Thierry Dupré

Sandrine Vuillaumier-Barrot

Isabelle Chantret

Hassane Sadou Yayé

Christiane Le Bizec

Alexandra Afenjar

Cecilia Altuzarra

Christine Barnérias

Lydie Burglen

Pascale De Lonlay

Sadou Yayé

Le Bizec

De Lonlay

F Feillet

S Napuri

S E H Moore

GDP-mannose: GlcNAc2-PP-dolichol mannosyltransferase deficiency (CDG Ik): 5 new patients and 7 novel mutations

Keywords: GDP-mannose: GlcNAc 2 -PP-dolichol mannosyltransferase (MT-1) deficiency, Congenital Disorders of Glycosylation type Ik (CDG Ik), protein N-glycosylation, ALG1-CDG

INTRODUCTION

Type I Congenital Disorders of Glycosylation (CDG I) are rare autosomal recessive metabolic disorders affecting protein N-glycosylation [START_REF] Jaeken | Congenital disorders of glycosylation: a rapidly expanding disease family[END_REF] . About 1000 cases have been identified worldwide. The diseases present with multisystemic signs, and their biochemical hallmark is the presence of hypoglycosylated serum glycoproteins [START_REF] Freeze | Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond[END_REF] . The underlying deficits in CDG I have been shown to affect steps in the biosynthesis of the lipid-linked oligosaccharide (LLO) precursor that is required for N-glycosylation [START_REF] Aebi | Congenital disorders of glycosylation: genetic model systems lead the way[END_REF] . LLO is generated by the successive additions of 1 residue of GlcNAc-P, 1 residue of GlcNAc, 9 residues of mannose and 3 residues of glucose to dolichol phosphate to generate Glc 3 Man 9 GlcNAc 2 -PP-dolichol. The oligosaccharide moiety of this LLO is transfered onto nascent polypeptides to yield Nglycosyl glycoproteins and dolichol pyrophosphate (dolichol-PP). The so called dolichol cycle is then completed by the regeneration of dolichol-P from dolichol-PP [START_REF] Burda | The dolichol pathway of N-linked glycosylation[END_REF] . Many proteins are required for dolichol recycling. So far, mutations in genes encoding 15 of them have been shown to underly CDG I. CDG I are subtyped according to the defective protein [START_REF] Jaeken | On the nomenclature of congenital disorders of glycosylation (CDG)[END_REF] .

Phosphomannomutase 2-deficiency (CDG Ia) is the commonest form of CDG I with more than 600 cases described world wide, and other CDG I subtypes appear to be much rarer [START_REF] Grunewald | Congenital disorders of glycosylation: a review[END_REF] .

Identification of the molecular bases of type I CDG is important because first, it enables the generation of antenatal tests for the affected families; second, mannose phosphate isomerase (MPI)-deficiency (CDG Ib) is treatable; and third, the establishment of potential genotype/phenotype relationships for the different CDG I subtypes could potentially facilitate future diagnostic procedures.

Here we report that skin biopsy fibroblasts from 5 patients with severe type I CDGlike clinical presentations reveal abnormal accumulations of the immature LLO intermediate GlcNAc 2 -PP-dolichol, and display less than 10% normal GDP-mannose: GlcNAc 2 -PPdolichol mannosyltransferase (MT-1) activity. Overall 9 mutations in ALG1 encoding MT-1 were identified. Seven novel mutations are described and the clinical presentations of these 5 MT-1 deficient (CDG Ik) patients are compared to those of the 5 previously described cases 7- 10

MATERIALS, METHODS AND PATIENTS

Patients -3 girls (patients P1, P2, P4) and 2 boys (patients P3, P5) were diagnosed with type I CDG of unknown molecular origin at the ages of 4 months, 16 months , 10 months, 18 months and 3 years and 7 months respectively. The diagnosis was made after Western Blot of serum proteins using blood samples collected onto paper as previously described [START_REF] Seta | Diagnostic value of Western blotting in carbohydrate-deficient glycoprotein syndrome[END_REF] . After parental consent a skin biopsy was performed on the forearm of each child. For gene studies, signed informed consent protocols were obtained from all parents.

Culture and metabolic radiolabeling of cultured skin biopsy fibroblasts -Skin biopsy fibroblasts from two control subjects and the 5 patients were prepared and cultivated 12 as previously described. Cells were metabolically radiolabelled for 30 minutes in RPMI 1640 medium containing 0.5 mM glucose and 2% dialysed foetal calf serum with either [2-3 H]mannose (21.5 Ci/mmol, Perkin Life Sciences, FR) or [6-3 H]glucosamine (37.7 Ci/mmol, Perkin Life Sciences, FR).

Recovery of metabolically radiolabeled LLO -Subsequent to radiolabelling, LLO were extracted from cells as previously described [START_REF] Chantret | Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase[END_REF] except that the CHCl 3 fractions from the CHCl 3 /methanol/water extracts were pooled with the 10/10/3 fractions before analysis.

Analysis of lipid linked oligosaccharides -Lipid extracts were dried down, subjected to acid hydrolysis (20 mM HCl) and released oligosaccharides were resolved by TLC using either system A: silica coated plastic sheets (Merck KGaA, DE) developed for 18 h in n-propanol: Mutation analysis -Genomic DNA was extracted from blood. RNA was isolated from fibroblasts or from fresh blood cells. Sequencing was performed with the BigDye terminator kit (Applied Biosystems, CA, USA) and analysed on an ABI PRISM 3130 sequencer (Applera, CA, USA). The ALG1 gene (NM_019109.4) was first sequenced on genomic DNA.

Primers were designed to amplify all 13 coding exons and flanking intronic sequences, with selected 3'ends matching the correct ALG1 sequence and not those of the ALG1 pseudogenes.

At the RNA level, primers were designed to amplify cDNA in five fragments from exon 1 to the 3'UTR region. Primer sequences are available on request.

To exclude common polymorphisms, 82 unrelated healthy individuals served as control subjects were sequenced in the region of each of the missense mutations (164 alleles Assay of mannosyltransferase-2 and -3 activities -Microsomes generated from HepG2 cells [START_REF] Dancourt | A new intronic mutation in the DPM1 gene is associated with a milder form of CDG Ie in two French siblings[END_REF] were incubated with 1 µM GlcNAc 2 -PP-dolichol and GDP-[ 14 C]mannose for 10 min and the resulting [ 14 C]LLO were extracted and purified as described above then incubated with 20 µg microsomal proteins derived from a control subject (Ctrl 2) and the 5 patients in either the absence or presence of 20 µM GDP-Man (Sigma) as described above. After extraction of reaction mixtures with organic solvents, radioactive components recovered from the CHCl 3 phase were further characterised after hydrolysis with 20 mM HCl. Released sugars were examined using TLC system A.

RESULTS

Patients and clinic -Clinical presentations of the five patients are detailed in the Table.

Pregnancies associated with patients P4 and P5 were uneventful whereas pregnancy induced maternal hypertension developed with those associated with P2 and P3, and for P1 and P3 Clinical biochemistry -Western blot analysis of the serum glycoproteins transferrin, haptoglobin, orosomucoid and α1-antitrypsin revealed the presence hypoglycosylated glycoforms in all patients (Figure 1A). In order to identify the molecular origins of this phenomenon, PMM activity, known to be depressed in CDG Ia, the commonest CDG I subtype [START_REF] Matthijs | Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome (Jaeken syndrome)[END_REF] , was measured in cell extracts from these patients, and in all cases found to be normal. shown to be indicative of a deficiency in GDP-mannose: GlcNAc 2 -PP-dolichol mannosyltransferase (MT-1) that adds the first mannose residue onto LLO [START_REF] Grubenmann | Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik[END_REF][START_REF] Kranz | Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I[END_REF][START_REF] Schwarz | Deficiency of GDP-Man:GlcNAc2-PPdolichol mannosyltransferase causes congenital disorder of glycosylation type Ik[END_REF] .

Metabolic radiolabelling of lipid linked oligosaccharides

Mutation analysis of ALG1

-MT-1 is encoded by ALG1, and the 13 exons and intron-exon boundaries of genomic ALG1 were sequenced using primers designed to discriminate between this gene and its documented pseudogenes. Where appropriate, cDNA was also sequenced.

Nine mutations were encountered in the genomic DNA prepared from the five patients (Figure 2 Patient P2 is homozygous with respect to the c.1129A>G (p.Met377Val) mutation that is predicted in silico to be deleterious. Allelic inheritance was confirmed in all cases by analysis of DNA obtained from the parents, or RNA in the case of patient P1.

Haplotype studies revealed a possible founder effect for the frequent p.Ser258Leu mutation observed in the three non-related families. In contrast, no specific common haplotype was associated with p.Arg276Trp and the c.1263G>A splicing mutation, that were each observed in two families (data not shown).

In vitro assays of microsomal GDP-Man mannosyltransferases -The mutations described above are not all predicted to perturb protein function so MT-1 activity was measured in the fibroblast cell lines. To this end, microsomes were prepared from cells from the 5 patients and incubated with GDP-[ 14 C]Man in either the absence or presence of GlcNAc 2 -PP-dolichol.

Microsomes derived from the control subject are able to incorporate substantial amounts of radioactivity into lipid components only when GlcNAc 2 -PP-dolichol is added to the incubation mixtures (Figure 3). By contrast, the microsome preparations generated from cells derived from the patients manifested less than 10% of control GlcNAc 2 -PP-dolicholdependent synthesis of radioactive lipid components (Figure 3A,B). In order to examine the radioactive products generated by microsomes derived from the 5 patients under our assay conditions, LLO-derived oligo-, and monosaccharides were resolved by TLC as shown in The ensemble of these assays indicates that only MT-1 is defective in the 5 patients.

Finally, the origin of the high GlcNAc 2 -PP-dolichol-independent incorporation of radioactivity into lipids by microsomes originating from patient P4 (Figure 3A) was investigated. Data shown in Figure 4C demonstrate that these microsomes generate substantially more dolichol-P-[ 14 C]Man than the other microsome preparations, and further experiments are being conducted in order to examine the origin of this phenomenon.

DISCUSSION

Here we report upon 5 CDG I patients whose skin biopsy fibroblasts manifest less than 10% normal MT-1 activity. Sequencing of ALG1 revealed 9 mutations, 2 of which (p.Ser150Arg 8 , p.Ser258Leu [START_REF] Grubenmann | Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik[END_REF][START_REF] Kranz | Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I[END_REF][START_REF] Schwarz | Deficiency of GDP-Man:GlcNAc2-PPdolichol mannosyltransferase causes congenital disorder of glycosylation type Ik[END_REF] ) have been demonstrated to be pathogenic. The c.1263G>A mutation leads to loss of exons 12 and 13 and a premature stop codon that causes a 69 amino acid truncation of the MT-1 protein. Interestingly, the underlying mutation in ALG1 of the temperature sensitive alg1-1 S. cerevisiae strain 18 also causes a C-terminal truncation, and it has been suggested that this region of the protein is important for its interactions with yeast MT-2 and MT-3 [START_REF] Gao | Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum[END_REF] . One or both of two novel mutations (c.740G>A/c.740+5G>A) at the exon6/intron6 splice site junction leads to skipping of exon 6 and generation of an enzyme with a 36 amino acid deletion. This deleted region contains two amino acids (Asp 216, Phe 222) that are conserved in yeast and mammals. Finally, 4 novel point mutations have been identified. The p.Met377Val mutation is predicted to be deleterious by all four of the pathogenicity software packages (supplemental online table). Met 377 is conserved in yeast and mammals and lies between 2 similarly conserved aspartic acid residues whose substitution affects yeast MT-1 activity [START_REF] Gao | Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum[END_REF] . Pathogenicity predictions for the p.Arg438Trp substitution are ambiguous but Arg 438 is adjacent to the C-terminal region that, in the yeast enzyme, affects interactions with MT-2 and MT-3 as described above. Likewise, pathogenicity predictions for the p.Arg276Trp and p.Gly145Asp substitutions are ambiguous but these residues are situated adjacent to the previously described p.Ser258Leu and p.Ser150Arg pathogenic mutations, respectively.

Five patients with CDG Ik were described in 2004 [START_REF] De Koning | Recurrent nonimmune hydrops fetalis associated with carbohydrate-deficient glycoprotein syndrome[END_REF][START_REF] Grubenmann | Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik[END_REF][START_REF] Kranz | Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I[END_REF][START_REF] Schwarz | Deficiency of GDP-Man:GlcNAc2-PPdolichol mannosyltransferase causes congenital disorder of glycosylation type Ik[END_REF] , and because no further case reports have appeared, the 5 patients described here double the descriptions of patients with this disease. CDG Ik seems to be associated with a severe clinical picture [START_REF] De Koning | Recurrent nonimmune hydrops fetalis associated with carbohydrate-deficient glycoprotein syndrome[END_REF][START_REF] Grubenmann | Deficiency of the first mannosylation step in the N-glycosylation pathway causes congenital disorder of glycosylation type Ik[END_REF][START_REF] Kranz | Congenital disorder of glycosylation type Ik (CDG-Ik): a defect of mannosyltransferase I[END_REF][START_REF] Schwarz | Deficiency of GDP-Man:GlcNAc2-PPdolichol mannosyltransferase causes congenital disorder of glycosylation type Ik[END_REF] . Four of the five originally described patients died in the first year of life. Of the five pateints described here, only one has died. By contrast, serious neurological complications are a constant feature with central hypotonia (4/5 our patients and 5/5 originally described patients) and epilepsy (5/5 and 5/5 cases, respectively) being particularly prominent. Occular complications ranging from absence of visual contact (2/5 and 1/4) to blindness (1/5 and 1/4) are noteworthy. With the exception of microcephaly described in 3/5 of the patients described here, dysmorphias are not constant and when present they appear to be variable. Recurrent infections and/or complications of the immune system were only noted in one of the presently described patients compared to 4/5 of the originally described cases. Another notable difference between the originally described cases and those described herein is the absence of hepatic and renal complications in the latter patients. The 773 C>T (p.Ser258Leu) mutation is present in all the previously described cases, and in two, homozygosity is associated with fatal outcome. In our study, this mutation was only found in patient P4 and occured in the heterozygous state. All other factors being equal, it might be that the p.Ser258Leu mutation may be more severe than the p.Met377Val mutation, present in the homozygous state in P2.

Despite these observations, with only 9 patients harbouring 11 different mutations it is difficult to establish potential genotype/phenotype relationships. All CDG Ik descriptions include severe neurological presentations that are also observed in other CDG I subtypes with the exception of CDG Ib where only visceral hepato-intestinal manifestations are observed [START_REF] De Lonlay | The clinical spectrum of phosphomannose isomerase deficiency, with an evaluation of mannose treatment for CDG-Ib[END_REF] .

In

FIGURE LEGENDS

Mutations found in genomic DNA corresponding to the exons and intron/exon junctions of the human ALG1 gene derived from patients. Genomic and or cDNA were prepared from blood leukocytes or cultured skin biopsy fibroblasts as described in Materials, Methods and Patients.

Apart from the maternally inherited synonymous mutation in exon 7 detected in patient 4, the genotypes of patients P1-5 are given in the Table . Where possible (P2-P5), allelic inheritance is indicated.

  acetic acid: water, 3/2/1, or system B: cellulose-coated plastic sheets (Merck KGaA, DE) developed in ethyl acetate: pyridine: water: acetic acid, 5/5/3/1 for 8-18 h. Radioactive components were visualised by fluorography after spraying the chromatograms with En 3 Hance spray (Perkin Life Sciences, FR). Radiolabeled di-N-acetylchitobiose ([ 3 H]GlcNAc 2 , BIOTREND GmbH, DE) was used as standard.

  foetal growth was retarded. At birth, neurological signs were noted: central hypotonia and psychomotor delay were present. All patients presented with at least one episode of epilepsy; ranging in severity from a unique treatable episode for patient P2 to multiple intractable seizures in patient P4. Exploration of neurological occurrences by EEG revealed more or less serious abnormalities for all children except P2. Using MRI, P2 and P5 presented normally at the time of examination. After MRI examinations at 18 days and 18 months, patient P1 revealed a progressive cerebellar hypoplasia. Patients P1, P3 and P4 presented with cortical atrophy. Finally, dysmorphias were found to be present to differing degrees in all children with microcephaly (-2.5 for P5) being noted for 4/5 patients. With the exception of P2 who presented with less severe neurological signs it is notable that the other children presented with ocular problems ranging from simple strabismus to partial blindness. Liver and kidney function appeared normal in all children, and when explored, haematological complications and coagulopathy were absent. Only patient P1 has died (respiratory insufficiency at 4 years and 9 months). These clinical pictures are compatible with those noted for type I CDG in which hypoglycosylation of serum glycoproteins is a hallmark.

  -In order to identify potential blocks in the biosynthesis of the LLO precursor required for N-glycosylation, skin biopsy fibroblasts derived from the 5 patients and 2 control subjects were first metabolically radiolabeled with [2-3 H]mannose. The use of this radioisotope allows detection of all LLO intermediates containing mannose (Man 1 GlcNAc 2 -PP-dolichol -Glc 3 Man 9 GlcNAc 2 -PPdolichol). Analysis of [ 3 H-Man]oligosaccharides released from LLO by mild acid hydrolysis

c

  Deduced from mRNA revealed no significant differences between the distribution of these components generated in cells from either the patients or control subjects (Figure1B, lower panel) Blocks in the second and third steps in LLO production potentially lead to the accumulation of GlcNAc-PP-dolichol and GlcNAc 2 -PP-dolichol and these intermediates can be detected after metabolic radiolabeling of cells with [ 3 H]glucosamine. Examination of [ 3 H]GlcNAclabelled oligosaccharides released from LLO by mild acid hydrolysis reveals a disaccharide that comigrates with standard di-N-acetylchitobiose (GlcNAc 2 ) in pathological but not normal cells (Figure1B, upper panel). An accumulation of GlcNAc 2 -PP-dolichol has previously been

Figure 2 (

 2 Figure 4A-C. When incubated with GlcNAc 2 -PP-dolichol and GDP-[ 14 C]Man, control microsomes yielded substantial quantities of components migrating as Man 1-5 GlcNAc 2 (Figure 4A), consistent with the capacity of MT-1, GDP-mannose: Man 1 GlcNAc 2 -PPdolichol mannosyltransferase (MT-2) and GDP-mannose: Man 3 GlcNAc 2 -PP-dolichol mannosyltransferase (MT-3) to generate [ 14 C]Man 1-5 GlcNAc 2 -PP-dolichol in a GlcNAc 2 -PPdolichol-dependent manner. Microsomes derived from cells of the 5 patients generate reduced amounts of all the [ 14 C]Man 1-5 GlcNAc 2 -PP-dolichol species (Figure4A). These results are compatible with but do not prove MT-1 deficiency. In order to rule out a general reduction in GDP-Man requiring mannosyltransferase activity in these microsome preparations, MT-2 and MT-3 that act subsequent to MT-1 in the dolichol cycle were examined (Figure4B). Microsomes were incubated with exogenously added [ 14 C]Man 1-2 GlcNAc 2 -PP-dolichol in either the absence or presence of GDP-Man. Results demonstrate that control and patient microsome preparations are similarly capable of elongating exogenously added [ 14 C]Man 1- 2 GlcNAc 2 -PP-dolichol to yield predominantly [ 14 C]Man 5 GlcNAc 2 -PP-dolichol (Figure 4B).

1

 1 Figure Legend 1 Serum glycoprotein hypoglycosylation and defective lipid-linked oligosaccharide biosynthesis in 5 patients with type I CDG -A. Western blot analysis of serum proteins derived from control subjects (Controls), patients P1-5, and a patient diagnosed with CDG Ia (Ia). Migration positions of normal transferrin (TFR), haptoglobin (HAP), orosomucoid (ORO) and alpha 1 antitrypsin (AAT) are indicated by arrowheads whereas hypoglycosylated isoforms of these glycoproteins are indicated by lines. B. Fibroblasts derived from control subjects 1 and 2 (different from above control subjects) and the 5 patients (P1-5) were radiolabeled with either [6-3 H]glucosamine ([ 3 H]GlcNH 2 , upper panel) or [2-3 H]mannose ([ 3 H]Man, lower panel) and oligosaccharides released from LLO by mild acid hydrolysis were examined by TLC using system B (upper panel) or system A (lower panel). The

Figure Legend 3

 3 Figure Legend 3 In vitro LLO biosynthesis in microsomes derived from fibroblasts of a control subject and patients -A. Increasing amounts of microsomes derived from control subject 2 (Ctrl 2) and the 5 patients (P1-5) were incubated with GDP-[ 14 C]Man in either the absence (-) or presence (+) of GlcNAc 2 -PP-dolichol (GN 2 -PP-dol) for 20 minutes. After stopping the reactions by the addition of organic solvents [ 14 C]LLO was quantitated by scintillation counting. The results were obtained from a single experiment. B. The capacity of microsomes to incorporate radioactivity into lipid components in a GlcNAc 2 -PP-dolichol-dependent manner was calculated from the 20 µg data point shown in A and expressed as a percentage of the control (Ctrl2).

Figure Legend 4

 4 Figure Legend 4Further evaluation of the mannosyltransferase activities in microsomes derived from cells of patients with CDG I -A. Microsomal MT-1, MT-2 and MT-3 mannosyltransferases generate [ 14 C]Man 5 GlcNAc 2 -PP-dolichol using exogenously added GlcNAc 2 -PP-dolichol and GDP-[ 14 C]Man. Using lipid fractions derived from the incubations described in Figure3, LLO were subjected to mild acid hydrolysis, and liberated oligosaccharides were resolved by TLC using system B. The abbreviations used are M 1 GN 2 , Man 1 GlcNAc 2 ; M 5 GN 2 , Man 5 GlcNAc 2 . B. To assay MT-2 and MT-3, a preparation of [ 14 C]Man-labelled LLO enriched in the MT-1 product Man 1 GlcNAc 2 -PP-dolichol was generated, and incubated with the different microsome preparations in the presence or absence of unlabelled GDP-Man. Oligosaccharides liberated from LLO were resolved using TLC system A. The abbreviations used are M 1-5 GN 2 , Man 1-

[ 2 MT- 3 A 1 M

 231 Man 1-2 GlcNAc 2 -PP-dol + GDP-Man Man 1-2 [ 14 C]Man 1-2 GlcNAc 2 -PP-dol + GDP-Man Man 3-4 [ 14 C]Man 1-2 GlcNAc 2 -PP-dol dol-P-[ 14 C]Man DPM synthase dol-P + GDP-[ 14 C]Man [ 14 C]Man 1 GlcNAc 2 -PP-dol + GDP[ 14 C]Man [ 14 C]Man 3 GlcNAc 2 -PP-dol + GDP[ 14 C]Man [ 14 C]Man 5 GlcNAc 2 -PP-dol MT-GlcNAc 2 -PP-dol + GDP[ 14 C]Man MT-

Table

  Comparison of the clinical presentations of five new cases of CDG Ik with those of the previously described cases

								Case
							Summaries
								Published	a
		P1	P2	P3	P4	P5
	Date of birth	2001	2006	2004	2005	2001
	Age of CDG I diagnosis	4 months	1 year 4 months	1 year 6 months	10 months	3 years 7 months
	Sex	F	F	M	F	M
	Complications	Foetal growth retardation.	Pregnancy-induced	Pregnancy-induced	No	Foetal growth retardation. 4/5	1/5
	during pregnancy		hypertension.	hypertension. Foetal growth retardation.
	Post delivery	Hypotonia.	Low blood pressure.	Hypotonia. Absence	Hypotonia.	Not reported	4/5
	complications		Vomiting.	of occular contact.
	Feeding difficulties	Yes	Yes	No	Yes	No	3/5
	Central hypotonia	Yes	Yes	Yes	Yes	Yes	5/5	3/5
	Psychomotor retardation	Yes	Yes	Yes	Yes	Yes	5/5
	Epilepsy	Multifocal epilepsy.	Once, treatable.	Multifocal epileps.	Intractable seizure.	Multifocal epilepsy.	5/5	5/5
	MRI	Evolutive cerebellar	Normal	Cortical atrophy.	Cortical and sub cortical atrophy.	Normal	3/5	2/5
		hypoplasia.		Demyelination.
	EEG	Abnormal	Normal	Abnormal	Abnormal	Abnormal	4/5
	Dysmorphias	Thin lips. Small triangular	Large cup-shaped ears.	No	Triangular face.	Thin lips. Large forehead.	4/5	4/5
		chin. Turned up nose.	Temporal narrowing of		Almond-shaped eyes.	Large mouth. Epicanthus.
			forehead. Depressed			Long smooth filtrum.
			nasal bridge. Small
			upturned nose. Thick
			lower eyelids. Short neck.
	Microcephaly	Yes		Yes		Yes	3/5	2/5
	Occular	Abnormal VEP b test	Normal	Abnormal VEP test.	Abnormal VEP test. Absence of	Abnormal VEP test.	4/5	3/5
	manifestations	Poor visual contact		Partially blind.	occular pursuit.	Strabism.
	Fatal outcome	Yes					1/5	4/5
	Maternal allele Paternal allele	p.Cys396X/p.Arg438Trp C	p.Met377Val p.Met377Val	p.Ser150Arg p.Ala211_Arg247del	p.Gly145Asp p.Ser258Leu	p.Cys396X p.Arg276Trp

a Data taken from references 7,8,9,17 ; b Visually Evoked Potential test;

  CDG Ih (ALG8 deficiency: OMIM 608104) and CDG Im (DK1 deficiency: OMIM 610768) neurological signs are accompanied by hepato-intestinal 20 and skin/heart 21 complications, respectively. In our French experience of CDG diagnosis, 131 families have now been demonstrated to be affected by CDG I. The majority of patients (81 families, 62%) are affected by CDG Ia and the next commonest forms of the disease are CDG Ib and CDG Ik with 8 families each, (6%),

	20.	Schollen E, Frank CG, Keldermans L, et al. Clinical and molecular features of three
		patients with congenital disorders of glycosylation type Ih (CDG-Ih) (ALG8
		deficiency). J Med Genet 2004;41(7):550-6.
	21.	Kranz C, Jungeblut C, Denecke J, et al. A defect in dolichol phosphate biosynthesis
		causes a new inherited disorder with death in early infancy. Am J Hum Genet
		2007;80(3):433-40.
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	closely followed by CDG Ic (ALG6 deficiency: OMIM 603147; 5 families, 4%). We have ABBREVIATIONS
	also diagnosed 3 families with CDG Ie (DPM1 deficiency: OMIM 603503), 3 families with Man, mannose; Glc, glucose; GlcNAc, N-acetylglucosamine; GlcNH 2 , glucosamine; Dol-P,
	CDG Ig (ALG12 deficiency: OMIM 607143), and one family each with CDG Ih and CDG dolichol-phosphate; ER, endoplasmic reticulum; LLO, lipid-linked oligosaccharide; SDS-
	Im. The 20 other families remain to be subtyped (CDG Ix: 15%). These statistics suggest that PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis.
	CDG Ik should be given more consideration when diagnostic strategies are prioritised based
	on apparent CDG I subtype frequencies.
	To summarise, 5 patients with severe type I CDG-like clinical presentations possessing an
	underlying MT-1 deficiency are described. The identification of these patients reveals that, in
	France, CDG Ik and CDG Ib are the most frequently diagnosed type I CDG after CDG Ia.

GlcNAc 2 . C. Dolichol-P-mannose synthase (DPM synthase) generates dolichol-P-

ACKNOWLEDGEMENTS

The authors' laboratories are supported by: The Mizutani Foundation; the GIS -Institut des maladies rares/INSERM funded French CDG Research Network; EUROGLYCANET (LSHM-CT-2005-512131), La Fondation pour la Recherche Médicale (FRM); institutional funding from INSERM. S.E.H.M. is the recipient of a Hospital/INSERM Contrat d'Interface.


Cécile Rouzier

Sandie Le Guédard-Méreuze

Konstantina Fragaki

Valérie Serre

Julie Miro

Sylvie Tuffery-Giraud

Annabelle Chaussenot

Sylvie Bannwarth

Céline Caruba

Elsebet Ostergaard

Jean-François Pellissier

Christian Richelme

Caroline Espil

Brigitte Chabrol

Véronique Paquis-Flucklinger

Pr Véronique Paquis-Flucklinger

The severity of phenotype linked to SUCLG1 mutations could be correlated with residual amount of SUCLG1 protein

Keywords: respiratory chain defect, mtDNA depletion, methylmalonic aciduria, SUCLG1 mutation, minigene expression system

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The mtDNA depletion syndromes (MDS) are a heterogeneous group of severe mitochondrial disorders of infancy and childhood, resulting from a decrease in mtDNA copy number in affected tissues [START_REF] Suomalainen | Diseases caused by nuclear genes affecting mtDNA stability[END_REF][START_REF] Alberio | Depletion of mtDNA : syndromes and genes[END_REF][START_REF] Spinazzola | Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk[END_REF] . They are inherited as autosomal recessive traits. To date, eight genes have been involved in MDS which can be classified in two genetic categories, those that affect mtDNA replication and those that affect the mitochondrial nucleotide pool [START_REF] Copeland | Inherited mitochondrial diseases of DNA replication[END_REF][START_REF] Saada | Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy[END_REF][START_REF] Bourdon | Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion[END_REF][START_REF] Bornstein | Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene[END_REF][START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF][START_REF] Spinazzola | MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion[END_REF][START_REF] Naviaux | POLG mutations associated with Alpers' syndrome and mitochondrial DNA depletion[END_REF][START_REF] Sarzi | Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion[END_REF] .

Mutations in the SUCLA2 and SUCLG1 genes have been found in MDS associated with encephalomyopathic presentations, moderate methylmalonic aciduria 12 , 13 and succinate-CoA ligase (SUCL) deficiency. SUCL is an enzyme located in the mitochondrial matrix, composed of an α subunit encoded by SUCLG1 and a β subunit encoded by either SUCLA2 or SUCLG2, resulting in an ATP-specific SUCL (A-SUCL) and a GTP-specific SUCL (G-SUCL) respectively. A-SUCL and G-SUCL are supposed to catalyse the reversible conversion of succinyl-CoA and ADP or GDP to succinate and ATP or GTP in the Krebs cycle [START_REF] Johnson | Characterization of the ATP-and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes incorporate the same alpha-subunit[END_REF] . Several studies have demonstrated that SUCL forms a complex with nucleoside diphosphate kinase (NDPK), which is involved in the salvage of deoxyribonucleotides [START_REF] Kowluru | Localizatio and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase[END_REF] . An absence of interaction with SUCL could lead to a decreased activity of NDPK and explain the mtDNA depletion observed in SUCL deficiency [START_REF] Ostergaard | Disorders caused by deficiency of succinate-CoA ligase[END_REF] .

To date, mutations in SUCLA2 have been identified in 12 families, including 8 families of the Faroe islands who have a common ancestor 12 , 17 , 18 . Most patients were normal at birth but later developed a Leigh-like disorder with dystonia and hearing impairment [START_REF] Chinnery | Mutations in SUCLA2: a tandem ride back to the Krebs cycle[END_REF] . Only four patients have been reported with SUCLG1 mutations. In one pedigree, the three patients carried a homozygous 2 bp deletion [START_REF] Ostergaard | Deficiency of the alpha subunit of succinate-Coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion[END_REF] . They had a severe lactic acidosis at birth and died within 2-4 days, suggesting that MDS linked to SUCLG1 mutations are more severe than those caused by SUCLA2 mutations. Recently though, an additional patient with a homozygous missense mutation in SUCLG1 who presented with a milder phenotype, similar to that of patients with SUCLA2 mutations, was reported [START_REF] Ostergaard | A novel misense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form with methylmalonic aciduria[END_REF] .

In this study, we describe two unrelated patients harboring novel pathogenic mutations in SUCLG1 and suggest that the severity of the phenotype is modulated by the presence of a residual amount of SUCLG1 protein. Clinical examination shows an extremely severe axial hypotonia with no active movements and very atrophic muscles. The patient is tube-fed through a percutaneous endoscopic gastrostomy and he needs permanent respiratory support by tracheotomy.

Patients and methods

Patients

OXPHOS spectrophotometric measurements

Spectrophotometric studies of OXPHOS complexes and citrate synthase were performed on fibroblasts and muscle crude homogenates, as previously described [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF][START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] .

Western blotting 50 µg of total protein extracts, obtained as previously described [START_REF] Bourdon | Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion[END_REF] , were separated on an acrylamide gel by SDS-PAGE and transferred to a PVDF membrane (Millipore, Saint-Quentin, France). Different primary antibodies have been used. A cocktail of anti-human total OXPHOS complex antibodies (Mitosciences, Eugene, USA) was used at 1/1000. For detection of the SUCLG1 protein, a rabbit polyclonal antibody was used at 1/1000 [START_REF] Ostergaard | Deficiency of the alpha subunit of succinate-Coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion[END_REF] . For SUCLA2 detection, a rabbit polyclonal antibody against amino acids 53-348 was used at 1/200 (Santa Cruz Biotechonology). Beta-tubulin detection was performed with a mouse monoclonal antibody at 1/10000 (Sigma-Aldrich, St Louis, USA). Anti-rabbit HRPconjugated secondary antibody was used at 1/20000 and signals were detected using an Enhanced Chemiluminescence system (ECL Plus, Amersham, Piscataway, USA).

Mitochondrial DNA analysis

Blood and tissue samples were obtained after parents had given informed consent.

Mitochondrial DNA quantification in muscle was performed by real-time quantitative PCR adapted from the method described by Sarzi et al. [START_REF] Sarzi | Mitochondrial DNA depletion is a prevalent cause of multiple respiratory chain deficiency in childhood[END_REF] . Primers and conditions are available on request.

Sequencing of SUCLG1, SUCLA2, SUCLG2 and NME4

The coding exons of SUCLG1 (NM_003849. PCR-RFLP analysis.

We searched for the presence of the c.509C>G mutation in 360 control chromosomes by PCR-RFLP analysis. This mutation abolishes an ApaI restriction site in exon 4 which was amplified with a forward primer in intron 3 : 5'-TGTCCTTTTCTTACCCCAAGA-3' and a reverse primer in intron 4 : 5'-GAGTTTTGAGGGTTTAAGGCA-3'. After ApaI digestion, 2 fragments of 292 and 280 bp are found in wild-type PCR controls. The 572 bp PCR fragment is not digested when the c.509C>G mutation is present.

In silico analysis

The model (residues 41 to 346) was obtained by comparative protein modeling methods and energy minimization using the Swiss-Model program. The 2.96 Å coordinate set for the pig enzyme in complex with GDP (pdb code 2fpg) was used as a template for modeling the human enzyme. Swiss-pdb Viewer 3.7 (http://www.expasy.org/spdbv) was used to analyze and visualize the structures.

RT-PCR analysis

Total RNA were extracted by using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA synthesis and PCR amplifications were performed using the SuperScript One-Step kit, RT-PCR with Platinum Taq (Invitrogen). Reverse transcription consisted of one cycle at 55°C for 30 min and 94°C for 2min and was followed by PCR amplification (primers and PCR conditions are available on request).

Ex vivo splicing assay

An overlap extension PCR strategy [START_REF] Heckman | Gene splicing and mutagenesis by PCR-driven overlap extension[END_REF] was used, due to the large size of SUCLG1 intron 1 (9.4 kb), to construct a chimeric minigene assembling SUCLG1 exon 1 with its upstream (498 bp)

and downstream (173 bp) intronic sequences (fragment 1) and exon 2 with its flanking intronic sequences (406 bp of intron 1 and 146 bp of intron 2) (fragment 2). The PCRs were performed from the genomic DNA of patient 1 using the Hot Start High Fidelity Phusion polymerase (Finnzymes). Sequences of primers are available upon request. The final fulllength product (1617 bp) was inserted between the XhoI/NheI restriction sites of the pcDNA3.1(+) mammalian expression vector (Invitrogen) using the T4 DNA ligase (Invitrogen) and validated by direct sequencing. Transient transfections in HeLa cells were carried out in 6-well plates using 6 μl of FuGENE6 (Roche) and 1 μg of wild-type or mutated minigene constructs according to the manufacturer's instructions. Experiments were carried out in triplicate and in at least two independent transfections. Cells were harvested 48 h post transfection. Total cytoplasmic RNA was extracted using the Nucleospin RNAII Kit (Macherey-Nagel). First-strand cDNA synthesis was performed from 1 μg of total RNA, with random hexamers as primers (Invitrogen) and Superscript II (Invitrogen). One tenth of the synthesized cDNA was used as a template for the subsequent PCR with the T7 forward vector specific primer and a gene-specific reverse primer (5'-CTGTTTGCCAGTGAAACCCTGG-3'). Amplification was performed using the Hot Start High Fidelity Phusion polymerase (Finnzymes) for 35 cycles, consisting of 10 sec at 98°C, 20 sec at 53°C and 50 sec at 72°C.

The products were resolved on 1.5% agarose gel. All PCR products were confirmed by DNA sequencing.

Results

Patient 1

Histological analysis of the muscle tissue showed intracellular lipid accumulation with numerous cox-negative and a few ragged-red fibers (not shown). Enzymatic spectrophotometric measurements of the individual respiratory chain complexes in muscle revealed a decrease of complex I, II, IV and V activity (Table 1A). Additionally, all activity ratios compared to complex IV, which best reflect the balanced respiratory enzyme activity [START_REF] Chretien | Reference charts for respiratory chain activities in human tissues[END_REF] , were severely abnormal in muscle. In fibroblasts, the specific activities of complex II, III, and IV were also decreased with an imbalance in the ratios between the complexes (Table 1B). In agreement with the enzyme measurements, western blot analysis demonstrated a marked reduction in the amount of specific proteins of complexes I, II, III and IV in patient's fibroblasts compared to control (Fig. 2A). Muscle was not available for western blotting analysis. The relative mtDNA copy number showed a decreased amount of mtDNA in muscle (11% of age-matched controls).

The clinical presentation including encephalomyopathy with mtDNA depletion and mild methylmalonic aciduria led us to perform mutation screening of genes involved in succinate-CoA ligase. No mutation was found in SUCLA2. Mutation analysis of the SUCLG1 gene showed that the patient was compound heterozygous for 2 novel mutations: c.509C>G (p.Pro170Arg) and c.97+3G>C (Fig. 2B). The involvement of SUCLG1 was confirmed by western blotting analysis which showed a total absence of SUCLG1 protein in patient's fibroblasts (Fig. 2C).

The c.509C>G mutation was inherited from the mother, and the healthy older sister was also heterozygous for the mutation. The missense mutation changes a highly conserved neutral proline into a polar charged basic arginine and was not found in 360 healthy French control chromosomes (Fig. 2D). The p.Pro170Arg mutation maps within the CoA-ligase domain and we carried out prediction studies of putative impact on SUCL-G structure and function. The high degree of identity (96%) between the pig GTP-specific succinyl CoA synthetase and SUCL-G α-subunits prompted us to predict the three-dimensional structure of the human enzyme. The missense mutation p.Pro170Arg is localized close to the catalytic histidine residue and the CoA-binding domain. The putative creation of an ionic bound with one of the neighbouring acidic residues could alter the flexibility of the loop connecting the two domains of the α-subunit (Fig. 3).

The second mutation c.97+3G>C, inherited from the father changes the highly conserved G nucleotide at position +3 of the donor splice site (5'ss) in intron 1. This base substitution was predicted to drastically decrease the intrinsic strength of this 5'ss by using three different we designed a splicing reporter minigene containing exon 1 and exon 2 of SUCLG1 with part of their flanking intronic sequences, containing a strong cryptic 5'ss predicted by the Human Splicing Finder (HSF) tool (http://www.umd.be/HSF/) which was located 314 bp upstream exon 1, to perform an ex vivo splicing assay (Fig. 4A). After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. No transcript retaining exon 1 could be detected in presence of the mutation. Indeed, the mutant minigene disclosed only a short fragment resulting from the complete skipping of exon 1 by the occurrence of a splicing event between the upstream cryptic 5'ss together with the acceptor splice site (3'ss) in intron 1 (Fig. 4A). In the wild-type context, two transcript species were detected. In both transcripts, the cryptic 5'ss was used either in combination with the 3'ss in intron 1 (transcript missing exon 1) as observed in the mutant or with a new cryptic exonic 3'ss (MaxEntScan score, 4.97) located 5 base pairs downstream of the established 5' border of exon 1 (transcript including exon 1) which preserves the translation initiation site (Fig. 4A). To further investigate in vivo the functionality of the two newly identified 5'ss and 3'ss, we performed a new RT-PCR analysis from both patient and control fibroblasts with a forward primer located upstream the cryptic 5'ss sequence. As observed in the wild-type construct, we confirmed the presence of two SUCLG1 mRNA isoforms in control fibroblasts resulting from alternative splicing of exon 1. Both isoforms contain an exonic sequence, upstream of exon 1, bound either to exon 1 (use of the cryptic exonic 3'ss) or to exon 2 (Fig. 4B). The same transcripts were found in patient's fibroblasts because of the presence of the wild-type allele. Hence we have evidenced the presence of a so-far unreported exonic sequence upstream of SUCLG1 exon 1, which is likely untranslated when bound to exon 1.

We have found a cDNA sequence corresponding to the second isoform lacking exon 1 in databases (BU600891; www.genome.ucsc.edu; www.ensembl.org). Nevertheless, the role of the putative corresponding gene product, which lacks the initiation AUG codon and the mitochondrial targeting sequence, has to be determined.

Patient 2

At 2 years of age, analysis of muscle showed numerous cox-negative fibers and lipid accumulation (not shown). On electron microscopy, abnormal enlarged mitochondria contained lipid droplets (Fig. 5A). Enzymatic spectrophotometric measurements showed markedly reduced activities for complexes I and IV (not shown) and we found a mtDNA depletion (18% of age-matched controls). No mutation was identified by sequencing the SUCLA2 gene. Mutation analysis of SUCLG1 showed that the patient was heterozygous for a c.448C>T (p.Gln150X) mutation (Fig. 5B), which was inherited from his healthy mother. We failed to identify a second mutation by SUCLG1 sequencing. Furthermore, we did not detect any sequence variation by sequencing the complete cDNA from fibroblasts. Immunoblot analysis of fibroblasts showed a decreased amount of SUCLG1 protein in patient compared with controls (Fig. 5C).

A-SUCL is a heterodimer composed of SUCLG1 and SUCLA2. We looked at the expression of SUCLA2 protein in our patients harboring SUCLG1 deficiencies. Western blotting in fibroblasts of patient 1, who harbors a complete absence of SUCLG1 protein, showed no expression of the SUCLA2 gene product (Fig. 5D). On the contrary, we only found a decreased amount of SUCLA2 when a residual amount of SUCLG1 protein is present (patient 2). These results are consistent with a destabilization of SUCLA2 in the absence of its heterodimer partner, SUCLG1.

Discussion

The first patients described with SUCLG1 mutations had a very severe phenotype at birth with death during the first days of life [START_REF] Ostergaard | Deficiency of the alpha subunit of succinate-Coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion[END_REF] . Patients with SUCLA2 mutations had a milder phenotype. They were generally normal at birth and later developed psychomotor retardation, severe muscle hypotonia, hyperkinetic movement disorders and sensorineural deafness 12 18 17 .

Recently presented with a "SUCLA2-like" phenotype [START_REF] Ostergaard | Deficiency of the alpha subunit of succinate-Coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion[END_REF][START_REF] Ostergaard | A novel misense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form with methylmalonic aciduria[END_REF] . All together, these results suggest that the severity of the phenotype is modulated by a residual amount of SUCLG1 enzyme.

Furthermore, we show that in fibroblasts a complete absence of SUCLG1 likely leads to degradation of SUCLA2, its heterodimer partner. It is reasonable to assume that an extremely severe phenotype with antenatal manifestations is observed when the absence of SUCLG1 leads to a complete abolition of both G-SUCL and A-SUCL activities. In patients with SUCLA2 mutations or SUCLG1 mutations that do not fully abolish SUCLG1 expression, some residual SUCL activity is present and the interaction with NDPK could be conserved thus NDPK activity.

Brain imaging in most patients with SUCLA2 mutations showed basal ganglia involvement closely resembling Leigh syndrome 12, 18 17 . Brain MRI data were available for only one patient with SUCLG1 mutations showing findings similar to that of patients with SUCLA2 mutations [START_REF] Ostergaard | A novel misense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form with methylmalonic aciduria[END_REF] . Our SUCLG1 patients also showed basal ganglia abnormalities. In addition, we found in both patients bilateral and symmetrical white matter involvement, which has not been reported previously. Albeit, neuroimaging of only a few patients has been reported, it

does not seem to be associated with mutations in a specific gene and both SUCLA2 and SUCLG1 genes have to be tested in patients with encephalomyopathic presentations associated with mild methylmalonic aciduria.

We have strong arguments for the pathogenicity of the three novel mutations that we detected. In patient 1, p.Pro170Arg affects highly conserved residue during evolution and was not found in 180 controls. Its location within the CoA-ligase domain and the prediction studies of its putative impact on SUCL-G structure and function are in favor of a deleterious effect. Indeed, this mutation may induce a local conformational change, altering the flexibility, proper interface interactions and ligand binding. In a more dramatical way, the p.Pro170Arg mutation may completely disrupt the local tertiary structure of the protein leading to a wrong folding which would explain why the protein is unable to form a complex with the β subunit. The second heterozygous mutation in patient 1, c.97+3G>C, was predicted to affect the 5'ss in intron 1. By using an ex vivo splicing assay, we demonstrated that this variant results in the skipping of exon 1 in all transcripts leading to the loss of the AUG initiation codon and the mitochondrial targeting sequence located in the first 40 amino acids of the SUCLG1 protein. Both ex vivo and in vivo assays on control fibroblasts showed the inclusion of an exonic sequence upstream of the exon 1 in the SUCLG1 transcripts, the role of which remains to be determined. Combined with the results of the immunoblot analysis showing the complete absence of SUCLG1 protein in the patient's fibroblasts, we demonstrate that the identified mutations result in the absence of a functional protein.

The heterozygous nonsense mutation p.Gln150X in patient 2 is expected to lead to the synthesis of a truncated protein which lacks the CoA-ligase domain. Unfortunately, we failed to identify a second mutation in SUCLG1, but we did not search for large deletions or deep intronic mutations. Sequencing of SUCLA2, SUCLG2 and NME4 (coding for the mitochondrial NDPK) revealed no mutation. Taken together, these findings and the severe decrease of SUCLG1 protein on immunoblot analysis are in favour of the involvement of SUCLG1 in the disease.

In conclusion, we confirm the role of SUCLG1 in encephalomyopathy with mtDNA depletion and mild methylmalonic aciduria. Overlap between the clinical presentations linked to SUCLG1 or SUCLA2 mutations should lead to screening of both genes in these phenotypes.

Titles and legends to figures

Figure 1 .

 1 Figure 1. Brain MR images of patient 1 (A-D) and patient 2 (E-H). A. Axial T1-weighted

Figure 2 .

 2 Figure 2. Identification of novel SUCLG1 mutations leading to G-SUCL defect in

Figure 3 .

 3 Figure 3. Modelled structure of the human SUCLG1 protein. A. The modelled α-subunit

Figure 4A .

 4A Figure 4A. Ex vivo analysis of the c.97+3G>C mutation. The genomic DNA segment tested

Figure 5A -

 5A Figure 5A-C. SUCLG1 defect in patient 2. A. Electron micrographs showing abnormal

  Patient 1 was the second child of unrelated healthy parents. Pregnancy and birth were unremarkable. At age 1 day, he was admitted to intensive care unit with severe hypotonia, respiratory failure and hypoglycemia. Laboratory investigations revealed a lactic acidosis (blood pH 6.76, normal 7.4; HCO 3

-4.8mmol/l, normal>18 mmol/l; plasma lactate concentration of up to 11.31mmol/l, normal<2.20mmol/l) and a high lactate/pyruvate ratio (33, normal<18). Lactate concentration in cerebrospinal fluid was elevated (22.68mmol/l, normal<3.10mmol/l). Measurement of acylcarnitine esters in plasma revealed increased levels of C3-carnitine (7.5µmol/l, normal<1µmol/l) and C4-dicarboxylic acylcarnitine, which reflect an elevated excretion of methylmalonic acid. The brain magnetic resonance (MR) imaging showed symmetric T2-hyperintense lesions of the basal ganglia and MR spectroscopy in single-voxel revealed a significant elevated lactate doublet peak within abnormal white matter (Fig.

1

). At age 7 months, psychomotor development was severely delayed with no head control, severe axial hypotonia and poor spontaneous movements. He presented with recurrent episodes of acidosis and respiratory failure. He died at age one year.

Patient 2 was the second son of unrelated healthy parents. Pregnancy and birth were unremarkable. At 3 months of age, he presented with severe hypotonia, feeding difficulties and failure to thrive. Metabolic findings were similar to those of the first patient (lactic acidosis and moderate excretion of methylmalonic acid). At 14 months of age, he presented with severe psychomotor retardation with lack of head control and sitting posture. Brain magnetic resonance imaging (MRI) is shown in figure

1

. At present, he is 12 years old.

  SSPNN: NNSPLICE 0.9 version) available at http://www.fruitfly.org and SpliceSiteFrame available at http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.htm). We first attempted to characterize the splicing outcome of this sequence variation by RT-PCR analysis from patient's fibroblasts, with a forward primer in exon 1 and a reverse primer in the last exon of SUCLG1 (exon 9). No aberrantly spliced mRNA could be identified and sequence analysis of the RT-PCR reaction detected only one allele with the 509 C to G mutation. Hence

	Network algorithm (
	splice-site	prediction	algorithms	(MaxEntScan	available	at
	http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html,	Splice-site	Neural

  , Ostergaard et al. described a patient with SUCLG1 mutations, presenting with a phenotype similar to that of patients with SUCLA2 mutations. Our patient 1 had a complete absence of SUCLG1 protein in fibroblasts. He presented with severe hypotonia and lactic acidosis at birth. He developed psychomotor development with recurrent episodes of acidosis leading to death at one year of age. Our patient 2 had a residual amount of SUCLG1 in

fibroblasts. The first symptoms appeared at 3 months of age and he is still alive at 12 years of age. Similarly, Ostergaard et al. described a severe disease in patients showing a complete absence of SUCLG1 while the patient who expressed small amount of SUCLG1 protein

Table 1 . Respiratory chain enzyme activities in muscle biopsy (A) and fibroblasts (B) of patient 1.

 1 Activities were measured spectrophotometrically. Results are expressed i) as absolute values for controls or patient (in nanomol of substrate per minute per milligram of protein) and ii) as activity ratios compared to complex IV. The abnormal values are in bold.

Acknowledgements

We thank Alexia Figueroa for technical help. This work was made possible by grants to V.P-F from the Association Française contre les Myopathies (AFM).

Licence

"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (


Cécile Christine Bellanné-Chantelot
email: christine.bellanne-chantelot@psl.aphp.fr

Saint-Martin Maria-Joao Ribeiro

Christelle Vaury

Virginie Verkarre

Jean-Baptiste Arnoux

Vassili Valayannopoulos

Sandrine Gobrecht

Christine Sempoux

Jacques Rahier

Cécile Saint-Martin

Jean-Christophe Fournet

Francis Jaubert

Yves Aigrain

Claire Nihoul-Fékété

Pascale De Lonlay

ABCC8 and KCNJ11 molecular spectrum of 109 patients with diazoxide-unresponsive congenital hyperinsulinism

Keywords: congenital hyperinsulinism, diazoxide, potassium channel, ABCC8, KCNJ11

come

Congenital hyperinsulinism (CHI; MIM #256450) characterized by an inappropriate over secretion of insulin from pancreatic β-cells is the most frequent cause of persistent hypoglycaemia with an estimated incidence of 1 / 50 000 births in most populations. [START_REF] Stanley | Hyperinsulinism in infants and children[END_REF][START_REF] Glaser | Genetics of neonatal hyperinsulinism[END_REF] The clinical challenge concerns patients who are unresponsive to the medical treatment, as severe and persistent hypoglycaemias lead to irreversible brain damage.

CHI is related to different genetic aetiologies. In rare cases, CHI results from anomalies of either the glucokinase (GCK), the glutamate dehydrogenase (GLUD1), shortchain L-3-hydroxyacyl-CoA dehydrogenase (HADH) genes (3 for review) or the hepatocyte nuclear factor 4A (HNF4A), the latter being associated with transient forms of CHI (4), and more recently the monocarboxylate transporter 1 (SLC16A1), which causes exercise-induced HI.(5) However, CHI is mostly associated with molecular defects of the β-cell ATP-sensitive potassium (K ATP ) channel genes, KCNJ11 and ABCC8, respectively encoding the channel subunits Kir6.2 and SUR1. [START_REF] Glaser | Genetics of neonatal hyperinsulinism[END_REF][START_REF] Thomas | Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy[END_REF][START_REF] Nestorowicz | A nonsense mutation in the inward rectifier potassium channel gene, Kir6.2, is associated with familial hyperinsulinism[END_REF][START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF][START_REF] Flanagan | Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism[END_REF] The management of patients depends on the responsiveness to medical therapy. The treatment of CHI is primarily based on the use of diazoxide, a K ATP channel agonist. Since ABCC8 and KCNJ11 defects can impair the channel response to its agonist, [START_REF] Dunne | Hyperinsulinism in infancy: from basic science to clinical disease[END_REF] another alternative is to use octreotide and frequent or continuous feeding. [START_REF] Stanley | Preoperative evaluation of infants with focal or diffuse congenital hyperinsulinism by intravenous acute insulin response tests and selective pancreatic arterial calcium stimulation[END_REF] Pancreatic surgery should be restricted to medically unresponsive CHI. It is therefore essential since their medical care will be different to classify patients correctly according to the two main histopathological forms of CHI. [START_REF] Rahier | The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B cells?[END_REF][START_REF] Goossens | Diffuse and focal nesidioblastosis. A clinicopathological study of 24 patients with persistent neonatal hyperinsulinemic hypoglycemia[END_REF][START_REF] De Lonlay-Debeney | Clinical features of 52 neonates with hyperinsulinism[END_REF] The focal form consists of abnormal pancreatic βcells restricted to a limited pancreatic area and can be cured by the targeted removal of the pathological tissue. In contrast, the diffuse form affects all the β-cells and thus requires near total pancreatectomy when patients are unresponsive to an intensive medical treatment.

Besides these two well-defined forms of CHI, some histopathological forms are not well-characterized, neither being typically focal nor typically diffuse, some of them presenting a mosaicism of their pancreatic β-cells resulting in an atypical form of CHI. [START_REF] Sempoux | A new morphological form of persistent hyperinsulinaemic hypoglycaemia of infancy: correlation with clinical and physiological data[END_REF][START_REF] Sempoux | Persistent hyperinsulinemic hypoglycemia of infancy: the pathologist's experience][END_REF][START_REF] Hussain | An ABCC8 gene mutation and mosaic uniparental isodisomy resulting in atypical diffuse congenital hyperinsulinism[END_REF] Focal and diffuse forms of CHI related to a K ATP channel defect involve distinct molecular mechanisms. Focal forms are sporadic defects resulting from two events: a paternally inherited KCNJ11 or ABCC8 mutation and the loss of the corresponding maternal allele in some pancreatic β-cells leading to a focal lesion. [START_REF] De Lonlay | Somatic deletion of the imprinted 11p15 region in sporadic persistent hyperinsulinemic hypoglycemia of infancy is specific of focal adenomatous hyperplasia and endorses partial pancreatectomy[END_REF][START_REF] Verkarre | Paternal mutation of the sulfonylurea receptor (SUR1) gene and maternal loss of 11p15 imprinted genes lead to persistent hyperinsulinism in focal adenomatous hyperplasia[END_REF] Diffuse forms are classically associated with an autosomal recessive mode of inheritance. [START_REF] Thomas | Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy[END_REF][START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF][START_REF] Thomas | The molecular basis for familial persistent hyperinsulinemic hypoglycemia of infancy[END_REF] Both focal CHI and recessively inherited CHI related to a K ATP channel defect are usually resistant to diazoxide. [START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF] By contrast, the rare diffuse forms described with a dominant inheritance are sensitive to diazoxide. [START_REF] Huopio | Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1[END_REF][START_REF] Thornton | Clinical and molecular characterization of a dominant form of congenital hyperinsulinism caused by a mutation in the high-affinity sulfonylurea receptor[END_REF] Altogether, ABCC8 and KCNJ11 mutations would account for about 50% of CHI patients.(2, 8) However, the proportion of K ATP channel mutations identified in CHI remains inaccurately defined since large studies have not been performed by sequence analysis, and most mutations were reported through case reports.(9) Furthermore, CHI cases related to ABCC8/KCNJ11 mutations were mostly described independently of their responsiveness to diazoxide.

Here, we describe the ABCC8 and KCNJ11 mutational spectrum in a large series of diazoxide-unresponsive CHI patients. We discuss the benefits/limits of the ABCC8/KCNJ11 genetic testing according to the radiological and histological diagnosis, and the clinical situations that warrant a molecular analysis to improve the clinical management of diazoxideunresponsive CHI patients.

METHODS

Patients

From 1999 to 2009, we studied 109 diazoxide-unresponsive unrelated CHI patients (57 boys, 52 girls) who were referred for clinical and/or radiological investigations or surgery to Necker-Enfants Malades Hospital (Paris, France). Most of them (92/109, 84.4%) were diagnosed during the neonatal period, whereas 16 patients were diagnosed between 1 and 6 months of life, and 1 girl at 22 months. The series includes 87 Euro-Caucasians (80%), 13

North-Africans, 3 Turkish, 3 Asians, 1 African and 2 from the Indian Ocean region.

Clinical diagnosis of CHI was based on the observation of recurrent non ketotic hypoglycemias (blood glucose concentration < 3 mmol/l) associated with concomitant inappropriate plasma insulin concentration.( 8) Non-responsiveness to diazoxide was defined as two confirmed blood glucose measurements lower than 3 mmol/l in a 24-hours period after at least five consecutive days of diazoxide therapy at 15 mg/kg/day (neonates) or 10 mg/kg/day (infants) divided in 3 oral doses. [START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF] Patients with syndromic CHI (e.g. hyperinsulinism-hyperammonemia (HI/HA) syndrome or CHI associated with overgrowth syndromes) were excluded, as each syndrome warrants its specific molecular testing and because they are diazoxide-sensitive. For all studied patients, we obtained the written informed consent in accordance with the French ethical guidelines. was assessed by analysis of intraoperative frozen samples, formalin fixed and paraffin embedded sections, as described. [START_REF] Rahier | The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B cells?[END_REF][START_REF] Sempoux | A new morphological form of persistent hyperinsulinaemic hypoglycaemia of infancy: correlation with clinical and physiological data[END_REF][START_REF] Suchi | Congenital hyperinsulinism: intraoperative biopsy interpretation can direct the extent of pancreatectomy[END_REF].

Radiological and histopathological diagnosis

Radiological

All the pancreatic tissue samples were analysed by two pediatric pathologists, at the time of surgery and before inclusion in this study. We subdivided CHI patients into three radiopathological groups. The first one consisted of 37 patients with a focal form of CHI, 35 of them underwent surgery. The second group included 64 patients with a diffuse form of CHI. In this group, patients were subclassified in "diffuse CHI" (27 operated patients, with pathological confirmation) and "suspected diffuse CHI" (37 patients with radiological diagnosis and no histological confirmation). The third group consisted of 8 operated patients with an atypical histopathological form as previously described. [START_REF] Hussain | An ABCC8 gene mutation and mosaic uniparental isodisomy resulting in atypical diffuse congenital hyperinsulinism[END_REF][START_REF] Sempoux | Persistent neonatal hyperinsulinism: new pathological findings which clarify the physiopathology of the syndrome and direct the therapeutic approach[END_REF] Median values of clinical characteristics were used for analysis. Categorical data were compared with Fisher exact test. A p value of less than 0.05 was considered of statistical significance.

Genetic analysis

The search for germline events was performed on genomic DNA extracted from peripheral lymphocytes. The study followed five steps:

(i) Sequencing of the coding sequence and exon/intron boundaries of KCNJ11 and ABCC8 genes and the ABCC8 promoter region in probands. In four patients, sequence analysis was also performed on pancreatic DNA extracted from frozen sections to search for somatic events.

(ii) Variant validation. First, we performed bioinformatic analysis using Polyphen, Sift and GVGD for missense variants; SpliceSiteFinder, NNSPLICE, GeneSplicer and MaxEntScan for splice site variants. Variants were considered as mutations when these algorithms predicted them to be deleterious. Second, the variants were excluded from 400 geographically-matched control chromosomes.

(iii) Cosegregation analysis in the parents. In case of a suspected de novo event, we genotyped 14 microsatellite markers (Individual Panel 15 for Linkage Mapping Set v2.5, Applied Biosystems) including 6 markers on chromosome 11 in both patient and parents.

(iv) In all probands except those with compound heterozygous mutations, search for ABCC8 genomic rearrangements by multiplex ligation-dependent probe assay (MLPA) (v) GCK screening in patients with no ABCC8 and KCNJ11 mutation.

Sequence analysis

KCNJ11 and ABCC8 sequencing. The sequence analysis included first the amplification by polymerase chain reaction (PCR) of the single exon of KCNJ11, the promoter region of ABCC8 and its 39 exons. The purified PCR products were sequenced in both directions using the ABI PRISM BigDye Terminator chemistry (Applied Biosystems). Sequencing reactions were run on an ABI3100 Genetic Analyzer and analyzed with the Seqscape software Version GCK sequencing. The sequence analysis of the coding exons of the pancreatic isoform of GCK (NM_000162.3) was performed for all patients with normal KCNJ11 and ABCC8 investigation.

All primer sequences and amplification conditions are available on request.

MLPA analysis

We used the SALSA MLPA KIT P117 ABCC8 (MRC Holland, The Netherlands) for the search for genomic rearrangements. The procedure was carried out according to manufacturer's instructions. Ligation products were separated on an ABI3730 Genetic Analyzer. The analysis was performed using GeneMapper, Version 4.0 (Applied Biosystems).

Single-exon deletions were checked by real-time quantitative PCR based on SYBR-Green I fluorescence.

Microsatellite marker analysis

A panel of 11 microsatellite markers from the chromosome 11-region around ABCC8 and KCNJ11 (D11S2071, D11S1363, D11S922, D11S2344, D11S2347, D11S1901, D11S419, D11S1397, D11S921, D11S902 and D11S1888) were analyzed on DNA extracted from leucocytes and pancreatic frozen sections. The analysis was performed using GeneMapper, Version 4.0.

RESULTS

We identified a K ATP channel defect in 89 out of 109 probands (81.6%), most of them involving the ABCC8 gene (79/89, 88.8 %). Two mutations were found in 28% (30/109) of the probands, which is therefore consistent with a recessive mode of inheritance (table 1).

Among them, 11/30 had a homozygous mutation, 5/11 belonging to a consanguineous family and 19/30 patients were compound heterozygotes. In 59 probands (59/109, 54%), a single heterozygous mutation was identified (table 1). The analysis of both parents for 41/59 showed 35/41 (85%) paternally inherited mutations and 1/41 maternally inherited mutation. De novo ABCC8 mutations occurred in 5/41 probands (12.2%). Lastly, no evidence for K ATP channel defect was found in 20/109 (18%) probands. A de novo GCK mutation was identified in one of these.

Characteristics of ABCC8 and KCNJ11 molecular defects

Eighty-nine probands were identified with K ATP channel defects. A total of 118 mutations were found, including 106 ABCC8 mutations (90%) and 12 KCNJ11 mutations (10%) (table 2). Ninety-four out of 118 were different mutations, 41 were previously reported. Out of the 53 (56%) new mutations, 47 (ABCC8, n = 40 and KCNJ11, n= 7, table 2) were only observed once in our series.

Eighty-one different ABCC8 point mutations were identified (table 2). Regarding their type, 39/81 (48%) were missense mutations whereas 42/81 (49%) were either truncating mutations predicted to generate premature stop codons (15 nonsense mutations, 8 out-of-frame mutations and 14 splice site defects) or in-frame small deletions/duplications (5/42). Most ABCC8 missense mutations (74%) were located in the nucleotide-binding domains (NBD1, amino acids 679-929 and NBD2, amino acids 1344-1578), particularly in the second domain whereas only 38% of truncating mutations affected those domains (14/37, Table 2).

Eleven different KCNJ11 mutations, 10 missense and 1 frameshift mutations were identified in 12 probands (table 2). One mutation, p.Gln128Arg, was located in the pore-forming helix; one was in an extra-cellular loop and affects a residue strongly involved in the interaction of K + along the pore (p.Arg136Cys) [START_REF] Robertson | Long-pore electrostatics in inward-rectifier potassium channels[END_REF]; other mutations were located in the cytoplasmic domains.

Patients with normal ABCC8 and KCNJ11 sequencing and those carrying a single ABCC8 heterozygous mutation were investigated in search for ABCC8 deletions. We detected by MLPA technique and confirmed by fluorescent quantitative PCR single-exon deletions of ABCC8 in two compound heterozygous patients: a deletion of exon 8 (p.Thr393_Gln444del52) and a deletion of exon 22 (p.Asp854_Trp899del46) (table 2 and Supplemental material). In order to exclude KCNJ11 deletions in negative patients, four recurrent polymorphisms located in the KCNJ11 coding sequence were analysed to rule out a possible hemizygosity. Ten patients were homozygous for the KCNJ11 gene. Gene deletion could be excluded for all of them using the fluorescent quantitative multiplex PCR method.

Correlation between the ABCC8/KCNJ11 genotype and the radiopathological diagnosis

Mutations were dispersed throughout the ABCC8 and KCNJ11 genes in focal forms as well as in diffuse or suspected diffuse forms. Most mutations were observed only once, thus very few mutations were identified in both focal and diffuse (c.428G>A, c.536A>G, c.1792C>T, c.3133_3152del20 and c.3644G>A) or suspected diffuse (c.1630+1G>T) forms (table 2). The 37/109 patients diagnosed with a focal form were all associated with a unique heterozygous mutation affecting the K ATP channel, 32 detected in ABCC8 and 5 in KCNJ11 (tables 1 and 2). Mutations were paternally inherited (24 out of 26 tested parents) or arose de novo (2/26). All were distinct, 46% of them (17/37) were missense mutations whereas 51% were truncating (19/37) and one led to a deletion of two amino acids (table 2). Among the 64/109 cases diagnosed with a diffuse form (confirmed by histology, 27/64 patients) or suspected to have a diffuse CHI (on PET or PVS examination, 37 patients), 47% (30/64) were either homozygotes (11/30) or compound heterozygotes (19/30), 34% (22/64) carried a single heterozygous mutation and 19% (12/64) had none (table 1). No significant difference was observed in the number (2, 1 and 0) of K ATP mutations identified between patients with a diagnosis of diffuse form proved by histology and those with a diagnosis of diffuse form based on radiological investigation only (table 1). In patients with a recessive form of CHI, 55% of K ATP mutations were missense mutations whereas 45% were truncating.

In contrast, truncating mutations were less frequently observed in patients carrying a single K ATP heterozygous mutation (23% vs. 45%).

Among the 22 patients with a single heterozygous K ATP channel mutation, 10 had a diagnosis of diffuse CHI confirmed by histology. For 5 of them, the segregation analysis of both parents showed that the mutation had arisen de novo in 2 and was paternally inherited in 3 (table 1).

To exclude a focal form, we analyzed 11 microsatellite markers in the pancreatic tissue of four patients compared to 2 patients with focal forms and 1 patient with a diffuse form due to 2 mutations. Our results showed a loss of the maternal allele in the two focal forms, as expected, while in the diffuse forms no difference was found between patients with one or two mutations (supplemental material). Futhermore, pathological comparisons between pancreatic specimens from diffuse cases with two mutations and those with one mutation showed the same features characterized by enlarged pancreatic β-cell nuclei throughout the entire pancreas. We also excluded a somatic coding mutation by sequencing ABCC8 and KCNJ11 of pancreatic DNA of these four patients.

In the 12 patients with diffuse form of CHI suspected by PET imaging and carrying a heterozygous ABCC8 mutation, segregation analysis of both parents for 10 cases showed that the mutation had arisen de novo in 1 case, was maternally inherited in 1 and paternally inherited in 8 (table 1). In the maternally inherited case, several episodes of hypoglycemiarelated seizures in infancy were reported in the mother, although she was asymptomatic at the age of 28. There is strong evidence that the identified mutation Gly716Asp is pathogenic: (i) it is located in the first nucleotide binding domain. (ii) the same residue was reported to be altered (Gly716Val) in a homozygous recessive form of CHI resistant to diazoxide [START_REF] Thomas | Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy[END_REF] and

(iii) the functional study of Gly716Val showed a reduced surface expression of the mutant channel. [START_REF] Yan | Congenital hyperinsulinism associated ABCC8 mutations that cause defective trafficking of ATPsensitive K+ channels: identification and rescue[END_REF] Among the 8 paternally inherited cases, no clinical symptom was reported in the fathers at the time of the child's diagnosis. Two families displayed the recurrence of severe CHI. For one of them, DNA was available from the affected sibling; the analysis of six microsatellite markers located in 11p15.1 showed that they did not share the same maternal haplotype and thus excluded a recessive inheritance (data not shown).

Last, no ABCC8/KCNJ11 mutation was found in 3 patients with a diagnosis of diffuse form and in 9 with diffuse form suspected by PET (table 1). One of the operated patients was subsequently found to carry a de novo GCK mutation, c.191C>T; p.Ser64Phe.

Finally, no germline ABCC8/KCNJ11 and GCK mutations were identified in the 8 patients with a histological diagnosis of atypical CHI. Paternal isodisomy at the ABCC8 locus was excluded at the pancreatic level for 3 of them. Since one patient had a monozygotic healthy twin, we also excluded the presence of a somatic mutation.

DISCUSSION

We have shown that mutations in the ABCC8 and KCNJ11 genes respectively encoding the SUR1 and KIR6.2 subunits of the K ATP channel are major causes of CHI refractory to diazoxide, as they account for 82% of all cases in a large series of 109 patients. This estimate is higher than those previously reported in several series in which a molecular aetiology was found in about 50% to 65% of cases. [START_REF] Glaser | Genetics of neonatal hyperinsulinism[END_REF][START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF][START_REF] Nestorowicz | Genetic heterogeneity in familial hyperinsulinism[END_REF][START_REF] Fournet | The genetics of neonatal hyperinsulinism[END_REF][START_REF] Fernández-Marmiesse | Mutation spectra of ABCC8 gene in Spanish patients with Hyperinsulinism of Infancy (HI)[END_REF] This discrepancy is probably due to differences in the clinical phenotype of patients, particularly their response to medical treatment, and in the sensitivity of the screening method used for the detection of mutations.

Our study shows that ABCC8 gene defects are the most important cause of diazoxideunresponsive CHI (89% of mutated patients). The mutational spectrum underlines the allelic diversity of ABCC8 and KCNJ11 mutations since 80% of identified molecular events were distinct in this series and half of them were not reported in the recent update of K ATP channel mutations [START_REF] Flanagan | Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism[END_REF] or in subsequent publications. A genomic rearrangement was identified in only two recessive forms of CHI, each one deleting a single exon, demonstrating the rarity of large deletions in CHI.

We classified CHI patients into three groups according to ABCC8/KCNJ11 mutation status (2 recessively inherited mutations, 1 single mutation and no mutation) and investigated the correlation between genotypes and histopathological or radiological diagnosis.

In 28% of patients (30/109), we found recessively inherited mutations, either homozygous or compound heterozygous, all associated with a diagnosis of diffuse form of CHI. Nearly two third of these patients were refractory to all medical treatment and consequently, required near-total pancreatectomy. These patients characterized by a severe clinical presentation were also frequently large for gestational age. Half of them had a birth weight above 4,000 g. In 54% of patients (59/109), we detected a single mutation in the coding sequence and boundary regions of ABCC8 or KCNJ11. We studied the allelic segregation in 41 families and found that 12% arose de novo, one was maternally inherited and 85% of mutations were paternally inherited. The 37 patients diagnosed with a focal form were all included in this group. This result emphasized the absence of genetic heterogeneity of focal forms of CHI which are exclusively related to functional abnormalities of the K ATP channel. This corroborates our previous results [START_REF] De Lonlay | Heterogeneity of persistent hyperinsulinaemic hypoglycaemia. A series of 175 cases[END_REF][START_REF] Verkarre | Paternal mutation of the sulfonylurea receptor (SUR1) gene and maternal loss of 11p15 imprinted genes lead to persistent hyperinsulinism in focal adenomatous hyperplasia[END_REF] and a recent study by Suchi et al. who found a K ATP channel gene mutation in 86% of patients with a focal form. [START_REF] Suchi | Molecular and immunohistochemical analyses of the focal form of congenital hyperinsulinism[END_REF] The 22 remaining patients had a diagnosis of either diffuse form (n=10) proved on histological examination or suspected diffuse form (n=12) based on PET-scan analysis. To explain the identification of a unique KATP mutation, we considered different hypotheses: (i) misdiagnosed focal forms, (ii) dominant forms of CHI, (iii) unidentified second recessive mutation and (iv) post-zygotic event. Firstly, we excluded the specific features of the focal forms at the histological and molecular levels. In the ten patients who had surgery, a diffuse lesion was confirmed by pathological examination of the pancreatic tissue showing a pattern dramatically different from a focal lesion. At the molecular level, the analysis of microsatellite markers excluded any loss of the maternal allele. Additionally, we and others have demonstrated the accuracy of the PET imaging on both focal and diffuse forms of CHI. [START_REF] Ribeiro | Functional imaging of the pancreas: the role of [18F]fluoro-L-DOPA PET in the diagnosis of hyperinsulinism of infancy[END_REF][START_REF] Ribeiro | The added value of [18F]fluoro-L-DOPA PET in the diagnosis of hyperinsulinism of infancy: a retrospective study involving 49 children[END_REF][START_REF] Hardy | Accuracy of [18F]fluorodopa positron emission tomography for diagnosing and localizing focal congenital hyperinsulinism[END_REF][START_REF] Hardy | Diagnosis and localization of focal congenital hyperinsulinism by 18F-fluorodopa PET scan[END_REF]. Altogether, our results did not support a bias related to the type of investigation since we observed a similar proportion of single mutations in diffuse forms in both operated (10/27) and non operated patients (12/37). Secondly, segregation analysis of 5 diffuse forms and 10 suspected diffuse forms showed that 3 and 8 were paternally inherited respectively. One suspected diffuse form had a maternally inherited mutation and corresponded to a dominant form. Except for this case, all patients bearing one mutation in a K ATP channel gene had parents with no clinical history of hypoglycaemia. In addition diabetes was not reported in parents and second-degree relatives of the probands, as originally described in dominant K ATP CHI. [START_REF] Huopio | Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1[END_REF] These two arguments, the recurrence of CHI in two siblings with asymptomatic parents and the fact that dominant inactivating ABCC8 or KCNJ11 mutation are usually associated with diazoxide-responsive forms of CHI [START_REF] Huopio | Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1[END_REF][START_REF] Pinney | Clinical characteristics and biochemical mechanisms of congenital hyperinsulinism associated with dominant KATP channel mutations[END_REF] are not in favour of a dominant inheritance that would explain the cases with a single K ATP heterozygous mutation. Nevertheless, these observation could be related to a non penetrance as reported for dominant forms of CHI and transient neonatal diabetes associated with K ATP channel mutations. [START_REF] Pinney | Clinical characteristics and biochemical mechanisms of congenital hyperinsulinism associated with dominant KATP channel mutations[END_REF][START_REF] Flanagan | Mutations in ATP-sensitive K+ channel genes cause transient neonatal diabetes and permanent diabetes in childhood or adulthood[END_REF] Thirdly, despite an exhaustive molecular analysis including the search for point mutations and for genomic rearrangements, we cannot exclude that rare variants located in intronic and regulatory regions which are not usually screened may be involved. However, the analysis of affected siblings showed that they did not share the same maternal haplotype and excluded a recessive inheritance. Lastly, another hypothesis may be the occurrence of a post-zygotic mutation. The recurrence of CHI in the aforementioned affected siblings could only be explained by the occurrence of independent post-zygotic events. Unfortunately, pancreatic material was not available for these patients. However, the exclusion of somatic point mutations in the pancreatic tissue of four patients was not in favour of the involvement of coding post-zygotic events as a general molecular mechanism.

Nevertheless, the sensitivity of sequence analysis would not allow identifying mutations restricted to the endocrine tissue or with a low level of mosaicism.

The predominance of paternally inherited mutations in suspected diffuse forms remains unexplained and needs to be confirmed on larger series. Interestingly, Fernandez-Marmiesse et al reported five diazoxide-unresponsive CHI patients who underwent surgery and whose histology did not show a focal lesion. All five inherited the mutation from their father. [START_REF] Fernández-Marmiesse | Mutation spectra of ABCC8 gene in Spanish patients with Hyperinsulinism of Infancy (HI)[END_REF] Epigenetic anomalies could be involved since ABCC8 and KCNJ11, which are not known to be imprinted genes, are located in the vicinity of the 11p15.5 imprinted region. DNA methylation abnormalities of the 11p15.5 locus are observed in Beckwith-Wiedemann syndrome, a congenital overgrowth syndrome that can be associated with hyperinsulinism. [START_REF] Rossignol | The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region[END_REF] Preliminary experiments performed in six patients did not show evidence of abnormal methylation at the H19, IFG2 and KCNQ10T1 loci in DNA extracted from leucocytes (data not shown). Further experiments should be done on pancreatic sections of these diffuse forms to exclude somatic epigenetic defects.

In the third and last group comprising patients with diffuse (n=3), suspected diffuse (n=9) or atypical (n=8) forms, neither a point mutation nor a large rearrangement in ABCC8/KCNJ11 was found. This third group of patients seems to have a less severe phenotype. Only 25% were large for gestational age and 40% were refractory to octreotide therapy. We screened the GCK gene since three de novo activating GCK mutations have been reported in patients unresponsive to diazoxide. [START_REF] Cuesta-Muñoz | Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation[END_REF][START_REF] Sayed | Extremes of clinical and enzymatic phenotypes in children with hyperinsulinism caused by glucokinase activating mutations[END_REF] We found one novel de novo GCK mutation which is predicted to be pathogenic and which altered an amino acid previously reported to be the target of a mutation in a patient with CHI. [START_REF] Christesen | Activating glucokinase (GCK) mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation[END_REF] This confirms the low prevalence of GCK activating mutations in severe forms of CHI. Even though the proportion of CHI patients with unexplained molecular aetiology was significantly decreased compared to previous studies, unknown genes remain to be identified in CHI refractory to diazoxide.

Finally, a mosaic paternal uniparental disomy (UPD) has been reported in an "atypical diffuse" form of CHI with a paternally inherited ABCC8 mutation. [START_REF] Hussain | An ABCC8 gene mutation and mosaic uniparental isodisomy resulting in atypical diffuse congenital hyperinsulinism[END_REF] Contrary to focal forms characterized by a paternal UPD of the 11p15.1-15.5 imprinted region, this atypical form was related to a paternal UPD observed both in DNA from leucocytes and pancreatic tissue but restricted to the 11p15.1 region. We excluded a similar molecular mechanism in our cases of atypical CHI.

In conclusion, 82% of patients with CHI unresponsive to diazoxide were related to ABCC8 or KCNJ11 mutations in our series. The molecular analysis alone provides an informative genetic diagnosis for the clinical management of CHI patients with recessively inherited pathogenic mutations. Genetic counselling and prenatal diagnosis may be offered to families with such previous CHI cases. In contrast, in patients with a single K ATP channel mutation, the molecular analysis should be systematically confronted with the parental segregation analysis and the PET imaging diagnosis. If the mutation is de novo or paternally-inherited and the PET diagnosis in favour of a focal form, the surgery will be offered for the resection of the focal lesion. But, when the PET imaging suspects a diffuse form, the molecular diagnosis has a limited added value for the clinical management of CHI patients. Maternal inheritance, n

TABLES

0 0 0 1 1 De novo, n 2 2 0 1 5
Absent in the mother $ , n

1 1 0 1 3
Unknown inheritance, n * Reported as c.3130_3149del20; $ Genetic status for diffuse forms; hmz: homozygous; c-htz: compound heterozygous; htz: heterozygous without information on the inheritance; htz p for paternally inherited, htz m for maternally inherited and htz novo for de novo mutations; ‡ D and F stand for diffuse and focal forms, respectively. When the diagnosis was based on histological data, H was added in lowercase. When only radiological diagnosis was available, the corresponding technique (PET, PVS) was indicated in lowercase.

  investigations were based on transhepatic catheterization of the portal and pancreatic veins with pancreatic veins sampling (PVS) for patients explored before 2005 and positons emission tomography (PET) performed with [ 18 F] fluoro-L-DOPA since 2005.(14, 23-26) In cases treated by surgery, the histological diagnosis (focal, diffuse or atypical form)

2. 2 (

 2 Applied Biosystems). The mutation nomenclature is based on the reference sequences KCNJ11 NM_000525.3 and ABCC8 NM_000352.3 corresponding to the L72808 isoform (1,582 amino acids) which incorporates the extra serine residue in exon 17.(9)

Table 1 :

 1 Relationships between the ABCC8/KCNJ11 genotype and the radiopathological diagnosis

Table 2 :

 2 Characteristics of ABCC8 and KCNJ11 mutations

Table 1 Correlations between the ABCC8/KCNJ11 genotype and the radiopathological diagnosis

 1

		Histopathological diagnosis	Radiological diagnosis #
		Focal	Diffuse	Atypical Focal (PET/PVS) Diffuse (PET/PVS)	Total
	n	35	27	8	2	37	109
	2 mutations, n (%)	0	14 (52%)	0	0	16 (43%) (12/4)	30 (28%)
	ABCC8/KCNJ11, n		14 / 0			13 / 3
	1 mutation, n (%)	35	10 (37%)	0	2 (2/0)	12 (32%) (12/0)	59 (54%)
	ABCC8/KCNJ11, n	32 / 3	8 / 2		0 / 2	12 / 0
	Paternal inheritance, n	22	3		2	8	35

Table 2 : Characteristics of ABCC8 and KCNJ11 mutations

 2

	10	4	0	1	15

ACKNOWLEDGEMENTS

We are very grateful to all physicians who referred CHI patients to us and provided all medical records: We thank Sylvie Rossignol for helpful discussions. We acknowledge the patients and their families for their participation to this study. This work was funded by the GIS-Institut des Maladies Rares.

Competing Interest: None to declare.

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