 defects 7 months 2 years No No No No No No No Sister at Brother at Related death subclinical hypothyroidism (TSH: 6.8 mUI/L at 5 years) hypothyroidism with thyroid gland in place Nl Nl Transient Transient Nl – – Nl Congenital Nl Nl Thyroid (11 years) (15 years) (12 years) myocardiopathy – Nl imaging (4 years) – Nl imaging – – Nl imaging Nl imaging – Hypertrophic Hypertrophic Heart - - symptoms puberty with 3 nodular testis (12.5 years) at 12 years, low testoster one at 13 years (11 years), two children (11 years), Nl menstrual cycle at 12 years, low testoster one at 15 years at 13 years 9 months at 12 years, Nl testicular function, surgery for varicocoele No pubertal Precocious Precocious Menarche Onset of puberty - Menarche Menarche Onset of puberty Onset of puberty Onset of puberty Gonads ↗ renin ( ↗ renin) adrenal crises with ↗ renin ↘ aldosterone (4, 7, 8 and 10 years) SW at 15 and 18 years years after an attempt to stop MC ttt renin) ttt ( ↗ renin) MC ttt No MC ttt No No MC ttt but MC ttt at 2 years No MC ttt but 4 MC ttt, SW at 12 MC ttt at 3 years ( ↗ Thirst for salt, MC Mineralocorticoid defect coma, SW convulsions asthenia, melanoderma, weight loss, SW fever, convulsions on ENT infections convulsions following gastroenteritis convulsions following gastroenteritis asthenia, melanoderma convulsions, SW convulsions, delay of diagnosis due to GC therapy for asthma melanoderma Hypoglycemic Hypoglycemic Hypoglycemia, Hypoglycemic Hypoglycemic Hypoglycemic Hypoglycemia, Hypoglycemic Hypoglycemic Hypoglycemia, Clinical data at age of diagnosis European Journal European of Endocrinology 9 13 8 19 10 1.5 (onset)/12 15 3 6 14 Age at diagnosis (months or years where indicated) 8 5 18 14 23 30 19 15 32 43 Age (years) F M F M F F F M M M Sex p.A863E p.A704Sfs*19 p.V665Pfs*29 p.M1?/p.M1? p.A863E/ p.A704Sfs*19/ p.V665Pfs*29/ p.R71*/p.R129* p.R129*/p.R129* p.R129*/p.R129* p.R71*/p.R71* NNT mutation NP_036475 yes Yes Yes No No Yes Yes No Consanguinity Clinical characteristics and follow-up of the patients with NNT mutations. Algerian Mauritian Moroccan Turkish French French/ Algerian Algerian French Origin 8 7 6 5 4b 4a 3 2b 2a 1 Table 2 Table Family

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Downloaded from Bioscientifica.com at 10/01/2021 04:43:07AM via free access European Journal of Endocrinology Clinical Study Table 2 (Continued).

Age at diagnosis (months or Clinical data at NNT mutation Age years where age of Mineralocorticoid Related Family Origin Consanguinity NP_036475 Sex (years) indicated) diagnosis defect Gonads Heart Thyroid death 9 Algerian Yes p.R379*/p.R379* M 10 22 (onset)/ Misdiagnosed at No Leydig cell Sub-Nl imaging at 6 Subclinical No 8 years 22 months adenoma years (LVEF at hypothyroidism (several (5 years) 75%) (TSH: 3.5 mUI/L at hyperthermic following by 5 years and 10.5 convulsions, precocious at 7 years), thyroid psychomotor puberty hormone retardation, treatment F Roucher-Boulez andothers sodium valproate ttt), all symptoms improved after glucocorticoid therapy initiated at 8 years 10a French No p.P437L/p.P437L M 57 4 years Melanoderma, Salt craving, MC ttt Nl puberty – – No asthenia 10b M 51 18 Melanoderma, Salt craving, MC ttt Nl testicular Nl imaging Nl No asthenia function at (51 years) 49 years defects NNT, adrenalandextra-adrenal 10c F 4* 16 Familial story, MC ttt – – – No deceased at 4 years 11a Algerian Yes p.R129*/p.R129* M 6 2 SW SW (2 months, Cryptorchidism Nl imaging (2 years) – Brother at 4 years), MC ttt (surgery) 8 months 11b M 8 4 years Hypoglycemia No Cryptorchidism Nl imaging (2 years) – Brother at (Nl at 3 years) (surgery) 8 months 12 French No p.R71*/del ex2-3 M 35 10 SW SW, MC ttt Bilateral TART, – Nl No azoospermia 13 Algerian Yes p.M337V/ F 9 8 Melanoderma, i↗ renin, ↘ No pubertal – Nl Sister at

Downloaded fromBioscientifica.com at10/01/202104:43:07AM p.M337V asthenia aldosterone symptoms 4 years (16 months, 12 years), salt, no MC ttt

Major abnormalities are in bold. For two patients (4a and 9), the age of diagnosis was late despite an earlier onset. –, not determined; M, male; F, female; Nl, normal; *, deceased; ttt, treatment; MC, 175:1 mineralocorticoid; GC, glucocorticoid; SW, salt wasting; ↘, decreased; ↗, increased; LVEF, left ventricular fraction; ENT, ear, nose, throat. www.eje-online.org 79 via freeaccess Clinical Study F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 80 defects

a Grantham score of 21. This variation was not listed in the databases (dbSNP, EVS or ExAC browser) and not found in 100 healthy controls from the Maghreb (Table 1).

Clinical data at onset and follow-up

Table 2 presents the clinical data and follow-up for the 18 patients reported. Only the predominant features are presented in the text.

Clinical presentation at age of diagnosis

Severe hypoglycemia, sometimes leading to coma, was the main symptom at age of diagnosis in all but two patients (numbers 11a and 12). That symptom was often associated with infections and melanoderma. This latter symptom, upon inquiry, was often reported to have been present Figure 3 before the hypoglycemia. Five out of the 13 families had Partial multiple amino acid alignment of NNT in human, experienced multiple deaths of other children; although bovine, mouse, Caenorhabditis elegans, Escherichia coli and not diagnosed at the time, those deaths too were probably Acetabularia acetubulum. Alignment was performed in due to adrenal insufficiency and severe hypoglycemia. ClustalW and edited with Genedoc. The mutant residues Patient 11a and 12 experienced salt wasting (SW) at p.M337V and p.A863E and corresponding amino acids are onset without hypoglycemia and three other patients shaded and show the conservation across all species. had a global adrenal insufficiency (patients 2b, 6, and 8) with SW. The median age at onset in our cohort was indirectly facilitate proton translocation by influencing 11.5 months (min–max: 1.5 months–4 years) (Fig. 4A and the centrally located H9, H10 and H13, in which the Table 2). Most cases were diagnosed belatedly around the proton channel is assumed to be located. In Escherichia first year of life (8–18 months), but some involving stress coli, mutations of certain residues in these regions result conditions were diagnosed earlier. A difference in age at European Journal European of Endocrinology in intermediate inhibitory effects (28). This mutation onset was detected between the subgroup with isolated may disrupt the conformational changes responsible for glucocorticoid deficiency and that with global adrenal interconversion of the open and occluded states (29). insufficiency. This was the case for both our cohort It may also play a role in coupling between the redox alone (Kruskal–Wallis test: P = 0.03379, Fig. 4B) and our state of the nucleotide and the proton movement in cohort aggregated with the data available in the literature the protein, as it is near the NADP(H) binding domain. (Kruskal–Wallis test: P = 0.003705, Fig. 4C). However, In in silico predictions, this mutation was most likely no difference in age at onset was found between the pathogenic using Align-GVGD class, probably damaging subgroup homozygous for non-truncated mutations and using Polyphen-2, deleterious using SIFT, and disease that homozygous for truncated mutations (Kruskal–Wallis causing using Mutation Taster. The high Grantham score test: P = 0.2172). of 107 is also concordant. This mutation is not reported in dbSNP, EVS or ExAC browser databases and has not been Follow-up found in 100 French Caucasian healthy controls (Table 1). As expected, the parents of patient 7 were heterozygous as At study end, the age of the 16 patients ranged from 4 to were his two healthy brothers. 57 years, permitting a long patient follow-up. The missense mutation, p.Met337Val, was identified –– Mineralocorticoid function: in the NAD(H) binding domain near the NAD(H) binding • Patient 3 had SW at age 15 then recurrence at 18, site. It should inhibit the hydride transfer from NADH to illustrating the importance of follow-up. Moreover, NADP+. The residue is highly conserved between species eight other patients (1, 2a, 4a–b, 10a–c, 13) had (Fig. 3). The mutation was predicted to be deleterious by all elevated renin and/or low aldosterone and needed of the mutation prediction tools mentioned above, despite mineralocorticoid or salt therapy.

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Downloaded from Bioscientifica.com at 10/01/2021 04:43:07AM via free access European Journal of Endocrinology – (P aggregated withthedataavailableinliterature deficiency withinourcohort(P glucocorticoid deficiency andthegroupwithsaltwasting ± *P Difference inageatpresentation(Kruskal–Wallis test, glucocorticoid deficiency;SW, saltwasting.(BandC) (A) AgeatpresentationforthepatientsofourcohortGD, Figure 4 – Clinical Study .075) (C). = 0.003705*) < Gonadotropic/genitalia function: • • • 0.05) betweenthegroupofpatientswithglucocorticoid hormone (LH):9 follicle-stimulating hormone(FSH)(luteinizing (TART). Hisazoospermiawasassociatedwithelevated were consistentwithtesticularadrenalresttumor age 18duringimagingstudiesforazoospermiaand For patient12,testicularinclusionsweredetectedat pubertal values. removed. Thereafter, testosteroneremainedatpre- analog therapywasdiscontinuedandtheadenoma growth hormone-releasinghormone(GNRH) Leydig cell adenoma. In this patient, a short revealinganinterstitial Patient 9hadsurgery undetectable gonadotropinsandhightestosterone. age 5,associatedwithtesticularnodules,lowor Two patients(7and9)hadprecociouspubertyat testes. forectopic surgery cryptorchidism andunderwent Patients 11aand11b,bothpresentedwith had noeffectonspermatogenesis. therapy didnotreducethe testicular inclusionand Increasing thedoseofhisglucocorticoid replacement normal (46,XY)withnoYchromosome microdeletion. was normal testosterone(5.8nmol/L). Hiskaryotype mUI/mL, FSH:18 = 0.03379*) (B);ourdata F Roucher-Boulez andothers mUI/mL), but mUI/mL), but – – This is in accordance with the literature, where the minimum suggesting theneedofastress totriggerthedisease(Fig. 4 A). conditions hadoccurred,that is,intercurrent infections, months). Some,however, werediscovered earlierifstress were diagnosedbelatedly, aroundthefirst year oflife(8–18 revealed by hypoglycemic convulsions. Most of the cases a severephenotype,withadrenalinsufficiencyoften pathogenic. OurpatientswithNNTmutationsdisplayed identified twonovelmissensemutations,whichshould be mutation similartop.Gly200SerinPalestine(12).We also Algerian families,suggestingthepossibilityofafounder in ourcohortwasp.Arg129*,whichidentifiedfour absence ofprotein.Themostfrequentmutationfound and mostledtoaprematuretruncatedproteinoran 26% ofthe50familiesstudied. insufficiency, whoweremembersof13families,thatis, in 18patients, 11 with FGD and7with global adrenal Here, wereportsevennewNNTmutationsidentified Discussion defects NNT, adrenalandextra-adrenal – – Heart function: Other functions: • • • • • • The mutations were distributed throughout the gene The mutationsweredistributedthroughoutthegene regurgitation. The left atrium was dilated (25 was measured at 15 The restingleftventricularoutflowgradient thickness measuredatthebasilarseptum:36 left age 23,showedatypicalandsevereasymmetrical A transthoracic echocardiography in patient 1, at ism withlowfreethyroxine(T hypothyroidism andpatient9hadhypothyroid- a thyroid gland in place. Patient 2b had congenital Two patients(2band9)hadhypothyroidismwith varicocele withepididymitis. Patient 1hadatesticularbiopsyatage31forleft with aleftventricularejectionfractionof75%. Patient 9atage6hadnormalheartfunction,but disorders forpatientswhoreachedadultage. or impairedglucosetolerance.Therewerenogrowth None ofthepatientspresentedpancreaticdysfunction hypoglycemia. mance or acquisitiondelays, possibly dueto severe and 9)wereexperiencingpooracademicperfor- all thepatientsbutfourofthem(patients3,5,7, We didnothaveinformationonsocialaspectsfor (4b, 11a,and13). tract infections Three patients had recurrent urinary no goiteratage7. elevated thyroid-stimulating hormone (TSH) with ventricular hypertrophy(maximalwall Downloaded fromBioscientifica.com at10/01/202104:43:07AM mmHg. There was no mitral 175:1 4 ) atage5andan www.eje-online.org mm mm). 81 via freeaccess 2 ).

Clinical Study F Roucher-Boulez and others NNT, adrenal and extra-adrenal 175:1 82 defects

age at diagnosis is 3 days (12) and the median age at onset is 7 and 9, gonadotropins were in the normal pre-pubertal 12 months for the 29 patients for whom data are available range and the increase in testosterone seemed to be due (9, 11, 12, 13, 15, 16). Unlike the data by Jazayeri et al. to secretion by autonomous nodules responsible for the (2015), our data suggest earlier onset in patients with global onset of puberty. Since the regression of puberty for patient adrenal deficiency compared with those with isolated 9 was due to either the removal of the adenoma or the glucocorticoid deficiency (Fig. 4B and C). The phenotypic short GNRH analog treatment, we cannot pronounce as to variability between patients having the same mutation or the central, peripheral or mixed origin of the precocious within the same family (family 2) suggests that there is no puberty. Reporting on a boy with a mutation in DAX-1/ correlation between genotype and phenotype. NR0B1, Domenice et al. (32) concluded that chronic It is clear that NNT mutations can result in global excessive ACTH levels may stimulate Leydig cells and lead adrenal deficiency. In the literature and comparably to 14 to gonadotropin-independent precocious puberty, a view of our patients, 5 families were recently described with shared by Hershkovitz et al. (13) as well. In contrast, the mineralocorticoid deficiency present at onset and 3 others testicular inclusions of our patient 12, associated with with elevated renin or electrolyte imbalance (11, 12, 13, azoospermia but normal testosterone values at age 18, 16). Our patient 3 had salt wasting at age 15, although although not reduced by glucocorticoid therapy, should be aldosterone requirements normally decrease throughout TART, often found in congenital adrenal hyperplasia (33). life (30). We observed phenotypic heterogeneity even To date, we have observed heart function impairment within the same family. Patient 11a presented salt wasting, (progressive hypertrophic cardiomyopathy) in only one whereas patient 11b had no mineralocorticoid deficiency. of our patients, but cannot exclude future cases because This emphasizes the need for careful monitoring of this of the mean age of our cohort and the subnormal imaging function, since some patients classified as FGD may of patient 9. This underlines the specific role of NNT in also have a slight mineralocorticoid defect. It has been heart tissue. In B6J-Sod2–/– mice, the presence of a normal shown that C57BL6/J mice carrying NNT mutations NNT allele preserves cardiac function, delays the onset of have disorganized zonae fasciculata with higher levels heart failure, and extends survival to the end of gestation of apoptosis (9). As aldosterone requirement decreases (24). In comparison, the suppression of NNT in zebrafish through life, the mineralocorticoid defect may be the results in ventricular malformations and contractile consequence of extended damage to all adrenal zona. dysfunctions (34). Moreover, in humans, relationships NNT has a role in the oxidative stress response between decreased NNT activity, modified mitochondrial and mutations in it may thus affect all tissues rich in redox regulation and cardiac failure have been reported. European Journal European of Endocrinology mitochondria. For this reason too, patients with NNT In the failing human heart, a partial loss of NNT activity mutations need to be closely monitored. Two in vitro studies adversely affects NADPH-dependent enzymes and the on the fibroblasts (12) and lymphocyte mitochondria (31) capacity to maintain membrane potential. This contributes of patients homozygous for missense NNT mutations to a decline in bioenergetic capacity, redox regulation and showed an increase in ROS levels, a decrease of ATP antioxidant defense, exacerbating oxidative damage to content, and impaired morphology of mitochondria with cellular proteins (26). A recent report of a heterozygous reduced mitochondrial mass and increased mitochondrial frameshift mutation of NNT in humans with left ventricular DNA deletion due to a lack of thymidylate biosynthesis noncompaction supports the assumption that NNT plays a (12, 31). The results from those two studies suggest that all major role in myocardium (34). However, Nickel et al. (35) tissues can be injured, as do our results from the follow-up demonstrated a completely opposing view. They reported of patients with extra-adrenal defects. that during heart pressure overload, NNT adopts a reverse Although NNT is widely expressed in adrenal, heart, mode contributing to oxidative stress from which mice kidney, thyroid, and adipose tissues, the most affected with mutation in NNT are protected. Those puzzling new tissue in our cohort appeared to be the gonads (9). Two insights may suggest that the functional mode (forward of our patients had cryptorchidism (patients 11a and or reverse) of NNT is dependent on the metabolic state. b) and two others (7 and 9) presented similar histories Nevertheless, TXNRD2 is in the same pathway of ROS involving, both at about 5 years of age, the development detoxification andTXNRD2 heterozygous mutations in of palpable nodules on the testicular surface or testicular humans have also been linked to dilated cardiomyopathy. enlargement followed by the onset of puberty with high Thus, for now, cardiac follow-up should be done (10, 36). testosterone levels. These last two cases are comparable to The thyroid gland, highly exposed to oxidative stress, that reported by Hershkovitz et al. (13). For our patients was the third most-affected organ in our cohort. Beyond

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Downloaded from Bioscientifica.com at 10/01/2021 04:43:07AM via free access European Journal of Endocrinology cells from H produced forTHbiosynthesisandantioxidantstoprotect NNT mutationsmaydisturbthebalancebetweenH who areunderunusualincreasedoxidativestress(39). of thyroiddysfunctioninpatientswithDownsyndrome inhibited (37,38).Anotherargumentistheprevalence ROS, thyroidperoxidaseandiodideorganificationare when thyroidcellsareexposedtosignificantamountof of peroxide.However, twostudieshavesuggestedthat biochemical reaction that depends on the formation biosynthesis ofthyroidhormone(TH)isanoxidative subclinical hypothyroidismhavebeenreported(16).The some hormonesynthesisdefect,twoothercaseswith our twopatientswithhypothyroidism,probablydueto perceived asprejudicingtheimpartiality oftheresearchreported. The authorsdeclarethatthereis no conflictofinterestthatcouldbe Declaration ofinterest mutations inboyswithPAI withorwithoutDSD. of PAI arebeingdescribed,forexample,STAR andCYP11A1 heterogeneity. We notethatmore and more‘atypical’cases way toreachadiagnosisindiseaseswithphenotype The analysisofmorethanonegeneatatimeispowerful NNT, appearstobeefficientforgeneticdiagnosis(16,42 ). ‘Patients and methods’, with a large panel of genes including for allpatients.TheMPSapproachdescribedinthesection fertility, heartandthyroidfunction,must bemaintained careful follow-up,especiallyformineralocorticoid,puberty, in FGD,butalsoglobaladrenalinsufficiency. Aboveall, authors, mutationsinNNTshouldbesearched not only cohort. Deducingfromourresultsandthoseofother etiologies inour‘atypical’congenitaladrenalinsufficiency NNT gene,whichwasoneofthemostfrequentmolecular tofurtherinvestigaterenalfunction. necessary tract infections.Thus,wefeelthatadditionalstudiesare that, threeofourpatientsexperiencedrecurrenturinary immunity againstbacterialcytoinvasion(41).Despite sensitive (40). Increased ROS usually plays a role in innate in contrasts withtheimpairedinsulinsecretionobserved metabolism havealsobeenimplicatedindiabetes.This NNT mutation, although defects in mitochondrial energy diabetes inhumanshasnotbeenreportedthesettingof growth and glucose metabolism. Glucose intolerance or may bemutated. cannot exclude that another gene involved in the thyroid with hypothyroidismwereconsanguineousandwe to THformationinhibition.Nevertheless,ourpatients Clinical Study NNT mutantmiceforwhichonlytheβ-cellsseemed In conclusion, we reported here mutations in the In conclusion,wereportedheremutationsinthe Other functionsinourpatientswerenormal,especially 2 O 2 -mediated oxidative damages, thus leading F Roucher-Boulez andothers 2 O 2

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Received 19 January 2016 Revised version received 18 April 2016 Accepted 29 April 2016

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