Ilenia ; Boria

Alan ; Beggs

E Garelli

H T Gazda

A Aspesi

P Quarello

E Pavesi

D Ferrante

J J Meerpohl

M Kartal

L Da Costa

A Proust

T Leblanc

M Simansour

N Dahl

A-S Fröjmark

D Pospisilova

R Cmejla

A H Beggs

M R Sheen

M Landowski

C Buros

C Clinton

L Dobson

A Vlachos

E Atsidaftos

J M Lipton

S R Ellisq

U Ramenghi

I Dianzani

THE RIBOSOMAL BASIS OF DIAMOND-BLACKFAN ANEMIA: MUTATION AND DATABASE UPDATE

Keywords: Diamond-Blackfan anemia, ribosomal protein, erythropoiesis, ribosome biogenesis Diamond-Blackfan anemia, ribosomal protein, erythropoiesis, ribosome biogenesis

Diamond-Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis.

By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes.

Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.

severe normochromic macrocytic anemia and reticulocytopenia, typically presenting in the first year of life. Patients generally show a decreased number of erythroid progenitors in their bone marrow [START_REF] Campagnoli | Molecular basis of Diamond-Blackfan anemia: new findings from the Italian registry and a review of the literature[END_REF]. The other bone marrow cell lineages are only rarely suppressed. Erythrocytes in DBA patients frequently express fetal hemoglobin (HbF) and erythrocyte adenosine deaminase (eADA) activity, a crucial enzyme of the purine salvage pathway, is elevated in 85% of cases [START_REF] Glader | Elevated red cell adenosine deaminase activity: a marker of disordered erythropoiesis in Diamond-Blackfan anaemia and other haematologic diseases[END_REF]. DBA is associated with an increased risk of malignancies, especially hematopoietic neoplasms and osteogenic sarcomas [START_REF] Vlachos | Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference[END_REF]. In 30% to 47% of cases patients show physical malformations involving head, thumb, heart and urogenital system [START_REF] Lipton | Diamond Blackfan anemia: new paradigms for a "not so pure" inherited red cell aplasia[END_REF]. Prenatal or postnatal growth retardation independent of steroid therapy is also often present.

The incidence of DBA is around 6 per 1 million live births [START_REF] Campagnoli | Molecular basis of Diamond-Blackfan anemia: new findings from the Italian registry and a review of the literature[END_REF]. Most cases are sporadic, but the disease can be inherited with an autosomal dominant pattern.

Penetrance is incomplete and expressivity widely variable, even in patients from the same family [START_REF] Campagnoli | Molecular basis of Diamond-Blackfan anemia: new findings from the Italian registry and a review of the literature[END_REF]. First-line therapy in DBA patients is steroid treatment.

Although 80% of patients have an initial steroid response, less than half the patients can be maintained on a safe and effective dose. Thus many of these initial responders may experience temporary or definitive steroid-resistance of dose limiting toxicity [START_REF] Vlachos | Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference[END_REF]. Patients who do not respond to steroids undergo chronic blood transfusions and need iron chelation to avoid secondary hemochromatosis. Preliminary data suggest that patients with DBA are more likely to develop iron overload than patients with thalassemia, another disease treated with chronic transfusions [START_REF] Roggero | Severe iron overload in Blackfan-Diamond anemia: a case-control study[END_REF]. Twenty percent of patients inexplicably achieve remission [START_REF] Lipton | Diamond Blackfan anemia: new paradigms for a "not so pure" inherited red cell aplasia[END_REF]. DBA can be treated successfully by allogeneic bone marrow or stem cell transplantation, but the mortality from infections, graft-versus-host disease and graft failure is significant, especially for unrelated donor transplants [START_REF] Roy | Bone marrow transplantation for Diamond-Blackfan anemia[END_REF][START_REF] Vlachos | Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference[END_REF].

The first DBA gene, ribosomal protein (RP) S19, was identified in 1999 [START_REF] Willig | Mutations in ribosomal protein S19 gene and Diamond Blackfan anemia: wide variations in phenotypic expression[END_REF] and is mutated in about 25% of patients [START_REF] Willig | Mutations in ribosomal protein S19 gene and Diamond Blackfan anemia: wide variations in phenotypic expression[END_REF][START_REF] Campagnoli | RPS19 mutations in patients with Diamond-Blackfan anemia[END_REF]. Mutations in an increasing number of other genes encoding RPs of the small (RPS24, RPS17, RPS7, RPS10, RPS26) and large (RPL35A, RPL5, RPL11) ribosomal subunits have been recently described in DBA patients [START_REF] Gazda | Ribosomal protein S24 is mutated in Diamond-Blackfan anemia[END_REF][START_REF] Cmejla | Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia[END_REF][START_REF] Farrar | Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia[END_REF][START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF][START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF]. All mutations are present on a single allele, pointing to autosomal dominant inheritance haploinsufficiency. DBA is unquestionably a ribosomapathy, a term initially proposed for dyskeratosis congenita [START_REF] Luzzatto | Dyskeratosis and ribosomal rebellion[END_REF]].

In eukaryotes, the ribosome is composed of four different ribosomal RNAs (rRNAs) and 79 ribosomal proteins. While 5S rRNA is transcribed by RNA polymerase III, 28S, 5.8S and 18S rRNAs are processed from a 45S precursor transcribed by RNA polymerase I. The maturation of pre-rRNA occurs in the nucleolus through a complex pathway involving both endo-and exonucleases that remove external and internal transcribed sequences (ETS and ITS). During these steps, the 45S pre-RNA associates with ribosomal proteins, ribonucleases, RNA helicases, small nucleolar RNPs (snoRNPs) and other accessory factors, to form 90S preribosomes. During the maturation process, the 90S pre-ribosome is separated into pre-40S and pre-60S subunits that are exported to the cytoplasm where maturation is completed [START_REF] Tschochner | Pre-ribosomes on the road from the nucleolus to the cytoplasm[END_REF]. Mature 40S subunits include 18S rRNA and 33 ribosomal proteins whereas mature 60S subunits contain 28S, 5.8S and 5S rRNAs and 46 ribosomal proteins. In humans there are several loci containing rRNA genes, but only one gene for each of the 79 ribosomal proteins. Molecular mechanisms underlying the causal effect between RP haploinsufficiency and anemia have not been elucidated. A generally recognized pathogenetic hypothesis implies defective ribosome biogenesis leading to apoptosis in erythroid progenitors. This mechanism has been named "ribosomal stress" and there are indications that it may be signalled through p53 [START_REF] Lipton | Diamond-Blackfan anemia: diagnosis, treatment, and molecular pathogenesis[END_REF]. Several RPs have a second function different from their roles as structural components of the ribosome. Defects in these extra-ribosomal functions might also contribute to the overall complexity of DBA phenotypes.

Mutations in DBA genes, along with their functional consequences and genotype-phenotype correlations, have been catalogued in the DBA Mutation Database, created by our group in 2008 and available via www.dbagenes.unito.it [START_REF] Boria | A new database for ribosomal protein genes which are mutated in Diamond-Blackfan Anemia[END_REF]. Here we report an update of the DBA Mutation Database. The updated database contains nine DBA genes (RPS19, RPS24, RPS17, RPS7, RPS10, RPS26, RPL5, RPL11, RPL35A) and 220 distinct mutations. It now includes information on molecular mechanisms involved in RP mutagenesis and more detailed information about inheritance. This update arises from the collaboration of Czech, French, German, Swedish, American and Italian DBA clinical and research groups.

VARIANTS

RPS19

The RPS19 gene (OMIM 603474; locus 19q13.2) was the first DBA gene that was discovered and is the most frequently mutated in patients. It comprises 6 exons and spans 11 kb. The first exon (372 bp) is not included in the coding DNA sequence (CDS) region, while the other five (435 bp) encode a protein of 145 amino acids (MW ~16 kDa).

Eighty-seven distinct mutations have been previously described in RPS19 gene [most reviewed in [START_REF] Campagnoli | RPS19 mutations in patients with Diamond-Blackfan anemia[END_REF]. We here report 42 additional mutations: 11 missense, 3 nonsense, 18 deletions and/or insertions, 10 splice-site defects ( At least 163 polymorphisms are listed in NCBI SNP database. We have identified 9 unpublished intronic allelic variants: c.71+174A>G, c.71+24A>G, c. 356+166G>T, c.411+6G>T, c.412-75A>G, c.356+153G>A, c.356+29T>C, c.356+229G>A and c.412-131T>C. Seven pseudogenes are annotated in the NCBI Gene database as 'inferred'.

RPL5

The human RPL5 gene (OMIM 603634; locus 1p22.1) consists of eight exons and spans 9.8 kb. The primary transcript is 1031 nt long and encodes a 297-aa protein (MW ~34.2 kDa) component of the 60S ribosomal subunit.

Heterozygous mutations in RPL5 gene have been reported in DBA patients [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF][START_REF] Cmejla | Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia[END_REF][START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF]. Gazda et al. showed mutations in 18 of 196 DBA probands (9%) and in six additional family members [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF], for a total of 24 individuals; Cmejla et al. studied 28 Czech families and identified sequence changes in 8 DBA patients from 6 families (21.4%) [START_REF] Cmejla | Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia[END_REF]; Quarello et al. reported mutations in 12 out of 92 (13%) unrelated Italian probands [START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF]. In this paper we are adding 10 new mutations found in 12 patients (Table 1) and two patients carrying two previously described mutations. The total number of patients with mutations in RPL5 is 58: 21 have a de novo mutation, 10 are familial cases. Thirty-nine mutations are distinct and are distributed as follows: 6 missense, 7 nonsense, 21 small deletions and/or insertions, 5 splice-site defects.

Ninety-five polymorphisms are listed in NCBI SNP database. There are multiple processed pseudogenes of RPL5 gene dispersed through the genome: three of them are annotated as 'validated' in NCBI Gene database. The RPL11 gene is located on chromosome 1 (OMIM 604175; locus 1p36.1-p35) and encompasses 6 exons spanning 4.6 kb. The RPL11 mRNA is 609 bp long and encodes a 178 amino acid protein (MW ~20.1 kDa).

RPL11

Twenty-three distinct mutations in 34 DBA patients have been previously described [Gazda et al., 2008;[START_REF] Cmejla | Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia[END_REF][START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF]; three new mutations are reported here for the first time (Table 1). The 26 mutations are classified as follows: 1 missense, 2 nonsense, 17 small deletions and/or insertions, and 6 splice-site defects. The total number of patients with mutations is 37: 12 carry a de novo mutation, whereas 7 are familial cases.

Fifty-seven polymorphisms are reported in NCBI SNP database and five pseudogenes are annotated in NCBI Gene database as 'inferred'.

RPL35A

The RPL35A gene (OMIM 180468) is located on chromosome 3q29-qter and comprises 6 exons spanning 5.6 kb. The predicted size of the primary transcript is 511 bp. The first exon (41 bp) is not included in the CDS region, while the other five (470 bp) encode a 110-aa protein (MW ~12.4 kDa). [START_REF] Farrar | Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia[END_REF] reported five mutations: one missense, one nonsense, one small deletion and two deletions of a complete allele. They are all considered pathogenic. The missense mutation c.97G>A, which creates a cryptic splice donor site within exon 3, was also found in the proband's father and sister, both showing isolated macrocytosis. Inheritance was not tested in the remaining cases [START_REF] Farrar | Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia[END_REF].

The RPL35A gene has 45 polymorphisms listed in NCBI SNP database and only one pseudogene annotated as 'validated' in NCBI Gene database.

RPS24

The RPS24 (OMIM 602412; locus 10q22-q23) gene comprises 6 exons and spans 8 kb. Its three isoforms are expressed as splice variants: S24a or variant 1 (615 bp), S24c or variant 2 respectively. The different isoforms show a tissue-specific pattern of expression [START_REF] Xu | The gene encoding human ribosomal protein S24 and tissue-specific expression of differentially spliced mRNAs[END_REF][START_REF] Gazda | Ribosomal protein S24 is mutated in Diamond-Blackfan anemia[END_REF].

Three RPS24 mutations in a total of 8 DBA patients [START_REF] Gazda | Ribosomal protein S24 is mutated in Diamond-Blackfan anemia[END_REF] have been previously reported in the DBA Mutation Database. Recently Quarello et al. and Badhai et al. showed two further changes in RPS24 gene: a small deletion and a missense mutation [START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF][START_REF] Badhai | Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond-Blackfan anemia[END_REF]. Additionally, we identified an unpublished splice donor variant (Table 1). In total 6 mutations are reported for this gene: one missense, two nonsense, one small deletion and two splice-site defects. In total, 12 patients were found mutated in this gene: only one has a de novo mutation, whereas 5 carry an inherited mutation.

Eighty-two polymorphisms are listed in NCBI SNP database and only one RPS24 pseudogene is annotated as 'validated' in NCBI Gene database.

RPS17

The RPS17 gene (OMIM 180472) is located on chromosome 15 (locus 15q). It encompasses five exons and spans 3.7 kb. The RPS17 mRNA is 562 nt long and encodes for a 135-amino acid protein (MW ~15.5 kDa). Two different sequence changes eliminating the natural start site for protein synthesis were found in this gene by [START_REF] Cmejla | Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia[END_REF][START_REF] Song | A novel initiation codon mutation in the ribosomal protein S17 gene (RPS17) in a patient with Diamond-Blackfan anemia[END_REF]. Gazda et al. [2008] identified a new mutation among 196 tested probands; it is a deletion of two nucleotides causing a frameshift. All mutations are de novo.

The RPS17 gene has 30 polymorphisms listed in NCBI SNP database and has two 'validated' pseudogenes in NCBI Gene database.

RPS7

The RPS7 gene (OMIM 603658; locus 2p25) consists of 7 exons spanning 5.6 kb. The predicted size of the primary transcript is 745 bp encoding a 194-aa protein (MW ~22 kDa). A donor splice-site mutation in intron 2 was found in a single DBA patient by Gazda and collaborators [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF]]. The inheritance was not tested.

Eighty-three polymorphisms are listed in NCBI SNP database for this gene and only one pseudogene is annotated as 'validated' in NCBI Gene database.

RPS26

The RPS26 gene (OMIM 603701) is located on chromosome 12 (locus 12q13) and has four exons spanning 2.32 kb. It results in a transcript of 699 bp encoding a 115-amino acid protein (MW ~12.9 kDa).

Recently, Doherty and collaborators identified 8 distinct mutations in 13 DBA patients: 5 carry a de novo mutation, whereas 3 are familial cases [START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF]. Mutations in this gene were identified in about 6.4% of the overall DBA population and are distributed as follows: 4 missense, one insertion and 3 splice-site defects.

The RPS26 gene has 64 polymorphisms listed in NCBI SNP database and 4 pseudogenes annotated as 'validated' in NCBI Gene database.

RPS10

The RPS10 gene (OMIM 603632; locus 6p21.31) encompasses 6 exons spanning 8.65 kb. The predicted size of the primary transcript is 636 bp encoding a 165-aa protein (MW ~18.8 kDa).

Mutations in this gene were identified in about 2.6% of the overall DBA population [START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF]. Specifically three distinct sequence changes in 5 DBA probands have been reported: one missense, one nonsense and one insertion. One mutation is de novo, whereas in the other cases the inheritance was not ascertained [START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF].

The RPS10 gene has 88 polymorphisms reported in NCBI SNP database and 3 'validated' pseudogenes listed in NCBI Gene database.

MUTAGENESIS MECHANISMS

Common mutagenesis mechanisms that generate point mutations include slippage, that causes small indels, and cytosine-guanine (CpG) dinucleotide methylation followed by spontaneous deamination, which causes G>A or C>T transitions. We evaluated the involvement of these mechanisms in DBA by comparing each mutant sequence to the wild-type gene sequence and by observing the context in which a mutation occurred. We found that 57 out of 87 indels in DBA patients are consistent with a slippage mechanism. The frequency of the different substitution classes is 64 transitions versus 50 transversions. Out of 64 transitions, we identified 15 mutations occurring within CpG dinucleotides.

A rare mechanism of mutagenesis is interlocus gene conversion arising from transfer of genetic information between highly homologous genes. Well-known diseases caused by this process are steroid 21-hydroxylase deficiency [START_REF] Morel | Gene conversions and rearrangements cause discordance between inheritance of forms of 21-hydroxylase deficiency and HLA types[END_REF] and Shwachman-Diamond syndrome [START_REF] Boocock | Mutations in SBDS are associated with Shwachman-Diamond syndrome[END_REF]. In these cases the donor sequence is a nearby pseudogene resulting from a recent duplication. To investigate the occurrence of pseudogene-mediated gene conversion in DBA, we retrieved pseudogene sequences of the most frequently mutated RPs (RPS19, RPL5 and RPL11) annotated as 'validated' and/or 'inferred' in NCBI Gene database; all of them were intronless. We aligned them with their respective RP gene sequences and looked at 5bp on each side of the mutation, searching for a correspondence between the pseudogene and the mutated sequence. We found only 6 mutations possibly due to gene conversion: 4 in RPS19, 1 in RPL5 and 1 in RPL11 (Table 2). The pseudogenes are all located on different chromosomes, as compared to the respective genes. Two mutations in RPS19 are identical to the corresponding sequence of pseudogene RPS19P2 located on chromosome 1. Obviously, we could not exclude that the same changes arose independently in the gene and in the pseudogene. In any case, our results show that gene conversion does not play a major role in generating mutations in RPS19, RPL5 and RPL11. In conclusion, the most frequent mutagenic mechanism observed in DBA patients appears to be slippage, followed by transitions occurring at CpG dinucleotides.

BIOLOGICAL RELEVANCE

Several studies have addressed the effects of DBA mutations showing that they can lead either to a reduction of RP mRNA or to the production of ribosomal proteins with defective stability and/or localization. In all these cases, mutations cause haploinsufficiency which in turn interferes with the biogenesis of the large or the small ribosomal subunit. These defects are due to aberrant rRNA maturation at different steps, depending on the affected RP [START_REF] Flygare | Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits[END_REF][START_REF] Choesmel | Impaired ribosome biogenesis in Diamond-Blackfan anemia[END_REF]Idol et al., 2008;[START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF][START_REF] Farrar | Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia[END_REF][START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF]. Here, we briefly revisit the biological function of each DBA RP and the effects of their mutations.

RPS19. RPS19 mutations are associated with a defect in the maturation of 18S rRNA resulting in the accumulation of 21S pre-rRNA precursors [START_REF] Flygare | Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits[END_REF][START_REF] Choesmel | Impaired ribosome biogenesis in Diamond-Blackfan anemia[END_REF]Idol et al., 2008]. All of these mutations cause loss of function and some have been functionally characterized. Extensive functional data were recently reviewed by Campagnoli et al. in this journal [Campagnoli et al., 2008]. Furthermore Crétien and collaborators reported the study of the subcellular localization of several RPS19 mutants fused to green fluorescent protein (GFP) [START_REF] Crétien | Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia[END_REF]. They observed impaired nucleolar localization and a marked decrease in levels of protein expression for the following mutants: p. [START_REF] Crétien | Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia[END_REF]. Another mutation was recently investigated for its functional consequences by Badhai and collaborators, who reported that primary fibroblasts from a DBA patient with a RPS19 acceptor splice-site mutation (c.72-2A>C) showed reduced proliferative capacity due to G1 arrest [START_REF] Badhai | Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond-Blackfan anemia[END_REF].

RPL5. RPL5 has been implicated in nucleocytoplasmic transport of 5S rRNA prior to its assembly into the large ribosomal subunit [START_REF] Steitz | A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells[END_REF]. RPL5 specifically binds to this rRNA through the domains located at both the amino terminus and the carboxyl terminus [START_REF] Michael | Distinct domains in ribosomal protein L5 mediate 5 S rRNA binding and nucleolar localization[END_REF]. It has been shown that the perturbation of ribosomal biogenesis by impaired rRNA synthesis, processing, or ribosome assembly, triggers the direct binding of RPL5 along with RPL11 and possibly RPL23 to MDM2. These interactions inhibit MDM2-mediated p53 ubiquitination and degradation, resulting in p53 activation [START_REF] Zhang | Signaling to p53: ribosomal proteins find their way[END_REF]. The pathogenic effect of RPL5 haploinsufficiency on ribosome biogenesis has been studied by Gazda and collaborators both in a knockdown cell model and in patient cells that harbored the following mutations: c. 67C>T, c.173delG, c.175_176delGA, c.[498_502delTGTGG;497_498ins40] [Gazda et al., 2008]. HeLa cells expressing small interfering RNAs (siRNAs) against RPL5 show decreased production of 28S and 5.8S mature rRNAs and accumulation of their precursors, in particular 32S and 12S. The same defect was observed in lymphoblastoid cells established from DBA patients. Moreover, RPL5 knockdown induces reduction of free 60S subunit and formation of half-mer polysomes [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF].

RPL11.

In yeast, Rpl11 forms a subcomplex with Rpl5 and 5S rRNA that is recruited into nascent ribosomes at an early step [START_REF] Zhang | Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes[END_REF]. In human cells, RPL11 appears to have a similar role in ribosome biogenesis but also functions to suppress the transcriptional activity of c-Myc and plays a feedback regulatory role in coordinating c-Myc level and activity with ribosomal biogenesis [START_REF] Dai | Feedback regulation of c-Myc by ribosomal protein L11[END_REF]. RPL11 also cooperates with RPL5 to inhibit the E3 ubiquitin ligase activity of MDM2, thus resulting in the accumulation of transcriptionally active p53 [START_REF] Zhang | Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway[END_REF]. Fumagalli et al. recently showed that RPL11-mediated p53 induction is a general response to defective 40S or 60S ribosome biogenesis in human cell lines [START_REF] Fumagalli | Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction[END_REF]]. The ubiquitin-like molecule NEDD8, that controls RPL11 stability and subcellular localization, plays an important role in the regulation of RPL11 signaling to p53 [START_REF] Sundqvist | Regulation of nucleolar signalling to p53 through NEDDylation of L11[END_REF]. In zebrafish rpl11 knockdown activates the p53 pathway and disrupts the normal embryonic development through a p53mediated apoptotic response [START_REF] Chakraborty | Loss of ribosomal protein L11 affects zebrafish embryonic development through a p53-dependent apoptotic response[END_REF]. Gazda and collaborators showed that mutations in RPL11, c.314_315delTT, IVS1+2t>c, IVS2-1g>a, IVS4+1g>t, lead to accumulation of the precursors of 28S and 5.8S rRNAs, similar to that of mutations in RPL5 [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF].

RPL35A. Farrar et al. studied RPL35A deficiency in UT-7/Epo and TF-1 cells by transduction with three different small hairpin RNAs (shRNAs) against Rpl35A mRNA. They observed decreased proliferation, increased apoptosis and reduced biogenesis of 60S subunits.

Metabolic rRNA labeling and Northern blot analysis revealed accumulation of 45S and 41S early precursors and decreased 12S and 7S pre-RNAs. An EBV-transformed lymphoblastoid cell line from a DBA patient with deletion of a complete allele also showed reduced 12S rRNA compared to healthy controls [START_REF] Farrar | Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia[END_REF].

RPS24. RPS24, like RPS19, is essential for the production of the small ribosomal subunit, as displayed by the reduction of 40S subunits and 80S monosomes in polysomal profiles of RPS24-depleted cells [START_REF] Choesmel | Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder[END_REF]. Lymphoblastoid cells from three patients with mutations in RPS24 (p.Gln106X, p.Arg16X, deletion N2-Q22) showed delayed maturation of 30S pre-rRNA with a corresponding decrease in 21S and 18S-E pre-rRNAs. Accumulation of the 30S pre-rRNA suggests that RPS24 is required for the maturation of both the 5′ and 3′ [START_REF] Badhai | Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond-Blackfan anemia[END_REF]. Moreover, Quarello et al. expressed FLAG-tagged RPS24 protein carrying the mutation p.Gln22del in HEK293 cells to study its subcellular localization. Although this mutant protein is less stable than the wild-type, it was able to reach the nucleolus [START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF].

RPS7. RPS7, like other RPs discussed above, interacts with MDM2 and regulates its E3 ligase activity on p53 [START_REF] Chen | Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function[END_REF]. RPS7 is itself a substrate of MDM2 and RPS7 ubiquitination enhances p53 response and facilitates cell death triggered by different stress signals [START_REF] Zhu | ratio (OR) and 95% CI from logistic regression; OR are drawn on a logarithmic scale[END_REF]. The pathogenic effect of the RPS7 mutation c.147+1G>A was studied in lymphoblastoid cells derived from the one patient. These cells show accumulation of 45S and 30S pre-rRNAs when compared to an unaffected sibling. Depletion of RPS7 by siRNA in HeLa cells confirmed a defect in 5'-ETS processing [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF].

RPS26. RPS26 can regulate its own expression by binding its pre-mRNA and suppressing its splicing [START_REF] Ivanov | Human ribosomal protein S26 suppresses the splicing of its pre-mRNA[END_REF]. Northern blot analysis showed that depletion of RPS26 in HeLa cells leads to the accumulation of 43S, 26S and 18S-E pre-rRNAs, pointing to defective cleavage at both ends of 18S. The same phenotype was present in lymphoblastoid cells derived from RPS26 mutated patients (c.1A>T, c.1A>G in two different probands, c.97G>A and IVS1+1g>c) [START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF]. RPS10. Analysis of RPS10 depleted HeLa cells and RPS10 mutated lymphoblastoid cells (c.260_261insC and c.337C>T in three different probands) revealed a pre-rRNA processing phenotype similar to RPS26 [START_REF] Doherty | Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia[END_REF].

CLINICAL RELEVANCE

For many years DBA was considered to be rarely inherited since most patients presented without any family history. Mutational analyses have clarified this observation: in our study, 125 of 197 patients whose family history was ascertained had de novo mutations, whereas the other 72 were familial. Thus, it is likely that the majority of DBA mutations arise spontaneously. This pattern may be due to reduced reproductive fitness of mutated patients.

Difficulties in completing pregnancy and an increase in stillborn offspring have been reported for those women with DBA who have conceived [START_REF] Faivre | High-risk pregnancies in Diamond-Blackfan anemia: a survey of 64 pregnancies from the French and German registries[END_REF].

Genotype-Phenotype Correlation

Genotype-phenotype correlations were evaluated in all patients reported in the DBA Mutation Database. They represent approximately 50% of the total number of DBA patients. Clinical description was not available for a few patients thus each analysis was calculated only for patients that had the specific clinical information.

The statistical analysis was performed by considering the following parameters: growth retardation, craniofacial malformation (other than cleft lip and palate), cleft lip and/or palate, upper limb abnormalities, flat thenar muscle, malformed thumb, triphalangeal thumb, heart anomalies, genitourinary anomalies, presence of any type of malformation including short stature, any type of malformation with the exclusion of short stature, multiple malformations, mental retardation, small for gestational age (SGA), response to steroids (Table 3).

Hematological information was not considered because clinical data reported in old publications were not updated. The risk connected with carrying a mutation in a specific RP relative to patients carrying mutations in the other eight RP genes studied was obtained by using logistic regression and odds ratio (OR) and 95% CI were calculated. P values less than 0.05 were considered statistically significant. Data were analyzed with the SAS software version 8.01. Data were not informative for some rarely mutated RP genes, namely RPS7, RPS17, RPS24 and RPS10 (Table 3). Results for RPS19, RPL5 and RPL11 are presented in Fig. 1.

As previously shown [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF][START_REF] Cmejla | Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia[END_REF][START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF], patients carrying mutations in RPL5 or RPL11 present more frequently with malformations. The risk of malformations of any type, including or excluding short stature, is 6.5 or 7.6-fold higher in RPL5-mutated and 4.5 or 2.7-fold higher in RPL11-mutated patients, respectively, than in patients with mutations in other genes. RPL5-mutated patients have a statistically higher risk of multiple malformations (OR 3.8). Each type of malformation evaluated in this study, with the exception of those of the genitourinary tract, is more frequent in patients with mutations in RPL5. Specifically, 21 out of 24 patients with cleft have mutations in RPL5. A cleft was shown in two patients who carried mutations in RPL11 and in one patient who carried a mutation in RPS26. Clefts have never been found in RPS19 patients. RPL5-mutated patients are SGA more frequently than patients with other mutations. Of 9 patients with SGA, 7 carry a RPL5 and 2 a RPL11 mutation. Although mutations in RPL11 are associated with an increased risk of any type of malformations, most of these are hand abnormalities (Table 3).

The gene currently associated with genitourinary malformations is RPL35A. Of 5 patients with mutations in RPL35A, 3 have genitourinary malformations. In contrast, only 16 of 249 patients with mutations in other RP genes have genitourinary malformations (Table 3). This difference in frequencies towards is statistically significant. Conversely, patients with mutations in RPS19 are less likely to have malformations of any type when compared with the other patients (OR < 1). This finding is also true when each type of malformation is considered independently. Patients mutated in RPS26 exhibit the lowest response to steroids (4 out of 10), whereas most DBA patients with mutations in other known RP genes respond to steroids at a higher frequency (92/125) (CI 0.06-0.90).

Interestingly, mental retardation is shown in only 9/270 patients. Eight of these have mutations in RPS19: four have large deletions at the RPS19 locus, two have translocations associated with deletions, one has a deletion of exons 1-3 and one has a splice-site defect (c.72-1G>A). The last patient has a frameshift mutation in RPL5 (c.169_172delAACA) and a complex malformation phenotype that includes myelomeningocele, cleft palate, and facial dysmorphism. Patients with mental retardation and large deletions/rearrangements in RPS19 are likely to show a contiguous gene syndrome [START_REF] Tentler | A microdeletion in 19q13.2 associated with mental retardation, skeletal malformations, and Diamond-Blackfan anaemia suggests a novel contiguous gene syndrome[END_REF]. In conclusion, we can say that mental retardation is not typically associated with mutations in ribosomal protein genes and when found in association with other clinical features of DBA is probably linked to contiguous genes.

Variable expressivity

Variable expressivity is shown for all RP gene mutations. Possible mechanisms underlying variable expressivity include an influence of modifier genes and environmental factors.

Stochastic factors are invoked in the case of non concordance for malformations in monozygous twins [START_REF] Campagnoli | Molecular basis of Diamond-Blackfan anemia: new findings from the Italian registry and a review of the literature[END_REF]. Potential modifier genes could be genes involved in modulating the level of expression of RP genes or other genes involved in ribosome biogenesis. A patient harboring mutations in two different RP genes, RPL5 and RPS24, was reported by [START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF]. In this case, the malformation phenotype was likely due to RPL5, since the patient carried hand malformation, often associated with RPL5 mutations. Moreover RPL5 mutation was de novo and the parent carrier of the RPS24 missense mutation did not show malformations. These aspects suggest that the RPS24 18 missense variant may be a silent mutation. Variations in the promoter or other regions have also been hypothesized to be phenotype modifiers [START_REF] Crétien | Genetic variants in the noncoding region of RPS19 gene in Diamond-Blackfan anemia: potential implications for phenotypic heterogeneity[END_REF].

DATABASE

The need for a comprehensive collection of all mutations in DBA genes, as well as of their functional consequences and clinical phenotypes, prompted us to create and maintain the DBA Mutation Database (http://www.dbagenes.unito.it) [START_REF] Boria | A new database for ribosomal protein genes which are mutated in Diamond-Blackfan Anemia[END_REF]. It is founded on the Leiden Open (source) Variation Database (LOVD) system that was upgraded to the latest version, LOVD 2.0 build 25, released in March 2010 [START_REF] Fokkema | LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach[END_REF].

The first version of the database included only three DBA genes, RPS19, RPS24 and RPS17, and 86 distinct disease-causing mutations. The database has been updated with the newly described DBA genes: RPL5, RPL11, RPL35A, RPS7, RPS26, RPS10, and now comprises a total of 220 distinct pathogenetic mutations, distributed as follows: 52 missense, 27 nonsense, 87 small deletions and insertions, 14 large deletion and rearrangements and 40 splice-site defects (Table 4). Out of the 134 newly added sequence changes, 78 were previously published while 56 are reported here for the first time. Overall, the database includes data for 355 patients, all carrying RP mutations.

According to the basic structure of the LOVD database scheme, each DBA gene has its own homepage providing general gene and database information, access to allelic variant tables, search tools for browsing data and links to external gene-related resources, such as NCBI SNP database, MIM, NCBI Entrez, HGMD (Supp. Figure S1). Furthermore links to schematic drawings showing the location of the pathogenetic variants in relation to the gene (Supp. Figure S2) and, when available, to the protein structures are included.

All mutations are described according to the Human Variation Society (HGVS) nomenclature [START_REF] Wildeman | Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker[END_REF].

All available data relative to each mutation are provided in the "Variants" section and include the exact molecular description at DNA and protein levels, the clinical features of the corresponding patients, literature references and details on the detection methods. Consistent with the functional classification proposed for RPS19 by [START_REF] Campagnoli | RPS19 mutations in patients with Diamond-Blackfan anemia[END_REF], information about functional consequences of mutations on mRNA and protein are reported.

Compared to the previous database version, it is now possible to specify the potential molecular mechanism leading to each allelic variant ("Molecular Mechanisms" column) and for each patient the clinical complications ("Complications" column). Furthermore, we substituted the "Occurrence" column with "Variant Origin" that describes the inheritance of the mutation in an exhaustive way. Further details can be found at the database web site.

A link to the DBA Mutation Database is provided for each gene in NCBI Gene database.

DIAGNOSTIC RELEVANCE

At the time of submission, 220 distinct mutations in 355 DBA patients have been identified in nine genes, all encoding ribosomal proteins. The difficulties in clinical diagnosis and the absence of biochemical assays make identification of the causative mutation clinically important. Identification of non-symptomatic carriers is mandatory when potential donors of hematological stem cells are evaluated within first degree relatives. Moreover, prenatal diagnosis may be requested by families with severely affected children. The RPS19 gene is the most frequently involved being mutated in 25% of patients. Mutations in RPL5 and RPL11 are frequently found in patients with malformations of upper limbs or face.

Malformations in general, also appear increased in patients with mutations in these genes. A

FUTURE PROSPECTS

The ribosomal basis of DBA is evident. So far about 50% of DBA patients may be characterized using the four most commonly mutated genes: RPS19, RPL5, RPL11, RPS26. It is expected that other RP genes may be mutated in the remaining patients. For this reason the DBA community has started a large project focused at sequencing each of the 79 RP genes in every DBA patient. However, sequencing is tedious and time-consuming. An easy and quick diagnostic assay would be of great help to clinical hematologists: the perfect assay should be able to diagnose all DBA patients, independently of the gene affected. A genetic or functional abnormality shared by all patients may be exploited to generate a diagnostic assay.

The definition of the molecular basis of DBA has also opened the road to molecular therapy.

Gene therapy looks promising since even a small increase of RP expression may be helpful to resolve the bone marrow failure thereby making this disease a reasonable target for this treatment [START_REF] Flygare | Gene therapy of Diamond Blackfan anemia CD34(+) cells leads to improved erythroid development and engraftment following transplantation[END_REF]. Treatment with leucine has been proven helpful in rare cases [START_REF] Pospisilova | Successful treatment of a Diamond-Blackfan anemia patient with amino acid leucine[END_REF] but large clinical trials are necessary to ascertain if its effect may be general or gene specific.

Accession Numbers

The GenBank accession number for human RPs are: NM_001022.

F o r P e e r R e v i e w

METHODS

MUTATION DETECTION AND PATHOGENICITY ASSESSMENT

Mutation detection has been performed using automatic sequencing. For each group details can be found on their previous papers [START_REF] Gazda | Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients[END_REF][START_REF] Cmejla | Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia[END_REF][START_REF] Quarello | Diamond-Blackfan anemia: genotype-phenotype correlation in Italian patients with RPL5 and RPL11 mutations[END_REF][START_REF] Proust | Ten novel Diamond-Blackfan anemia mutations and three polymorphisms within the rps19 gene[END_REF].

Generally, the complete coding sequence and exon-intron boundaries have been sequenced to eliminate the possibility of the presence of other mutations. Verification has been performed in keeping with the journal's requirements. In particular, all mutations have been confirmed

on a second sample and on a second PCR product. In absence of biochemical data, pathogenicity of missense mutations have been established by at least two methods. In turn, the following methods were used: linkage to disease in a family, concurrent appearance of the phenotype with a de novo mutation, or determination that the mutation is absent among at least 50 normal individuals (100 alleles) or involves a highly conserved amino acid. In some cases the Polyphen algorithm has been used. Otherwise pathogenicity is reported as uncertain in the Database.

  anemia (DBA, OMIM 105650) is a rare inherited disease characterized by

  ) and S24b or variant 3 (633 bp), encoding proteins of 130, 133 and 131 amino acids,

  rRNA. Primary fibroblasts obtained from a DBA patient with an RPS24 start codon mutation (c.1A>G) showed reduced proliferation and abnormal expression of cell cycle regulatory proteins

[

  den Dunnen and Antonarakis 2000] and their pathogenicity was established according to the Initiative/HGVS (Supplemental Material). Mutation nomenclature has been checked with Mutalyzer program

  types of malformations or with multiple malformations should be screened first for these genes.

  retardation; SR: steroid response. (*) Total number of patients whose clinical data were available.

FIGURES

  FIGURES

Table

  RPS19 mutations are reported and they are carried by 219 patients: 82 of these are de novo and 45 are inherited. The inheritance was not ascertained in the remaining cases.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	). Overall 129 distinct
	6

  The URLs of resources cited in this work are the following: Single Nucleotide Polymorphisms database, http://www.ncbi.nlm.nih.gov/SNP/ Entrez Gene database, http://www.ncbi.nlm.nih.gov/gene/ Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim The Human Gene Mutation Database, http://www.hgmd.cf.ac.uk PolyPhen, http://genetics.bwh.harvard.edu/pph/

	Web Resources
	F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R e v i e F F o r o r P P e r R e v i e w e i v e R e e e r 122x84mm (200 x 200 DPI)
	w w w w w w
	3 for RPS19,
	NM_000969.3 for RPL5, NM_000975.2 for RPL11, NM_000996.2 for RPL35A,
	NM_033022.3 for RPS24, NM_001021.3 for RPS17, NM_001011.3 for RPS7, NM_001029.3
	for RPS26 and NM_001014.3 for RPS10.

Table 1 .

 1 Newly reported mutations in RPS19, RPL5, RPL11 and RPS24. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

	Page 29 of 44			Human Mutation
	Mutated	Patient	Exon/Intron	cDNA Mutation	Predicted amino	Mutation	Malformations	Growth	Steroid	Inheritance
	Gene	(Gender)			acid change	Type		Retardation	Response
	RPS19	Ps19_1(F)	Ex 2	c.2T>A	p.Met?	Missense	na	na	na	familial
		Ps19_2(F)	Ex 2	c.10_13delGTTA	p.Val4LeufsX2	Deletion	none	no	yes	sporadic
		Ps19_3(F)	Ex 2	c.14delC	p.Thr5MetfsX2	Deletion	na	na	na	unknown
		Ps19_4(M)	Ex 2	c.28_29insT	p.Asn10IlefsX41	Insertion	na	na	na	sporadic
		Ps19_5(F)	Ex 2	c.34_47del	p.Gln12SerfsX34	Deletion	Dystrophy	na	na	unknown
		Ps19_6(M)	Ex 2	c.49G>C	p.Ala17Pro	Missense	na	na	na	sporadic
		Ps19_7(NA)	Ex 2	c.58G>C	p.Ala20Pro	Missense	na	na	na	unknown
		Ps19_8(F)	Ex 3	c.83T>G	p.Leu28Arg	Missense	none	no	na	unknown
		Ps19_9(M)	Ex 3	c.88delG	p.Val30SerfsX46	Deletion	Microcephaly, micro-retrognathy,	na	na	familial
							hypertelorism, cafe au lait spots
		Ps19_10(M)	Ex 3	c.93delC	p.Glu32AsnfsX44	Deletion	Thumb	no	no	unknown
		Ps19_11(M)	Ex 3	c.103dupG	p.Asp35GlyfsX16	Insertion	none	no	no	unknown
		Ps19_12(M)	Ex 3	c112A>T	p.Lys38X	Nonsense	none	no	no	sporadic
		Ps19_13(M)	Ex 3	c.156G>A	p.Trp52X	Nonsense	Low hairline, cafe au lait spots	na	na	unknown
		Ps19_14(M)	Ex 3	c.172G>C	p.Ala58Pro	Missense	none	yes	no	de novo
		Ps19_15(F)	Ex 4	c.178A>C	p.Thr60Pro	Missense	none	no	na	sporadic
		Ps19_16(M)	Ex 4	c.187_189insCAC	p.His63dup	Insertion	Flat nose, low hairline, mitral vale	na	na	sporadic
							and tricuspid valve insufficiency
		Ps19_17(NA)	Ex 4	c.195C>G	p.Tyr65X	Nonsense	na	na	na	unknown
		Ps19_18(NA)	Ex 4	c.195C>G	p.Tyr65X	Nonsense	na	na	na	unknown
		Ps19_19(F)	Ex 4	c.203_204insG	p.Gly69TrpfsX85	Insertion	na	na	na	sporadic
		Ps19_20(M)	Ex 4	c.212G>A	p.Gly71Glu	Missense	na	na	na	sporadic
		Ps19_21(F)	Ex 4	c.281G>T	p.Arg94Leu	Missense	none	na	na	sporadic
		Ps19_22(F)	Ex 4	c.284delG	p.Gly95AlafsX16	Deletion	ASD	na	na	unknown
		Ps19_23(M)	Ex 4	c.289_290insAGGC	p.Lys97ArgfsX58	Insertion	Dysplastic aortic valve	na	na	unknown
		Ps19_24(F)	Ex 4	c.296_297delTG	p.Val99GlyfsX54	Deletion	na	na	na	unknown
		Ps19_25(M)	Ex 4	c.301C>T	p.Arg101Cys	Missense	na	na	yes	unknown
					John Wiley & Sons, Inc.

F: female; M: male; na: not available; VSD: ventricular septal defect; ASD: atrial septal defect.

Table 2 .

 2 Pathogenetic point mutations in RP genes that are homologous to pseudogene sequences. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

								Page 32 of 44
	Gene	DNA Mutation	Mutation	Exon/Intron	Gene sequence	Mutated sequence	Pseudogene	Pseudogene name
	name		Type				sequence	(locus)
	RPS19	c.384_385delAA	Deletion	Ex 5	GGACAAAGAGAT	GGACA--GAGAT	GGACA--GAGAT	RPS19P2 (1p13.2)
		c.403G>A	Missense	Ex 5	GAATCGCCGGA	GAATCACCGGA	GAATCACCGGA	RPS19P2 (1p13.2)
		c.191T>C	Missense	Ex 4	GCACCTGTACC	GCACCCGTACC	GCACCCGTACC	RPS19P4 (5q11.2)
		c.166C>T	Nonsense	Ex 3	ACACGCGAGCT	ACACGTGAGCT	ACACGTGAGCT	RPS19P7 (10q11.21)
	RPL5	c.535C>T	Nonsense	Ex 6	CCAAACGATTC	CCAAATGATTC	CCAAATGATTC	RPL5P34 (22q13.2)*
	RPL11	c.94_97delAGAC	Deletion	Ex 2	GAGACAGACTGACG GAGAC----TGACG GAGAC----TGACG RPL11P5 (12q24.31)

* Note that this variation is carried also by other pseudogenes, but in a slighty different context (data not shown).

Table 3 .

 3 Clinical data relative to all DBA patients reported in the DBA Mutation Database.

	Gene Name	Patients with Mutations*	Patients with Malformations	Malformations no Short Stature	Face	Cleft Lip and/or Palate	Upper Limb	Flat Thenar	Thumb	Triphalangeal Thumb	Heart Malf.	Genitourinary Anomalies	Mental Retardation	Small for Gestational Age	Multiple Malf.	GR SR
	RPS19	166	55	53	27	0	12	2	11	3	14	7	8	0	15	41 39
	RPL5	50	42	41	24	21	28	10	18	12	15	3	1	7	14	6 19
	RPL11	36	26	24	5	2	19	8	14	6	6	3	0	2	2	2 19
	RPL35A	5	4	4	1	0	0	0	0	0	1	3	0	0	1	na 4
	RPS26

Table 4 .

 4 Summary of the pathogenic variants in DBA Mutation Database. We have here considered the association with an individual patient irrespective to the fact that a mutation could recur in the same family.

	Type of
	Mutation

John Wiley & Sons, Inc.Human Mutation

John Wiley & Sons, Inc.

Acknowledgements

We would like to thank Dr. Ivo Fokkema for his support in improving the quality of the database. This work was partly funded by the following grants: Telethon (to ID), DBA Foundation (to ID, to HTG), Italian Ministry of University and Research (to ID and UR), Gruppo di Sostegno DBA Italia (to ID and UR), the National Institutes of Health (USA) R01 AR044345 (to AHB), R01 HL079571 and The Feinstein Institute for Medical Research General Clinical Research Center M01 RR018535 (to JML, AV, EA), the Pediatric Cancer Foundation (to JML), the Ministry of Education, Czech Republic, grant MSM 6198959205, the Ministry of Health, Czech Republic grants 00023736 and NT11059 (to DP and RC), the Federal Ministry of Education and Research, Germany, grants 01GM0312 and 01GM0847 (to JJM).

We also thank the Daniella Maria Arturi Foundation for supporting communication among DBA researchers.


T Lehrnbecher
email: thomas.lehrnbecher@kgu.de

A Attarbaschi

M Duerken

J Garbino

U Gruhn

S Kontny

R Lüer

J Phillips

H J Scholz

T Wagner

A H Wiesel

Groll

B Gruhn

R Phillips

J Scholz

H J Wagner

A H Groll

Posaconazole salvage treatment in paediatric patients: a multicentre survey

  L'archive ouverte pluridisciplinaire

Posaconazole (Noxafil®) is a novel oral second generation antifungal triazole. Due to its extended activity, posaconazole is widely used in adult patients. While a paediatric dosage has not been defined, posaconazole is occasionally being used in children. Here we report the results of a multicentre retrospective survey on safety and efficacy of posaconazole salvage therapy in children.

Patients eligible for inclusion were <18 years of age, had received at least one dosage of posaconazole, and had begun posaconazole therapy prior to the start of the survey. The compound was administered orally at dosages individually determined by the responsible physician until occurrence of intolerance or maximum efficacy on the basis of refractory infection, intolerance of or contraindications to standard therapies, or as best individual therapeutic option. The study was approved by the local ethics committee of Münster, the site of one of the principal investigators (AHG). Invasive fungal infections and response to treatment were classified according to the published EORTC/ MSG criteria and a previous clinical trial of antifungal salvage therapy, respectively [START_REF] De Pauw | Revised definitions of invasive fungal disease from the European organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (EORTC/MSG) consensus group[END_REF][START_REF] Maertens | Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy[END_REF]. Clinical adverse events were recorded and graded according to current Common Toxicity Criteria [START_REF]Common terminology criteria for adverse events v3.0 (CTCAE)[END_REF]. For statistical comparisons of continuous data, the Mann-Whitney U test was used. A P value of <0.05 was considered as significant.

The survey identified 15 patients (9 girls, 6 boys; median age 10 years, range 3.6-17.5) who received posaconazole as salvage treatment for proven (n=9) or probable (n=6) invasive fungal infection (7 cases with zygomycosis, 4 cases with invasive mold infection, 2 cases with invasive aspergillosis, and 2 cases with chronic disseminated candidiasis) (Table 1). To the best of our knowledge, this is the largest case series of the use of posaconazole in children with proven and probable invasive fungal infection. Most patients had acute leukaemia (10 cases), one patient each suffered from non-Hodgkin lymphoma, aplastic anaemia, solid tumour, soft tissue trauma, chronic granulomatous disease, and diabetes mellitus. Three patients were allogeneic haematopoietic transplant recipients. Nine patients had been exposed to therapeutic dosages of glucocorticosteroids and nine patients had a period of profound neutropenia (≤500/µl) within the four weeks prior to the start of posaconazole. All patients had received systemic antifungal therapy prior to treatment with posaconazole (median duration 22 days, range 4-319), in most cases with at least two antifungal agents in combination or sequentially. Six patients received posaconazole as single agent, and nine patients in combination with other systemic antifungal compounds (3 cases with amphotericin B, 1 case with caspofungin, and 5 cases with amphotericin B and caspofungin). The median duration of treatment with posaconazole was 32 days (range 4-262); the median daily dosage was 21 mg/kg (95%CI 17-25; range 4.8-33.3). Seven patients received posaconazole at a daily dosage of 600 mg or less (6 patients <13 years of age), 7 patients at a daily dosage of 800 mg (3 patients <13 years of age), and one 10-year old at a daily dosage of 1200 mg. Plasma levels of posaconazole were not determined in the majority of the patients due to the lack of drug monitoring for posaconazole in most centres.

In none of the patients was treatment with posaconazole discontinued due to clinical or laboratory adverse events (AEs). Irrespective of cause, 22 clinical AEs were observed in 11 patients (73%). Most AEs (18/22; 8 patients) were of grade I/II; 4/22 recorded AEs were grade III (3 patients). There was no relationship between incidence of AEs and dosage of posaconazole. The most commonly recorded AEs were fever, nausea and/or vomiting, abdominal pain, diarrhea, headache, and skin eruptions. Increases in laboratory hepatic and renal function parameters during therapy were frequent in this population of severely immunocompromised children usually receiving multiple concomitant drugs. However, the mean of the last available values during treatment of liver transaminases, bilirubin, alkaline phosphatase and creatinine were not significantly different from baseline. Overall, both pattern and extent of adverse events observed in this survey do not appear to be substantially different from those reported in adults [START_REF] Cornely | Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia[END_REF][START_REF] Langner | Posaconazole in the management of refractory invasive fungal infections[END_REF][START_REF] Ullmann | Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease[END_REF].

When analysing efficacy of posaconazole salvage therapy, complete or partial responses were observed in 60% of the patients: in 4/7 patients with zygomycosis, in 3/4 patients with invasive mold infections, in 1/2 patients with invasive aspergillosis, and in 1/2 patients with CDC (Table 1 In summary, the results of our analysis indicate that posaconazole may be safe and efficacious in the salvage management of invasive fungal infections in immunocompromised children and adolescents. However, the data of ongoing dose-finding pharmacokinetic studies are urgently needed before posaconazole can be recommended in children as salvage treatment for invasive fungal infections.

  ). Overall survival at 3 months post start of Although we recognise that the assessment of efficacy is curtailed by the retrospective nature of this analysis and different indications, the rate of complete or partial responses in our survey was comparable to adults receiving posaconazole salvage therapy[START_REF] Greenberg | Posaconazole as salvage therapy for zygomycosis[END_REF][START_REF] Raad | Novel antifungal agents as salvage therapy for invasive aspergillosis in patients with hematologic malignancies: posaconazole compared with highdose lipid formulations of amphotericin B alone or in combination with caspofungin[END_REF][START_REF] Van Burik | Posaconazole is effective as salvage therapy in zygomycosis: a retrospective summary of 91 cases[END_REF][START_REF] Walsh | Treatment of invasive aspergillosis with posaconazole in patients who are refractory to or intolerant of conventional therapy: an externally controlled trial[END_REF].

	treatment was 73% (11/15).
	Table 1 Responses to posaco-nazole salvage therapy	Infection	Number of patients
			CR	PR	SD	NR	Survival
	The numbers of proven/probable invasive fungal infections are	Invasive zygomycosis (4/3) Invasive mold infection (4/0)	3 2	1 1	1 -	2 1	5/7 2/4
	given in parenthesis	Invasive aspergillosis (1/1)	-	1	-	1	2/2
	CR complete response, PR par-	Chronic disseminated candidiasis (0/2)	1	-	1	-	2/2
	tial response, SD stable disease, NR non-response	All (9/6)	6	3	2	4	11/15

  Conflict of interest TL has received grants from Gilead; he is a consultant to Gilead, Merck, Sharp & Dohme and Schering-Plough, and served at the speakers' bureau of Astellas, Gilead, Merck, Sharp & Dohme and Schering-Plough. AHG has received grants from Gilead and Merck, Sharp & Dohme; he is a consultant to Astellas, Gilead, Merck, Sharp & Dohme and Schering-Plough, and has served at the speakers' bureau of Astellas, Gilead, Merck, Sharp & Dohme, Pfizer, Schering-Plough and Zeneus/Cephalon. All other authors have nothing to declare. Funding None to declare.


Claude Ecoffey
email: claude.ecoffey@chu-rennes.fr

Frederique Lacroix

MD Elisabeth Giaufré

MD Gilles Orliaguet

MD Philippe Courrèges

Prof Claude Ecoffey

Claude Ecoffey Md

MD Frédéric Lacroix

Epidemiology and morbidity of regional anaesthesia in children: a follow-up one-year prospective survey of the French-Language Society of Paediatric Anaesthesiologists (ADARPEF)

Keywords: Audit, Complications, Regional anesthesia Pediatrics: children; Regional anesthesia: epidemiology, complications F

come

Epidemiology and morbidity of regional anaesthesia in children : a follow-up one-year prospective survey of the

French-

Introduction

In 1996, the ADARPEF (French-Language Society of Paediatric Anaesthesiologists) published a 1 yr prospective study evaluating the importance and the incidence of complications of regional anesthetics (RA) in pediatrics [START_REF] Giaufré | Epidemiology and morbidity of regional anesthesia in children: A one-year prospective survey of the French-Language Society of Pediatric Anesthesiologists[END_REF]. The study collected data from 24,409 regional blocks including 60% of central blocks (mainly caudal). Complications were rare (00.9%), minor and all resulting from central blocks. Accordingly, the authors encouraged anaesthesiologists to preferentially perform peripheral nerve blocks when appropriate.

Twelve years later, the ADARPEF saw a need to design a second study providing practitioners with an updated understanding of both the epidemiology and the morbidity of RA in children.

Methods

The ADARPEF board developed a 1 yr multicenter, anonymous, prospective and observational study based on data collected from 3 separate forms provided to participating hospitals: one for identification, a 2 nd for details, and a 3 rd for complications. A senior exclusive paediatric anaesthesiologist whose topic was RA was established as coordinator. A senior paediatric anaesthesiologist (≥ 50% activity in children) was established to the local person with responsibility in each participating hospital.

Identification

The local responsible used the identification to describe the characteristics of his hospital, particularly administrative affiliation (public, private, university, teaching...), number of pediatric beds, geographical location. As soon as it was completed, the form was sent to coordinator who used it to give each hospital an ID number.

Epidemiology

The epidemiological study took place in four steps:

(i) Step 1: in each participating hospital, each anaesthesiologist provided extemporaneous details from each patient he anesthetized.

(ii) Step 2: the local coordinator recorded overall data on an electronic form with the ID number separating pure GA, RA associated with GA and isolated RA. RA was assigned to a central blocks group including spinals and epidurals (caudal, sacral, lumbar, thoracic) or in a peripheral nerve blocks group. Catheter placements were identified in each group. Patients were classed into 7 age groups: ex-preterm aged < 1 mo; full-term aged < 1 mo, ex-preterm aged l-6 mo, full-term aged l-6 mo, infants and toddlers aged 6 mo-3 yr, children aged 6-12 yr, adolescents aged 12-18 yr.

(iii) Step 3: the coordinator was sent the details forms monthly. He used the ID number to check and classify the data before entering the whole in a database hiding the ID numbers. In this way, data became anonymous for everybody.

(iv) Step 4: overall results were analysed.

Morbidity

Morbidity was studied 1 yr long on the basis of outcome. Any adverse event was reported by the practitioner(s) caring for the patient using a form with the ID number of the hospital, the patient being unidentified. The coordinator received the form and was the only one able to identify the hospital using the ID number. The form described the technique and equipment used, the type of drug and the administered dose, the specific of the adverse event, the were requested to phone the anaesthesiologist in case of any disturbing symptom.

Hospitalized children were followed up daily until discharge. When a symptom persisted on discharge, one (or more) specific consultation, per example with a neurologist if there was a neurological deficit, was organized to assess the evolution, the coordinator being sent he complication form either when full recovery was found or at the end of 1 yr.

Analysis

Results are calculated as percentages of overall RA and given in terms of anaesthetics rather than in terms of patients. Upper 95% confidence bound (CI 95%) was used each time that necessary. In case of comparison, P < 0.05 was considered significant.

Results

Identification

The study involved 47 hospitals: 37 university hospitals, 6 non-university public hospitals, and 4 private hospitals. The size of the hospitals varied from 100 to 2000 beds including 25 to 400 paediatrics beds. Thirty-nine facilities were located in France, 3 in Belgium, and 1 each in Canada, Italy, Switzerland and Tunisia. Forty-one hospitals included a specific paediatric department, whereas 6 hospitals treated children and adults together in the same unit. Peripheral blocks represented 66 % of RA. Upper limbs blocks accounting for 10% of peripheral were mainly axillary (43% of all upper limb blocks), fingers (13%), and infraclavicular (11 %) blocks, while "others" were isolated medial or radial, interdigital, posterior or anterior interosseous and posterior ulnar blocks (table 3). Lower limbs blocks accounting for 19% of peripheral were femoral (20 % of all lower limb blocks), ilio-fascial (16%), posterior popliteal sciatic (15%) and lateral sciatic (12%) blocks, while "others" were mainly blocks of toes, posterior tibial and saphenous blocks (table 4). Trunk and face blocks consisted mainly of ilio-inguinal (37% of all trunk and face blocks), penile (35%) and pudendal nerve blocks while face blocks included 1,118 blocks (5%) of nerves supplying head and neck, mainly infraorbital nerve block for cleft palate repair, the remaining being mainly subtenon, retrobulbar, auricular and laryngeal (table 5).

Epidemiology

The study recorded 2726 (9% of all RA) catheter placements (table 6). No data on duration of use of catheters or on their possible home use was collected. Placements concerned 23% of lower limbs blocks (mainly popliteal sciatic and psoas compartmental), 15% of central blocks (a catheter being inserted in 88% of lumbar/thoracic epidurals, 71% of sacrals, 1% of caudals, and 0.8% of spinals), 6% of upper limbs blocks (mainly axillary), and 1% of trunk and face blocks, (mainly thoracic paravertebral).

Local anaesthetics used in this study were ropivacaine 85%, bupivacaine 8%, lévobupivacaine 4%, mepivacaine 2% and lidocaine 1%. Mepivacaine and lidocaine were used only in peripheral blocks. Spinal anaesthesia was performed mainly with hyperbaric bupivacaine.

Morbidity

One hundred and seventy five complications forms were collected. Most concerned central blocks (n=112, 64%). One hundred and thirty four were eliminated as inappropriate, including No one resulted in either sequelae or harm 1 yr later. Their incidence was 0.12% (CI 95% = 0.09-0.17). After upper extremity and facial blocks (unable to be an alternative to central blocks) had been subtracted from peripheral RA for a valid comparison, the incidence was founded significantly higher for central (0.29%; CI 95% = 0.21-0.43) than for peripheral RA (0.29%; CI 95% = 0.03-0.10) Table 7 presents age-group distribution of complications. The incidence was significantly higher in the low age group (0.4% in 3,860 infants aged < 6 months versus 0.1% in 27, 272 children aged > 6 months). No complication was directly related to the use of wrong equipment. No medico-legal concern was recorded. The incidence of complications directly related to the use of catheters is 0.14% versus 0.13% in techniques without catheter.

One patient (11.5 yr) had two complications related with multiple attempts at performing a lumbar epidural. Two complications forms were completed: the first form reported an unrecognised dural tap while the second form reported a total spinal anaesthesia following the local anaesthetic (LA) injection through the lumbar catheter. Total spinal was recognized in recovery room because the patient was unable to breathe spontaneously. The patient did not experience any other disability. Controlled ventilation continued, the recovery was uneventful 4 h later.

Two 2 ex-preterm (1 and 3 months respectively) had an inadvertent extension of a spinal anaesthetic, both due to a rising of their lower limbs during operative installation, In terms of toxicity, convulsions occurred in 1 not anesthetised patient (9 yr) given an axillary single shot of ropivacaine in a normal dose (aspiration test negative, test dose not done).

Recovery was full under classical pharmacological treatment, i.e. oxygen by face mask and 4mg/kg thiopental. Plasma level of LA was not performed. The surgery (fracture reduction) was done.

Fifteen patients (age ranged from 1.5 mo to 17 yr) underwent cardiac toxicity of LA (0.05%; CI 95% 0.03-0.08). The involved blocks were 1 axillary, 6 single shot and 1 continuous caudals, 1 penile + ilio-inguinal, 4 lumbar and 2 thoracic epidurals. LA was ropivacaine (5 cases) or bupivacaine (10 cases), given in adequate doses were except 1 drug error, 0.75% ropivacaine caudally given instead of 0.2% details (aged 21 mo). Injections consisted of 9 single shots through a needle, 4 primary and 2 secondary injections through a catheter. A test dose with epinephrine was performed and considered uneventful in 6 patients. Specifics were either single changes in electrocardiogram (2 patients) or transient arrhythmias (13 patients including 1 bradycardia in a 8 yr old boy given both penile and ilio-inguinal blocks in combination). No one required active treatment. Plasma levels of LA were not performed.

In terms of trauma, 2 patients (6 mo and 3 yr respectively) scheduled for one day surgery underwent an ilio-inguinal block resulting in colonic puncture. They recovered without sequelae after watching, fasting, fluids, and antibiotics in ward.

Ten dural taps (0.10%; CI 95% = 0.05-0.19) were recorded (age ranged from 1 mo to 13 yr, the one previously reported in association with an inadvertent total spinal being excluded).

Six of them occurred during caudal blocks in babies, 3 during lumbar and 1 during thoracic epidurals. No post-dural puncture headache was noted and preventive analgesia was given in 4 patients only. Five nerve injuries were suspected. The 1 st one was an isolated Claude Bernard Horner syndrome catheter remaining 18 h after a thoracic epidural was stopped (aged 12 yr). The 2 nd one was a deficit of the external popliteal nerve persisting 48 h after a lumbar epidural was stopped (aged 13 yr). The 3 rd one consisted of tingling associated with hypoesthesia in the femoral area following an ilio-fascial block and disappearing for 48 h (aged 11 yr). The 4 th one (aged 8 yr) consisted of unilateral tingling in one thigh during 3 weeks following a caudal that was noted as uneventful. The last one was a minor intermittent dorsal pain following a lumbar epidural and requiring intermittent paracetamol 11 mos long (aged 12 yr). All of them recovered fully without serious harm.

Four patients had a minor complication: one inflammation of a lumbar epidural puncture point locally treated (aged 2 yr), one ilio-inguinal block performed at the wrong side (aged 5.5 yr), one breakage of femoral catheter requiring removal under LA (aged 12 yr), one pleural displacement of a thoracic paravertebral catheter (aged 12 yr). The displacement was diagnosed because RA had become inefficient. No other signs were noted. The catheter was removed without other treatment.

Discussion

Regional anaesthesia represents 22% of all anaesthesias recorded by the study, similar to the incidence founded by the previous ADARPEF' study after removal of 5,306 local anaesthesias (Bier blocks, tracheal blocks, infiltrations, field blocks) that are not recorded by the current survey. That is also close to the incidence of 20 -25% of RA founded in the whole population (adults/children together). Overall complications have the same low rate that found in the study 12 yrs ago (local anaesthesias recorded in the 1 st ADARPEF' study being removed). In the same way, adverse effects are mainly related with central blocks; most of the blocks being performed under GA, the practitioners were deprived of clinical signs of One essential question concerns the accuracy of the data, particularly the reported complications. Some of them could be not recorded, whereas the occurrence of fatalities cannot be hidden. However, the coordinator was intensively connected to each local responsible, phoning or e-mailing him whenever he had doubts. Several anaesthesiologists collected the data at the same time in the same hospital, inevitably adding a mutual review to the double control performed both by the local responsible and the coordinator. Despite an insignificant percentage of minor complications could have been missed after discharge, the procedure allows to regard overall data collection as efficient and to consider the risk of missing severe complications as almost non-existent.

Epidemiology

The widespread geographical distribution of the hospitals involved in the study is representative of the French paediatric anaesthesia in private and public hospitals. The significant amount of data illustrates clearly a transition in practice from predominantly central block to an increased number of peripheral nerve blocks including catheter techniques.

Since the previous ADARPEF study, the overall incidence of central blocks has decreased dramatically, collapsing even in children aged < 3 years (however the group of age undergoing central blocks most frequently), while the incidence of peripheral blocks increased strongly. Trunk blocks represent their largest part, characterized by the emergence of techniques that were not clearly identified by the previous ADARPEF study (ilio-inguinal, para-umbilical, pudendal, thoracic and lumbar paravertebral blocks). Facial blocks are a new and widely used practice for facial and reconstructive surgery, particularly in cleft lip repair [START_REF] Simion | Postoperative pain control for primary cleft lip repair in infants: is there an advantage in performing peripheral nerve blocks?[END_REF].

The study records a significant number of catheter placements, in central as well in peripheral RA, most of them being neuraxial. Indeed, neuraxial continuous epidural analgesia Continuous peripheral nerve blockade emerged as safe and effective in adults. Then several prospective studies demonstrated their benefits after orthopaedic procedures in children. In 2001, placement of a brachial plexus catheter for pain control was far less common in children than in adults [START_REF] Tobias | Brachial plexus anaesthesia in children[END_REF]. Today, peripheral blocks allow to provide postoperative pain relief in the greatest part of orthopaedic surgery using RA techniques [START_REF] Dadure | Continuous epidural block versus continuous popliteal nerve block for postoperative pain relief after major pediatric surgery in children: a prospective, comparative randomizedstudy[END_REF][START_REF] Ganesh | Continuous peripheral nerve blockade for inpatient and outpatient postoperative analgesia in children[END_REF][START_REF] Dadure | Continuous peripheral nerve blocks for postoperative analgesia in children: feasibility and side effects in a cohort study of 339 catheters[END_REF], and to treat complex regional pain syndrome in adolescents [START_REF] Dadure | Continuous peripheral nerve blocks at home for treatment of recurrent complex regional pain syndrome I in children[END_REF].

These results confirm a retrospective report from a single institution (10,929 RA performed on a 17 yr period) revealing both a dramatic decrease of central blocks, and the emergence of continuous postoperative analgesia via perineural catheters [START_REF] Rochette | A review of pediatric regional anesthesia practice during a 17-year period in a single institution[END_REF], peripheral blocks emerging as routine practice in children at the end of the 90's following both peer recommendations [START_REF] Giaufré | Epidemiology and morbidity of regional anesthesia in children: A one-year prospective survey of the French-Language Society of Pediatric Anesthesiologists[END_REF] and evolution of devices.

Morbidity

Previous case reports of spinal cord injury resulting from thoracic epidural [START_REF] Allison | Paraplegia after thoracotomy under combined general and epidural anesthesia in a child[END_REF], sacral osteomyelitis [START_REF] Wittum | Sacral osteomyelitis after single-shot epidural anesthesia via the caudal approach in a child[END_REF] and bowel traumas requiring surgery resulting from ilio-inguinal blocks [11; 12] revealed that RA in children is not without risk, even in case of peripheral blocks. anaesthesiologists with maximal precautions. Moreover, this study has confirmed the low complications rate despite the performance of 96% on general anaesthesia or heavy sedation; indeed, due to the significant differences between children and adults with respect to selfcontrol and the ability to communicate effectively, the usual recommendation is that general anaesthesia or heavy sedation should not be considered an absolute contraindication to regional anaesthesia in children (15). Central RA has the highest incidence of complications (7 times higher that peripheral).

The incidence remains low despite an increase since the 12 last years. Complications never reach the severity founded by a UK audit [16] on paediatric epidurals (10,633 epidurals performed for 5 yr) reporting permanent residual neurologic deficit in a child aged 3-mo (1-yr follow-up), 2 epidural abscesses, 1 meningism, 1 post-dural puncture headache requiring active blood patching, 1 drug error resulting in cauda equina syndrome and 5 severe neuropathy/radiculopathy resolved over a period of 4-10 mo using a pharmacological therapy in a Pain Clinic. It is difficult to explain this difference of severity in complications, excepted by the longer duration and the many more cases of the UK survey, i.e. 1,500 versus 10,000.

The study records a very low overall morbidity of peripheral RA, almost 6 times lower that in central RA. Despite 2 colonic punctures, that should encourage anaesthesiologists to use peripheral rather than neuraxial (including caudal) blocks as often as possible when appropriate.

The use of catheters does not seem to increase the occurrence of complications, even if considering that the cardiac toxicity following a secondary injection through a catheter could be due to an inadvertent displacement of the catheter.

LA toxicity, resulting in 1 case of convulsions only while the UK survey reported 2 respiratory arrests and 1 seizure, did not require lipid treatment as reported in a child [17].

Some complications (at less drug error, wrong side, lower limbs raising resulting in

Conclusion

This survey involving a large population shows that the importance of RA in pediatrics is unchanged for 12 yr, but that a clear transition in practice from central to peripheral blocks and continuous techniques has occurred. Some new techniques have emerged. The incidence of complications is almost unchanged and remains low, low age and central blocks increasing the risk. Most of the recorded complications were minor, occurred at the beginning of the procedure in the operating theatre, had a short duration and did not result in permanent harm. This safety profile means that RA remains an excellent way to provide postoperative analgesia, despite a risk of complications increasing in children aged < 3 yr.

Anaesthesiologists must be encouraged to continue to choose peripheral instead of central blocks each time it is possible because a lower morbidity. A future ADARPEF' study should be designed for few years to assess the impact of ultrasonography on paediatric RA. designed a one-year prospective, multicenter and anonymous study in order to update both epidemiology and morbidity of regional anaesthesia in children.

F o r P e e r R e v i e w

F o r P e e r R e v i e w

F o r P e e r R e v i e w

Methods: From November 2005 to October 2006, data from participating hospitals were recorded using an identification form, a data recording form and a complication form.

Information collected included the characteristics of the hospitals, the number and type of regional anaesthetics, the age of the involved children as well as the incidence and type of complications.

Results: Data collected in 47 institutions included 104,612 pure general anaesthesias, 29,870 general anaesthesias associated with regional blocks and 1,262 pure regional blocks. Central blocks accounted for 34 % of all RA. Peripheral blocks (66 %) were upper or lower limb blocks (29 % of peripheral blocks), trunk and face blocks (71 %). In children aged ≤ 3 yr, the percentage of central blocks was similar to the peripheral ones (45% versus 55), while in older children, peripheral blocks were more than four times used than peripheral ones.

Complications (41 involving 40 patients) were rare and usually minor. They did not result in any sequelae. The study revealed an overall rate of complication of 0.12%; CI 95% [0.09-0.17], significantly 6 times higher for central than for peripheral blocks.

Conclusions: Due to the low rate of complications, regional anaesthetic techniques have a good safety profile and can be used in order to provide postoperative analgesia. In addition, the results should encourage anaesthesiologists to continue to use peripheral instead of central (including caudal) blocks as often as possible when appropriate.

Key words: Pediatrics: children; Regional anesthesia: epidemiology, complications

  management and the patient outcome, including neurological and medico-legal implications if relevant. Parents of children discharged the day of surgery received a call the next day and/or

  doses, missing in localisation or puncture, blood reflux through needles or catheters, subcutaneous injections without lesions, catheters inadvertently removed, folded or capped, inefficient blocks accessed by increase in heart rate at surgical incision, urinary retention, and one complaint 6 months after a femoral blockade with motor block of three days duration not diagnosed despite a 2 days post operative follow-up in ward... At the end, 41 forms involving 40 patients were validated. They occurred mostly in the operating theater.

  resulting in isolated apnoea requiring controlled ventilation < 12 h long without sequelae.

  LA toxicity or of nerve injury.

  the best techniques to obtain pain relief in children (particularly postoperative pain relief in younger). Perineural catheters became usual practice, first in hip and foot surgery.

  The study founds that complications are rare, most often requiring extra observation or minor treatment. Most of them occur early in the operating theater. No complications were as a result of equipment failure. Only one child includes pathologies lasting months, requiring a treatment and/or having a major impact both on child's physical and emotional status and on the quality of life of family. Neurologic deficits have good outcome in children as previously reported[13; 14]. As reported by literature, complications are more frequent (4 times in the current study) in children aged < 6 months that in children aged > 6 months, despite the fact that the youngest patients are probably managed by the most experienced paediatric

  spinal anaesthesics, drug error and a part of cardiac toxicity) were avoidable. It is thus possible to improve the safety of paediatric RA provided basic precautions are followed.

  Michalon (university), La Rochelle Hôpital Laennec (public), Le Havre Hôpital Jacques Monod (public), Lille Hôpital Jeanne de Flandre (university) and Hôpital Saint Vincent de Paul (private), Limoges Hôpital Dupuytren (university), Lyon Hôpital Femme-Mère-Enfant (university), Marseille Hôpital Privé Clairval (private) and Hôpitaux Timone enfants and Hôpital Nord (university), Montpellier Hôpital Lapeyronnie (university), Nancy Hôpital Brabois Enfants (university), Nantes Pôle Mère-Enfant (university), Nice Fondation Lenval (private) and Hôpital Archet (university), Orléans Hôpital Porte Madeleine (public), Paris Hôpital Armand Trousseau Hôpital du Kremlin-Bicêtre Hôpital Necker-Enfants Malades and Hôpital Saint-Vincent de Paul (universities), Pau Hôpital François Miterrand (public), Poitiers Pôle Femme-Mère-Enfant (university), Québec Centre Pédiatrique Laval (Canada), Reims Hôpital American Memorial Hospital (university), Rennes Hôpital Pontchaillou (university), Rouen Hôpital Charles Nicolle (university), Saint Brieuc Hôpital Y Le Foll (public), Saint-Etienne Hôpital Nord (university), Strasbourg Hôpital Hautepierre (university), Toulouse Hôpital Purpan (university), Tours Hôpital Clocheville (university), Tunis Hôpital d'Enfants (Tunisia), Turin Regina Marghertia Children's Hospital (Italy), Villeneuve Saint Georges Centre Hospitalier (public).

  nerve block. Paediatr Anaesth. 2006; 16: 993-6. 12. Amory C, Mariscal A, Guyot E, Chauvet P, Leon A, Poli-Merol ML. Is ilioinguinal/iliohypogastric nerve block always totally safe in children? Paediatr Anaesth. 2003; 13:164-6. 13. Ecoffey C, Samii K. Neurologic complication after epidural anesthesia in a 15-yearold boy. Ann Fr Anesth Reanim 1990; 9: 398. 14. Zeidan A, P. Narchi, E. Goujard, D. Benhamou. Postoperative nerve irritation syndrome after epidural analgesia in a six-year-old child. Br J Anaesth 2004; 92: 146-8. 15. Bernards CM, Hadzic A, Suresh S, Neal JM. Regional anesthesia in anesthetized or heavily sedated patients. Reg Anesth Pain Med. 2008; 33: 449-60. 16. Llewellyn N, Moriarty DA. The national pediatric epidural audit. Pediatr Anesth 2007; 17: 520-33 17. Ludot H, Tharin JY, Belouadah M, Mazoit JX, Malinovsky JM. Successful resuscitation after ropivacaine and lidocaine-induced ventricular arrhythmia following posterior lumbar plexus block in a child. Anesth Analg. 2008; 106: 1572-4.

Table 1 :

 1 Different regional block procedures according to patient's age (n = 31,132)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	blocks	%
	Epidurals	82	227	428	1,082	4,495	3,311	473	10,098 32.4
	Spinals	9	9	38	40	43	60	188	387	1.3
	Others central	0	0	0	4	1	23	43	71	0.3
	Upper limbs	1	2	5	36	454	1,099	484	2081	6.7
	Lower limbs	2	12	14	62	529	1,540	1,665	3,824	12.4
	Trunk, abdomen 22	154	288	1,063	4,506	6,185	612	12,830	41
	Face, head	5	71	49	155	471	756	334	1841	5.9

Table 2 :

 2 Details of the different central blocks according to patient's age (n = 10,556)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	Blocks	%
	Caudals	76	187	402	951	4,141	2,699	37	8,493	27.2
	Sacral	0	2	1	4	12	35	4	58	0.2
	Lumbar	5	33	18	93	240	450	344	1,183	3.8
	Thoracic	1	5	7	34	102	127	88	364	1.2
	Spinals	9	9	38	40	43	60	188	387	1.3
	Others central 0	0	0	4	1	23	43	71	0.3

Table 3 :

 3 Details of the different upper limb regional block according to patient's age (n = 2,081)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	Blocks	%
	Parascalene	0	0	0	1	7	75	43	126	0.4
	Infraclavicular	0	0	0	2	14	141	81	238	0.8
	Axillary	0	0	2	10	158	543	188	901	2.9
	Mild humeral	0	0	0	0	16	24	19	59	0.2
	Ulnar	0	0	0	0	0	9	13	22	0.1
	Wrist	0	0	0	4	49	56	27	136	0.4
	Finger	0	1	0	9	119	104	37	270	0.9
	Others	1	1	3	10	91	147	76	329	1

Table 4 :

 4 Details of the different lower limbs block according to patient's age (n = 3,824)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	Blocks	‰
	Psoas compartment	0	0	0	0	7	65	31	103	0.3
	Femoral	0	0	1	5	46	291	413	756	2.4
	Ilio-fascial	0	1	3	5	51	268	274	602	1.9
	Parasacral	0	0	0	0	4	4	3	11	0.03
	Sciatic Labat	0	0	0	1	12	18	15	46	0.1
	Sciatic anterior	0	0	0	0	3	1	1	5	0.1
	Sciatic subgluteal	1	1	0	5	11	30	29	77	0.2
	Sciatic lateral	0	0	0	10	95	193	150	448	1.4
	Sciatic posterior popliteal	0	0	0	6	98	215	273	592	1.9
	Sciatic lateral popliteal	0	3	3	9	49	152	183	399	1.3
	Ankle	0	1	0	0	5	13	17	36	0.1
	Others	1	6	7	21	148	290	276	749	2.4

Table 5 :

 5 Details of the different trunk and face blocks according to patient's age (n = 12,830)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	Blocks	%
	Rectus sheath	9	70	54	215	328	460	88	1224	3.9
	Ilio inguinal	12	73	225	750	1779	2462	203	5504	17.7
	Penile	0	3	7	70	2015	2801	221	5117	16.4
	Pudendal	1	2	2	19	343	410	59	836	2.7
	Lumbar paravertebral	0	0	0	3	6	10	6	25	0.08
	Thoracic paravertebral	0	1	0	3	24	28	25	81	0.3
	Intercostal	0	5	0	3	11	14	10	43	0.1
	Others (including facial blocks) 5	71	49	155	471	756	334	1841	5.9

Table 6 :

 6 Details of the different blocks with catheter according to patient's age (n = 2,726)

		0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr	Total
	Technique	premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799	Blocks	%
	Caudals	0	3	7	5	53	39	7	114	0.4
	Sacral	0	2	0	4	12	23	1	42	0.1
	Lumbar	5	28	15	83	216	368	303	1,108	3.5
	Thoracic	1	5	5	32	100	116	82	341	1.1
	Spinals	0	0	0	0	0	0	3	3	0.01
	Others centrals	0	0	0	0	0	1	19	20	0.06
	Parascalene	0	0	0	0	0	7	9	16	0.05
	Infraclavicular	0	0	0	0	3	16	19	38	0.1
	Axillary	0	0	0	2	8	47	19	76	0.2
	Psoas compartment	0	0	0	0	5	50	15	70	0.2
	Femoral	0	0	0	1	6	57	124	188	0.6
	Ilio-fascial	0	0	2	0	4	57	53	116	0.04
	Parasacral	0	0	0	0	2	3	1	6	0.02
	Sciatic subgluteal	0	0	0	0	1	13	11	25	0.08
	Sciatic lateral	0	0	0	7	44	28	31	110	0.3
	Sciatic posterior popliteal	0	0	0	0	37	108	153	298	0.9
	Sciatic lateral popliteal	0	0	0	0	8	30	28	66	0.2
	Lumbar paravertebral	0	0	0	1	5	8	6	20	0.6
	Thoracic paravertebral	0	1	0	3	21	20	16	61	2
	Intercostal	0	3	0	1	3	2	1	10	0.03
	Others	0	6	0	6	27	15	10	64	0.2

Table 7 :

 7 Incidence of complications according to the age (n = 41). The ADARPEF (French-Language Society of Paediatric Anaesthesiologists)

	Complications	0-30 days	0-30 days	1-6 mo	1-6 mo	6 mo -3 yr	3-12 yr	> 12 yr
		premature	full term	premature	full term
		n = 121	n = 475	n = 822	n = 2,442	n = 10,499	n = 12,974	n = 3,799
	% of studied population	0.4	1.5	2.6	7.8	33.7	41.7	12.2
	Relative % of complications	2.4	2.4	7.3	17.1	17.1	39	14.6
	% of complications in the group 0.8	1	0.02	0.3	0.06	0.13	0.05

Acknowledgements

The authors acknowledge the enthusiasm and commitment of the centres involved in the survey : Amiens Hôpital Nord (university), Angers Hôpital Larrey (university), Antony Hôpital Privé (private), Besançon Hôpital Saint Jacques (university), Bordeaux Hôpital Pellegrin Enfants (university) and Polyclinique du Tondu (private), Bruxelles Cliniques Universitaires Saint Luc (Belgium) and Hôpital Reine Fabiola (Belgium), Caen Hôpital Femme-Enfant-Hématologie (university), Charleroi Hôpital Civil (Belgium), Clermont-Ferrand Hôtel Dieu (university), Genève Hôpital des Enfants (Switzerland), Grenoble Hôpital