Aimee Dc Paulussen
email: aimee.paulussen@mumc.nl

Alexander Stegmann
email: sander.stegmann@mumc.nl.

Marinus J Blok

Demis Tserpelis

Crool Posma-Velter

Yvonne Detisch

Eric Ejgl Smeets

Annemieke Wagemans

Jaap Jp Schrander

Marie-José H Van Den Boogaard

Grazia ; Mancini

Aimée Dc Paulussen

Alexander Pa Stegmann

Jasper Van Der Smagt

Arie Van Haeringen

Irene Stolte-Dijkstra

Wilhelmina S Kerstjens- Frederikse

Grazia M Mancini

Marja W Wessels

Raoul Cm Hennekam

Maaike Vreeburg

Joep Geraedts

Thomy De Ravel

Jean-Pierre Fryns

Hubert J Smeets

Koenraad Devriendt

Constance Trm Schrander- Stumpel

PhD A D C Paulussen

PhD A P A Stegmann

MLL2 mutation spectrum in 45 patients

Keywords: Kabuki Syndrome, MLL2, histone methyl transferase, mutation Kabuki syndrome, MLL2, mutation, histone methyl transferase

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

ABSTRACT: Kabuki Syndrome (KS) is a rare syndrome characterized by intellectual disability and multiple congenital abnormalities, in particular a distinct dysmorphic facial appearance. KS is caused by mutations in the MLL2 gene, encoding an H3K4 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. Direct sequencing of all 54 exons of the MLL2 gene in 45 clinically well-defined KS patients identified 34 (75.6%) different mutations. One mutation has been described previously, all others are novel. Clinically, all KS patients were sporadic, and mutations were de novo for all 27 families for which both parents were available. We detected nonsense (n=11), frameshift (n=17), splice site (n=4) and missense (n=2) mutations, predicting a high frequency of absent or non-functional MLL2 protein. Interestingly, both missense mutations located in the C-terminal conserved functional domains of the protein. Phenotypically our study indicated a statistically significant difference in the presence of a distinct facial appearance (p=0.0143) and growth retardation (p=0.0040) when comparing KS patients with an MLL2 mutation compared to patients without a mutation. Our data double the number of MLL2 mutations in KS reported so far and widen the spectrum of MLL2 mutations and disease mechanisms in KS. ©2010 Wiley-Liss, Inc.

INTRODUCTION

Kabuki syndrome (KS) (or Niikawa-Kuroki syndrome, MIM# 147920) is a rare syndrome, characterized by intellectual disability and distinct dysmorphic features, which was originally described by two independent groups in Japan [START_REF] Kuroki | A new malformation syndrome of long palpebral fissures, large ears, depressed nasal tip, and skeletal anomalies associated with postnatal dwarfism and mental retardation[END_REF][START_REF] Niikawa | Kabuki make-up syndrome: a syndrome of mental retardation, unusual facies, large and protruding ears, and postnatal growth deficiency[END_REF]. The estimated prevalence of KS was estimated at 1:32,000 in the Japanese population. Dominant clinical findings include a characteristic facial appearance, developmental delay and growth retardation and additional features such as hypodontia and persistent fetal fingerpads [START_REF] Adam | Kabuki syndrome: a review[END_REF][START_REF] Matsumoto | Kabuki make-up syndrome: a review[END_REF]. The unique facial characteristics observed in KS include elongated palpebral fissures and eversion of the lateral one third of the lower eyelid, an arched or nicked eyebrow with sparseness of hairgrowth in the lateral half, relatively large ears with simplified morphology, a depressed nasal tip and a full lower lip. Recently, a gene causing KS was identified through exome sequencing, reporting mutations in the histone methyl transferase (HMT) gene MLL2 in 66% of 53 patients with Kabuki syndrome [START_REF] Ng | Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome[END_REF]. MLL2 is a member of the so called mixed lineage leukemia (MLL) family of HMT proteins, of which MLL1 (previously called ALL1) has been extensively studied because of it's frequent involvement in chromosomal rearrangements in acute myeloid and/or lymphatic leukemia [START_REF] Meyer | The MLL recombinome of acute leukemias[END_REF]. MLL2 is the paralog of MLL1, arisen through gene duplication, and is transcribed into a 5,537 amino acid residue protein (NCBI reference sequence NP_003473.3). The MLL protein family is part of the SET domain containing group of HMTs in humans, which is evolutionary homologous to the Drosophila trithorax protein (TRX) [START_REF] Dillon | The SET-domain protein superfamily: protein lysine methyltransferases[END_REF][START_REF] Qian | SET domain protein lysine methyltransferases: Structure, specificity and catalysis[END_REF]. SET domains are highly conserved in evolution and are the essential catalytic protein subunits that execute the histone lysine methylation which is the core function of HMTs. MLL proteins require complex formation with other proteins, which allows recruitment to target genes and facilitates the catalytic function of the protein's SET domain. MLL proteins add methyl groups specifically to the fourth lysine residue of the lysine side chains of histone H3, referred to as H3K4 methylation which is a transcriptional activation mechanism. Histone methylation in general thus constitutes an evolutionary conserved control mechanism for (epigenetic) gene regulation. Multiple MLL containing protein complexes have been identified in non-redundant constellations despite considerable sequence and functional domain similarities among them [START_REF] Ansari | Mixed lineage leukemia: roles in gene expression, hormone signaling and mRNA processing[END_REF][START_REF] Eissenberg | Histone H3 lysine 4 (H3K4) methylation in development and differentiation[END_REF]. MLL proteins in general are involved in embryogenesis and development most notably through regulation of HOX gene expression and their interaction with nuclear receptors suggests a role in steroid-hormone controlled signaling. MLL2 is involved in regulation of cell adhesion, growth impairment and cell motility [START_REF] Issaeva | Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth[END_REF]). Interestingly, methyl transferase pathways in general have been implicated before in the pathology of MR syndromes such as Kleefstra Syndrome (MIM# 610253) which is caused by mutations in the EHMT1 gene, an H3K9 methyl transferase, and Schinzel-Giedion syndrome (MIM#: 269150) which is caused by mutations in the SET1BP1 gene that encodes a protein known to bind to SET domains (Hoischen et al., 2010;[START_REF] Kleefstra | Further clinical and molecular delineation of the 9q subtelomeric deletion syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype[END_REF].

MATERIALS AND METHODS

Patient cohort

The patient cohort consists of 45 index patients (31 female, 14 male) with Kabuki syndrome from the Netherlands (n=38) and Belgium (n=7), diagnosed by pediatricians and clinical geneticists. For the current mutation screen, all patient files were reviewed for inclusion by two clinical experts (CSS, YD) using the guideline:

"Management of Kabuki Syndrome, a Clinical Guideline" published online (http://www.dyscerne.org/dysc/guidelines. In order to assess a potential genotype-phenotype correlation detailed photographic evidence of the KS characteristic facial appearance of the patients was evaluated by two independent clinical experts (JS, MV) without prior knowledge of MLL2 mutation carrier status. A subset of patients have been extensively studied [START_REF] Schrander-Stumpel | Kabuki syndrome: Clinical data in 20 patients, literature review, and further guidelines for preventive management[END_REF] and all have undergone routine cytogenetic evaluation showing normal karyotypes at the 550-band resolution levels in the past. The majority of the cohort has also been tested on various SNP-microarray and array-CGH platforms which, with the exception of one patient (Table 1, nr.10) did not result in the identification of any clinically significant de novo CNVs (data not shown) in line with reports by others [START_REF] Kuniba | Molecular karyotyping in 17 patients and mutation screening in 41 patients with Kabuki syndrome[END_REF]. Patient 10 has been reported before, because of a de novo deletion of exon 5 of the MACROD2 gene, identified by array-CGH [START_REF] Maas | The C20orf133 gene is disrupted in a patient with Kabuki syndrome[END_REF]. Written permission for the studies was obtained from each of the patient's parents or caretakers and the study protocol was approved after evaluation by the local Medical Ethics Committee of the Maastricht University Medical Center (MUMC + ).

PCR and sequence analysis

DNA was extracted from each proband and their parents (if available) from two independent peripheral blood samples following standard procedures. Mutation screening of the complete coding region of the MLL2 gene was performed by direct sequence analysis in both directions using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit and the ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Primer sets for all 54 exons spanning the MLL2 coding region, including intron-exon boundaries, were either designed by Applied Biosystems (AB) for VariantSEQr SNP discovery or were custom designed. All primers were tagged with M13 extensions to customize further sequence-analysis. All PCR's were performed using a PE9700 thermal cycler. Full details of primer sequences, amplicon lengths and PCR conditions are available on request from the authors. Sequences were aligned with the known MLL2 coding sequence (NCBI reference sequence accession NM_003482.3). Mutation analysis was performed using Mutation Surveyor V3.30 (©SoftGenetics, PA, USA). Mutations identified were confirmed on a DNA sample extracted from the second independent blood sample from the proband if available. The pathogenicity of the mutations was based on a number of genetic criteria: de novo occurence, predicted non-functionality of mutated proteins, location, type and conservation of mutated aminoacids. In silico prediction of functional consequences was performed using Align GVGD (http://agvgd.iarc.fr/agvgd_input.php), SIFT (http://blocks.fhcrc.org/sift/SIFT.html), PolyPhen (http://genetics.bwh.harvard.edu/pph/) and MutationTaster (http://www.mutationtaster.org/). Effects on splicing were investigated in silico using http://www.fruitfly.org/seq_tools/splice.html, http://www.cbs.dtu.dk/services /NetGene2/, http://www.genet. sickkids.on.ca /~ali/splicesitefinder.html and http://rulai.cshl.edu/tools/ESE/). All parents available were tested for the mutations found in their affected child by sequence analysis. Non-paternity was excluded in case of de novo mutations by multiplex VNTR analysis using the AmpFST® Identifiler PCR Amplification kit (Applied Biosystems, Foster City, CA) following the manufacturers protocol.

RESULTS AND DISCUSSION

In total, we detected 34 (75,6%) heterozygous mutations (25 female, 9 male) in 45 index KS patients. The type of mutations as well as their inheritance are presented in Table 1. In line with the report from Ng et al. [START_REF] Ng | Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome[END_REF], the majority are truncating mutations (nonsense or frameshift mutations), although in our study the number of frameshift mutations was relatively high and the number of nonsense mutations low. In our study we also report the first splice-site mutations (n=4), which adds exon skipping to the spectrum of mechanisms leading to potential abrogation of full-length protein expression. One of the mutations we detected (c.16360C>T (p.Arg5454X), Table 2) was also reported in the original study by Ng et al., all others were novel and private. The 34 mutations detected were randomly distributed across the gene with an expected clustering in some of the larger exons (Table 2, Fig. 1). Table 2 presents an overview of the MLL2 mutations detected, together with the mutations reported by Ng et al. In our cohort two patients carry a missense mutation that each result in amino acid changes at the C-terminal end of the protein in highly conserved protein domains (FYRN and SET ). As both missense mutations were de novo in nature and the majority of the in silico programs predict these mutations to be pathogenic, it is highly likely that they are causal and affect the function of the protein in a negative way. Additionally, we detected four splice junction mutations affecting the consensus site positions at -1 (patient nr. 26), -2 (patients 7, 27) and +5 (patient 25) , respectively (Table 2, mutation in orange Fig 1). The first three are predicted to result in exon skipping, of exons 47, 48 and 23, the fourth one (c.13999+5G>A) predicts a profound change, but not necessarily skipping of exon 42. This variant was however also de novo in nature, and therefore we suspect it to be causal. RNA studies will be performed to determine whether exon skipping is the causal mechanism for all these splice-site mutations. 28 out of 34 mutation carriers represent truncating mutations, either nonsense (n=11) or frameshift (n=17). Although merely three of these mutations localize to one of the proteins functional (conserved) domains, functionally all 28 result in the loss of either the entire C-terminal cluster of functional domains (n=26) or significant parts of it (n=2) (Fig. 1). In these cases, truncating mutations abrogate the possibility of complex formation and recruitment and ultimately of methyl transfer onto target lysine residues. As several of the mutations are located within the 50-54 nucleotide perimeter of exon-exon junctions, it is suspected that these will lead to nonsense-mediated decay of mRNA prior to protein synthesis.

Table 3 lists the variants and polymorphisms we detected (n=23) in the coding regions and intron-exon boundaries of the MLL2 gene in all patients. Five were located in introns, ten were silent variants and eight were missense variants. Twelve of these variants were detected more than once in the KS population and eleven of these were listed as SNP's. Of the remaining eleven, two were intronic, four were silent and five were missense variants (no listing in the SNP database). The latter five variants were all inherited from an asymptomatic parent, four were identified in patients with an already detected pathogenic mutation and the majority of in silico programs predicts them to be benign. As such, these variants are unlikely to be causative for KS (Table 3). * = detected together with pathogenic mutation

In a preliminary genotype-phenotype correlation study, we compared key clinical features of KS in patients with an MLL2 mutation to those without an MLL2 mutation in our cohort (Table 4). The evaluation of facial characteristics was performed by two independent clinical experts (JS, MV) without prior knowledge of MLL2 mutation carrier status. In our cohort KS patients with a MLL2 mutation were statistically different from patients without a MLL2 mutation with respect to the presence of the unique facial characteristics and growth retardation (threshold -3SD) above the age of 3 years. The other tested characteristics (hypodontia, fingertip pads, hypotonia and hyperlaxity) were equally frequent in both groups. Although larger groups of patients and more elaborate phenotypic characteristics should be evaluated, this study indicates that patients with an MLL2 mutation may represent a clinical subgroup within the KS patient population.

As pointed out by Ng et al. yet other genes might cause KS and could be identified in patients with KS but without a detectable MLL2 mutation. Likewise, MLL2 microdeletions (or microduplications) involving, for example entire exon(s), could still be present in the latter. Although many patients included in this study were assessed previously on various microarray platforms, aberrations at the exon deletion scale could have been missed due to resolution constraints. These patients will be studied by higher resolution microarrays or by semi-quantative PCR based approaches, focusing on the MLL2 region.

The ramifications of MLL2 mutations for early developmental processes, leading to the severe phenotype observed in KS need to be addressed in functional studies following these initial mutation screens. An experimental model such as a mouse Mll2 gene knock-out would provide valuable information but has yet to be developed. Since there is considerable confusion in the literature it needs to be pointed out here that the existing mouse Mll2 knock-out is actually the equivalent of a human MLL4 knock-out due to a recent nomenclature switch that results in mouse Mll2 now being the ortholog of human MLL4 [START_REF] Glaser | Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development[END_REF]. The emerging spectrum of MLL2 mutations to which this study adds 33 novel mutations further provides insight into Kabuki syndrome's etiology. Specific functional studies can now be designed to understand the disease's underlying mechanisms. As stated above, methyl transferase pathways have been implicated before in the pathology of some intellectual disability syndromes. Given the apparent non-redundancy of HMTs within their respective families and their equally non-redundant modes of gene regulation, a specific pathogenicity for each of the pathways involved make HMTs viable targets for further mutational analyses in the very divergent spectrum of, as of yet, unresolved intellectual disability syndromes.

  in green Fig 1.

Figure 1 .

 1 Figure 1. Schematic diagram of 34 mutations detected in the MLL2-gene in Kabuki syndrome patients. Upper figure: genomic structure of the MLL2 gene including 54 exons. Coding exons in orange, untranslated region in blue. Mutations in: red; frameshift mutations (n=17), orange; splice-site mutations (n=4), blue; nonsense mutations (n=11), green: missense mutations (n=2). Lower figure: MLL2 protein structure with conserved protein domains: PHD = plant homeodomain finger, HMG = high mobility group, FN = "FY-rich" domain, N-terminal region, FC = "FY-rich" domain, C-terminal region, SET = SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain, PSET = PostSET domain.

Table 1 .

 1 Frequency of reported MLL2 mutation types in Kabuki Syndrome

	Type of	Frequency			no parents
	mutation	(n)	de novo	inherited	available
	Nonsense this study	11	9	0	2
	Ng et al.	20	7	1	12
	Frameshift this study	17	14	0	3
	Ng et al.	7	3	0	4
	Missense this study	2	2	0	0
	Ng et al.	5	2	0	3
	Splice site this study	4	2	0	2
	Ng et al.	0	0	0	0

Table 2 .

 2 MLL2 mutations detected in 34/45 KS index patients (bold) and as reported by Ng et al.The MLL2 sequences were compared to the reference sequence of MLL2 (GenBank accession number NM_003482.3) with nucleotide numbering starting at the first adenine of the translation initiation codon ATG. NA = not available, (M) mother not a carrier, (F) father not a carrier. * c.8641_8646delCGGCACinsGACCTCAATGTCTCTGGACCAGG, ** c.10114_10126delA GCTCACAGCCAG.

						Human Mutation			Page 6 of 8
	1
	2
	3
	4
	5 6 7	nr. 1	Family number 8312	Exon	Mutation c.1301delT	AA change p.Leu434GlnfsX496	Mutation type F	Inheritance de novo	Reference This study
	8				c.1324delC	p.Pro442HisfsX487	F	NA	Ng et al. 2010
	9 10 11	2 3 4	13070 8299 12812		c.2110delG c.2272delG c.2558_2559delCT c.3585_3586insA	p.Asp704ThrfsX226 p.Glu758SerfsX171 p.Arg853ProfsX2 p.Pro1196ThrfsX11	F F F F	de novo de novo de novo de novo	This study This study This study Ng et al. 2010
	12	5	15969		c.3889delC	p.Arg1297ValfsX33	F	de novo	This study
	13 14 15	6 7	8274 8433	-	c.4219_4222delTACT c.4843C>T c.4956_4957insG c.5320-2 A>G	p.Tyr1407ValfsX9 p.Arg1615X p.Glu1654X	F N N S	de novo NA NA NA	This study Ng et al. 2010 Ng et al. 2010 This study
	16	8	17802		c.5585delA	p.Lys1862SerfsX14	F	de novo	This study
	17 18	9	12810		c.5875_5891dup17 c.5912delG c.6010C>T	p.Glu1965GlyfsX88 p.Ser1971ThrfsX76 p.Gln2004X	F F N	NA de novo de novo	Ng et al. 2010 This study Ng et al. 2010
	19				c.6295C>T	p.Arg2099X	N	de novo	Ng et al. 2010
	20 21 22	10 11 12	17809 13915 17804		c.6595delT c.7933C>T c.8200C>T c.8311C>T	p.Tyr2199fsX65 p.Arg2645X p.Arg2734X p.Arg2771X	F N N N	de novo de novo de novo de novo	Ng et al. 2010 This study This study This study
	23				c.8488C>T	p.Arg2830X	N	NA	Ng et al. 2010
	24 25 26	13 14 15	11103 17803 8358		c.8641_8646delins* c.9223dupT c.9329delG c.9961C>T	p.Arg2881AspfsX35 p.Ser3075PhefsX3 p.Arg3110ProfsX9 p.Arg3321X	F F F N	NA de novo NA (M) NA	This study This study This study Ng et al. 2010
	27	16	16163		c.9770dupA	p.Lys3250GlufsX43	F	de novo	This study
	28 29 30	17	8313		c.10114_10126del13** c.10599_10630del32 c.10738C>T c.11149C>T	p.Ser3372CysfsX16 p.Val3534GlnfsX11 p.Gln3580X p.Gln3717X	F F N N	NA (M) NA NA de novo	This study Ng et al. 2010 Ng et al. 2010 Ng et al. 2010
	31 32 33	18 19 20	17733 8346 8355		c.11497delC c.11707C>T c.11722C>T c.11794_11797delCAAC	p.Arg3833GlyfsX48 p.Gln3903X p.Glu3908X p.Gln3932SerfsX46	F N N F	de novo de novo de novo de novo	This study This study This study Ng et al. 2010
	34	21	13202		c.11944C>T	p.Arg3982X	N	de novo	This study
	35 36 37	22	8453		c.12164_12165delCT c.12241C>T c.12697C>T c.12703C>T	p.Pro4055ArgfsX6 p.Gln4081X p.Gln4233X p.Gln4235X	F N N N	de novo NA de novo NA	This study Ng et al. 2010 Ng et al. 2010 Ng et al. 2010
	38	23	16501		c.12969dupA	p.Pro4324ThrfsX10	F	de novo	This study
	39 40 41	24	17808		c.13390C>T c.13450C>T c.13580A>T c.13606C>T	p.Gln4464X p.Arg4484X p.Lys4527X p.Arg4536X	N N N N	de novo NA inherited NA	Ng et al. 2010 This study Ng et al. 2010 Ng et al. 2010
	42	25	8438	-	c.13999+5G>A		S	de novo	This study
	43 44 45	26 27 28	11212 8393 8348	--	c.14516-1G>C c.14644-2A>G c.14710C>T c.14845_14848dupCCTC	p.Arg4904X p.Leu4950ProfsX9	S S N F	NA (F) de novo NA de novo	This study This study Ng et al. 2010 This study
	46 47 48	29 30	8347 8817		c.14878C>T c.15079C>T c.15195G>A c.15217C>T	p.Arg4960X p.Arg5027X p.Trp5065X p.Gln5073X	N N N N	de novo de novo de novo NA	This study This study Ng et al. 2010 Ng et al. 2010
	49				c.15326G>T	p.Cys5109Phe	M	de novo	Ng et al. 2010
	50 51 52	31	8423		c.15444_15445delTT c.15536G>A c.15618T>G c.15629A>G	p.Phe5149CysfsX9 p.Arg5179His p.Tyr5206X p.Tyr5210Cys	F M N M	NA de novo de novo de novo	Ng et al. 2010 Ng et al. 2010 Ng et al. 2010 This study
	53				c.15641G>A	p.Arg5214His	M	NA	Ng et al. 2010
	54 55 56	32 33 34	12811 17805 13108		c.16018C>T c.16019G>T c.16283G>A c.16360C>T	p.Arg5340X p.Arg5340Lys p.Gly5428Asp p.Arg5454X	N M M N	de novo NA de novo NA	This study Ng et al. 2010 This study This study, Ng et al. 2010
	57				c.16391C>T	p.Thr5464M	M	NA	Ng et al. 2010
	58				c.16501C>T	p.Arg5501X	N	NA	Ng et al. 2010
	59
						John Wiley & Sons, Inc.

Table 3 .

 3 MLL2 variants/polymorphisms detected in the 45 KS index patients

	Exon/	Variant	AA change	inherited	nr of	rs number
	Intron				patients
	int3	c.401-36T>G	-		1
	10	c.2074C>A*	p.Pro692Thr	maternal	1
	10	c.2220G>T	p.Gly740Gly		1
	10	c.2438C>T	p.Pro813Leu		3	rs75226229
	11	c.2826C>T	p.Ile942Ile		23	rs2241726
	int12	c.4020+13C>G	-		1
	int13	c.4131+83insT	-		21	rs72518091
	31	c.6573G>A	p.Thr2191Thr		2
	31	c.7144C>T	p.Pro2382Ser		2	rs3741626
	31	c.7378C>T*	p.Arg2460Cys	maternal	1
	31	c.7479G>T	p.Gly2493Gly		29	rs10747559
	31	c.7670C>T*	p.Pro2557Leu	paternal	1
	34	c.9726C>T	p.Ala3242Ala		1
	34	c.10192A>G*	p.Met3398Val		1	rs75937132
	35	c.10233C>T	p.Asp3411Asp		1
	35	c.10256A>G*	p.Asp3419Gly	paternal	1
	38	c.10671A>G	p.Pro3557Pro		7	rs61942218
	39	c.10836G>A	p.Gln3612Gln		23	rs3782357
	39	c.12510A>G	p.Pro4170Pro		22	rs3741622
	39	c.13071G>C	p.Arg4357Ser	maternal	1
	int39	c.13531-62_65del4	-		4
	41	c.13689C>T	p.Pro4563Pro		5	rs11168830
	int52	c.16412+16delG	-		23	rs57079239

Table 4 .

 4 Distribution of Kabuki syndrome clinical features in MLL2 mutation carriers and non-carriers

	Syndrome	MLL2 mutation	no MLL2 mutation p-value 1
	Feature	(n = 34)	(n = 11)
	Characteristic face 2	29/34	5/11	0.0143
	Growth retardation 3	24/25	5/10	0.0040
	Hypodontia	24/27	4/6	0.2162
	Fingertip pads	31/33	7/9	0.1957
	Hypotonia	32/33	9/9	(1.0)
	Hyperlaxity	32/33	9/9	(1.0)

1 = two sided Fisher's exact test, significance threshold at P<0.05; 2 defined by elongated palpebral fissures and eversion of the lateral one third of the lower eyelid, an arched or nicked eyebrow with sparseness of hairgrowth in the lateral half, relatively large ears with simplified morphology, a depressed nasal tip and a full lower lip. 3 = threshold -3SD and assessed above 3 years of age; boldface. = statistically significant (p<0.05).

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

We thank all families that agreed to participate in the study. We are indebted to the Dutch Kabuki Patient Association (Netwerk Kabuki Syndroom) and its president mrs. Els Vergouwen-Knapen, M.Sc. for active support and cooperation. We are grateful to ms. Nadine van Veenendaal and mrs. Sera Langenveld for administrative assistance.


Giuseppe Saverio

Caterina Parisi

Samantha Boldrin

Roberto Andreis

Rodolfo Ferretto

Marina Fuser

Vinicio Malena

Sandro Manfrin

Renzo Panese

Luca Scaggiante

Dori

Saverio G Parisi
email: saverio.parisi@unipd.it

Caterina Boldrin

Samantha Andreis

Roberto Ferretto

Rodolfo Fuser

Marina Malena

Vinicio Manfrin

Sandro Panese

Renzo Scaggiante

Luca Dori

Sarmati

Maria A Biasolo

Emanuele Nicastri

Massimo Andreoni

Mario Cruciani

Giorgio Palù

MD Giuseppe

KSHV DNA viraemia correlates with low CD4+ cell counts in Italian

Keywords: KSHV-DNA, HIV, immunodeficiency, homosexual relationship immunodeficiency, homosexual relationship. 24

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

To evaluate the relevance, virological and immunological markers of Kaposi sarcoma 3 herpesvirus 8 (KSHV) viraemia in Italian male patients at the time of diagnosis of infection 4 with HIV-1, 481 men infected with HIV were recruited consecutively. The presence of KSHV 5 DNA was evaluated in peripheral blood mononuclear cells (PBMCs) and in plasma and 6 correlated with demographic and viro-immunological parameters. 7

Seventy-four patients had KSHV DNA detected in PBMCs. By univariate analysis, the 8 presence of KSHV DNA was associated significantly with unprotected homosexual 9 relationships (p=0.003) and it was significantly higher in patients with CD4+ cell <350 10 (p=0.025). By multivariate analysis, homosexual relationships were associated independently 11 with KSHVDNA in PBMCs (OR: 3.25; 95% CI: 1.1-9.7; p=0.035). Among the 74 patients 12 with KSHVDNA detected in PBMCs, plasma samples from 60 were analysed and 33 were 13 positive for KSHV DNA. The CD4+ cell counts and percentages were significantly lower in 14 patients with KSHV DNA in both PBMCs and plasma as compared to patients with only 15 KSHV DNA in PBMCs (p=0.006 and p=0.019, respectively). Among the patients with KSHV 16 DNA detected in PBMCs, all 13 patients with CD4+ cells count <200 had detectable levels of 17 KSHV in their plasma. By multivariate analysis adjusted for the epidemiologic and 18 virological parameters, low CD4+ cell count was the only independent variable associated 19 with the presence of KSHV DNA in plasma (OR, 0.001; 95% CI, <0.001-0.001; p=0.03). 20

In HIV-positive antiretroviral therapy-naïve males, KSHV active replication as detected by 21 KSHV DNA in plasma was associated significantly with low CD4+ cell count. 22 Introduction 1 2 Kaposi's Sarcoma-associated herpesvirus (KSHV) is linked causally to primary effusion 3 lymphoma, multicentric Castleman disease, and all epidemiological forms of Kaposi's 4 sarcoma (KS) [START_REF] Boshoff | Kaposi's sarcoma associated herpesvirus[END_REF]. The prevalence of KSHV varies between countries 5 and populations. Variable routes of transmission, both sexual and non-sexual, have been 6

described [START_REF] Schulz | Kaposi's sarcoma-associated herpesvirus: a new human 18 tumor virus, but how?[END_REF][START_REF] Hengge | Update on Kaposi's sarcoma and other HHV8 associated diseases. Part 1: 16 epidemiology, environmental predispositions, clinical manifestations, and therapy[END_REF]. In areas with low seroprevalence, 7 KSHV appears to be acquired predominantly through sexual contact, and sex between men 8 may be an important route of transmission [START_REF] Schulz | Kaposi's sarcoma-associated herpesvirus: a new human 18 tumor virus, but how?[END_REF]. Familial clustering of 9 KSHV is observed frequently in endemic regions, where nasal secretions and saliva may be 10 the sources of KSHV transmission [START_REF] Plancoulaine | High titers of anti-human 1 herpesvirus 8 antibodies in elderly males in an endemic population[END_REF][START_REF] Andreoni | High seroprevalence of antibodies to human herpesvirus-8 in Egyptian 8 children: evidence of nonsexual transmission[END_REF][START_REF] Rezza | Prevalence and risk factors for human 12 herpesvirus 8 infection in northern Cameroon[END_REF]. 12 Variable seroprevalence of KSHV has been reported among subjects infected with HIV 13 (Campbell et al., 1999;[START_REF] Laney | Repeated measures study of human herpesvirus 8 (HHV-8) DNA and antibodies in 3 men seropositive for both HHV-8 and[END_REF]). In Italy, its overall prevalence in HIV-14 seropositive people in 1997-98 was demonstrated to be 31.4%, an increase from the 1987-88 15 prevalence of 14.6%, suggesting a spread of infection among HIV-seropositive subjects, and 16 possibly from them to the HIV-negative community, by sexual and non-sexual routes (Parisi 17 SG, Sarmati L et al., 2002). [START_REF] Chang | Identification of Herpesvirus-like DNA sequences in AIDS-associated Kaposi's 20 sarcoma[END_REF] HIV and KSHV are able to influence each other, and there is in vitro evidence that suggest 19 the ability of one virus to facilitate the replication of the other. HIV activates lytic cycle 20 replication of KSHV, and KSHV lytic antigens interact with HIV Tat to increase the cell's 21 susceptibility to HIV infection [START_REF] Zeng | Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle 3 replication of Kaposi's sarcoma-associated herpesvirus: role of JAK/STAT signaling[END_REF][START_REF] Varthakavi | Human immunodeficiency 11 virus type-1 activates lytic cycle replication of Kaposi's sarcoma-associated herpesvirus 12 through induction of KSHV Rta[END_REF]. In vivo, HIV- 1 22 infection has been associated with the presence of KSHV in any mucosal surface, and the 23 presence of KSHV DNA in PBMCs is predictive of the development of KS in patients 24 infected with HIV [START_REF] Moore | Kaposi's sarcoma-associated herpesvirus infection prior to onset of 8 Kaposi's sarcoma[END_REF][START_REF] Taylor | Shedding of 7 Human Herpesvirus 8 in oral and genital secretions from HIV-1-seropositive and seronegative 8 Kenyan women[END_REF][START_REF] Engels | 1 Detection and quantification of Kaposi's sarcoma-associated herpesvirus to predict AIDS-2 associated Kaposi's sarcoma[END_REF]. KSHV-DNA 25 was more detected frequently in people infected with HIV and in the oropharynx and 26 4 peripheral blood of Ugandans with endemic and epidemic KS [START_REF] Johnston | 21 Impact of HIV infection and Kaposi sarcoma on human herpesvirus-8 mucosal replication and 22 dissemination in Uganda[END_REF]. KSHV 1 DNA is detectable in KS tissues [START_REF] Chang | Identification of Herpesvirus-like DNA sequences in AIDS-associated Kaposi's 20 sarcoma[END_REF], in PBMCs from patients with KS and 2 multicentric Castleman disease, and in PBMCs and effusion fluid samples from patients with 3 primary effusion lymphoma [START_REF] Marcelin | Relationship between the quantity of Kaposi 24 Sarcoma associated Herpesvirus (KSHV) in peripheral blood and effusion fluid samples and 25 1[END_REF]. Among KS patients, KSHV DNA has 4 been detected in both plasma and PBMCs, and often no consistent difference between 5 biological compartments is detected [START_REF] Lin | Effects of chemotherapy in AIDS-associated non-Hodgkin's lymphoma 11 on Kaposi's sarcoma herpesvirus DNA in blood[END_REF]. KSHV-infected cells were detected at a 6 very low frequency in the inguinal lymph nodes of HIV-1-seropositive subjects without 7 KSHV-associated diseases [START_REF] Campbell | 5 Persistence of Kaposi sarcoma-associated herpesvirus (KSHV)-infected cells in KSHV/HIV-6 1-coinfected subjects without KSHV-associated diseases[END_REF], suggesting that the latent infection of 8 lymph node cells provides a mechanism for the persistence of KSHV in KSHV/HIV-1-co-9 infected people. 10 Although effective HIV suppression has been correlated with the regression of KS after 11 antiretroviral therapy [START_REF] Martinez | Remission from Kaposi's Sarcoma on HAART is associated with 3 suppression of HIV replication and is indipendent of protease inhibitor therapy[END_REF], the persistence of KS despite seemingly 12 effective antiretroviral therapy (e.g., an undetectable HIV load) is not rare (Krown et al., 13 2008). However, the interactions between immune responses, KSHV replication, and their 14 relative contribution to KS have not been well characterised to date. 15

The aim of the study was to evaluate the prevalence of KSHV DNA viraemia in male patients 16 from northeastern Italy with chronic and acute HIV-1 infections at the time of HIV diagnosis. 17 The eligibility criteria included being of Italian origin, >18 years of age, and having 8 antiretroviral-drug-naïve status. 9

Blood samples were submitted to the Laboratory of Virology at the University of Padua, 10 stored within 6 hours from collection, and analysed subsequently. 11 A primary or recent HIV infection was defined by the presence of either of the following: (i) a 12 negative or indeterminate HIV antibody-ELISA associated with HIV RNA-positive plasma; 13 or (ii) an initially negative test for HIV antibodies followed by positive serology within 18 14

months. 15

The HIV RNA plasma viral load assessment (using Roche Cobas AmpliPrep-Cobas TaqMan 16 HIV-1, version 1 assay, F. Hoffmann-La Roche, Diagnostics Division, Basel, Switzerland) 17 and HIV genotypic analysis to detect subtype were performed as described previously (Parisi 18 et al., 2007). 19 CD4+ cell counts and percentages were determined at enrolment. Blood collected in EDTA 20 was separated into plasma and cells by Ficoll-Paque Plus density gradient centrifugation. 21 Aliquots of plasma and dry pellets of 2 x 10 6 PBMCs were stored at -80°C until use. 22

KSHV DNA analysis 23

The extraction and purification of DNA from cells and plasma samples was performed using 24 the QIAmp Blood kit (Qiagen, Inc., Chatsworth, CA). KSHV DNA from cell and plasma 25 samples was detected using a real-time polymerase chain reaction (RT-PCR; with TaqMan 26 previously [START_REF] White | Quantitation of Cell-Free and Cell-Associated Kaposi's 24 Sarcoma-Associated Herpesvirus DNA by Real-Time PCR[END_REF]. The nucleotide sequence targeted by the primers and 2 probes is conserved highly among the three major subgroups of KSHV [START_REF] Poole | Comparison of genetic variability at multiple loci across the 6 genomes of the major subtypes of Kaposi's Sarcoma-Associated Herpesvirus reveals evidence 7 for recombination and for two distinct types of open reading frame K15 alleles at the right-8 hand end[END_REF]. 3 During all DNA extractions and purifications, precautions were taken to reduce the risk of 4 false-positive results. 5

Cellular HIV DNA quantitation 6

The Real-Time TaqMan protocol published by Viard and colleagues [START_REF] Viard | Impact of 5 years of maximally successful highly active antiretroviral 16 therapy on CD4 cell count and HIV-1 DNA level[END_REF] was 7 used to quantify the HIV DNA copy number in PBMCs. The cell line 8E5, containing one 8 copy of integrated HIV DNA in each cell, was used to build a standard curve. The sensitivity 9 of the test was 13.3 copies/10 6 PBMCs. 10

Statistical analysis 11

The variables initially taken into account were age, risk factors for HIV infection (men who 12 had sex with a man vs. other risk groups), primary HIV infection, HIV subtype (B subtype vs. 13 other subtypes), CD4+ cell count, HIV RNA, and HIV DNA load. Among these, age was a 14 quantitative variable, while primary HIV infection, HIV subtype, and the presence of drug 15 resistance mutations were binary (yes/no) variables and were coded as 1/0. CD4+ cell count, 16

HIV RNA, and HIV DNA were both quantitative and discrete variables. 17 Univariate analyses were performed using all of the covariates, taking them both (when 18 appropriate) as continuous variables, as well as categorising by clinically relevant values. Chi 19 square and Kruskal-Wallis tests were used to assess differences between patient groups with 20 reference to categorical and continuous variables, respectively. The significance level was set 21 at 0.05 and all P values were two-tailed. To obtain an adjusted analysis while accounting for 22 all possible risk factors, a multiple logistic regression model was used. Age, HIV-RNA level, 23 and all of the significant variables in the univariate analysis were included in the multivariable 24 model. 25 Of the 481 patients enrolled, in 43 cases (8.9%) it was not possible to define their risk for HIV 3 infection. Of the remaining 438 patients, 282 were homosexual, 90 were heterosexual and 66 4 were intravenous drug abusers. Additionally, 102 patients had a primary HIV infection and 5 379 patients had a chronic infection. 6

Patients with chronic HIV infections were older (41+11.4 vs. 37.6+10.2 years, p=0.008) and 7 had CD4+ cell counts (413.8+273.4 vs. 603.9+297.5 cells/µl, p<0.001) and percentages 8 (21.2+10.2 vs. 26.6+10.5%, p<0.001) significantly lower than the patients with primary 9 infections. 10 Table 1 presents the population of patients infected with HIV studied according to the 11 presence of KSHV DNA in PBMCs and different epidemiological and viro-immunological 12 parameters. [START_REF] Lorenzen | HHV-8 DNA in blood and the development of 15 HIV-associated Kaposi's sarcoma in the era of HAART--a prospective evaluation[END_REF] Of the 481 enrolled patients, 74 (15.4%) had KSHV DNA detected in their PBMCs. By 14 univariate analysis, unprotected homosexual relationships and a CD4+ cell count <350 15 cells/µl were associated significantly with the presence of KSHV DNA in PBMCs (p=0.003 16 and p=0.025, respectively). 17

Patients with primary HIV infections had a lower (but not significantly lower) rate of KSHV 18 DNA positivity Table 2 presents the univariate analysis of the population of HIV/KSHV co-infected male 1 patients studied in relation to the simultaneous presence of KSHV DNA in PBMCs and 2 plasma. 3 Among the 74 subjects with KSHV DNA detected in PBMCs, the presence of KSHV DNA in 4 plasma was analysed for 65 subjects from whom plasma specimens were available. Of the 65 5 patients, 38 (58.4%) also had KSHV DNA-positive plasma. Overall, five patients had an 6 ultimate diagnosis of KS and were excluded from the evaluation. Among the 60 non-KS 7 patients, 33 (55%) had KSHV DNA detected in both PBMCs and plasma; 28 (85%) of these 8 patients had a chronic HIV infection and 5 had a primary HIV infection. The CD4+ cell 9 counts and percentages were significantly lower in patients with detectable levels of KSHV 10 DNA in both PBMCs and plasma compared to patients with KSHV DNA detectable only in 11 PBMCs (p=0.006 and p=0.019, respectively). Moreover, among patients with KSHV DNA 12 detected in PBMCs, all 13 subjects with CD4+ cell counts <200 cells/µl also had KSHV DNA 13 detectable in plasma. 14

In the multivariate analysis (Table 3) adjusted for the different epidemiologic and virological 15 parameters, CD4+ cell count was the only independent variable associated with the presence 16 of KSHV DNA in the plasma of co-infected HIV/KSHV patients (OR, 0.001; 95% CI, 17 <0.001-0.001; p=0.03). 18

A subgroup of 20 patients with KSHV DNA detected in PBMCs and plasma were re-analysed 19 for the presence of KSHV DNA in PBMCs and plasma in a follow-up study. 20 Nine subjects still had KSHV DNA detectable in PBMCs and plasma after a mean time of 19 21 months. Six of these subjects were still highly active antiretroviral therapy (HAART)-naïve 22 and three were on HAART, two of whom were HIV plasma viraemic at the time of the second 23 analysis. Of the 14 patients who were successfully HAART-treated (<50 HIV RNA 24 copies/ml), 11 were KSHV DNA-negative in PBMCs and plasma after a mean time of 17 25 months. 26 The presence of KSHV DNA in a cohort of patients at the time of their initial diagnosis of 3 HIV infection was studied. Out of the 481 evaluated subjects, 74 (15.3%) were found to have 4 detectable levels of KSHV DNA in their PBMCs. Approximately half of the subjects found to 5 be PBMC-positive had concomitant active KSHV replication in their plasma. 6

The presence of KSHV DNA in PBMCs is predictive of the development of KS in HIV-7 infected patients, and KSHV DNA has been detected in up to 70% of HIV-related KS cases 8 [START_REF] Whitby | Detection of Kaposi sarcoma associated 20 herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi's 21 sarcoma[END_REF][START_REF] Moore | Kaposi's sarcoma-associated herpesvirus infection prior to onset of 8 Kaposi's sarcoma[END_REF]. Previous studies have reported variable results on the 9 prevalence of KSHV DNA in HIV-1-infected people without KS. Laney and colleagues found 10 KSHV DNA in the PBMCs of 28.9% of patients without KS, a rate almost double that of the 11 cohort in this study [START_REF] Laney | Repeated measures study of human herpesvirus 8 (HHV-8) DNA and antibodies in 3 men seropositive for both HHV-8 and[END_REF]. By contrast, Campbell et al (1999) found that 18% of 12 KSHV/HIV-1 co-infected people without KS have detectable KSHV DNA in their PBMCs. [START_REF] Lorenzen | HHV-8 DNA in blood and the development of 15 HIV-associated Kaposi's sarcoma in the era of HAART--a prospective evaluation[END_REF] The lower prevalence in the cohort of this study may be due to the distribution of risk factors 14 for HIV, with only 67% of our subjects being homosexual compared to >90% in the previous 15 studies. The median CD4+ cell values were lower in the studies by Campbell and by Laney 16 than they were in the cohort of this study. 17

In this study, patients with detectable KSHV DNA had lower CD4+ cells count compared to 18 patients without detectable KSHV DNA. Using multivariate analysis, CD4+ cell count was 19 found to be correlated negatively with active KSHV replication. By contrast, any correlation 20 between KSHV DNA detection and either HIV RNA, HIV DNA viral load, or HIV subtype 21 was not found. The lack of a correlation between the presence of KSHV DNA and HIV 22 viraemia seems to confirm previous observations that assign a greater role to the immune 23 response (in particular the NK-type response) and CD4+ cell recovery rather than to HIV 24 replication in the control of KS in AIDS patients [START_REF] Sirianni | NK cell activity 22 controls human herpesvirus 8 latent infection and is restored upon highly active antiretroviral 23 therapy in AIDS patients with regressing Kaposi's sarcoma[END_REF][START_REF] Cattelan | Acquired immunodeficiency syndrome-related Kaposi's sarcoma 15 regression after highly active antiretroviral therapy: biologic correlates of clinical outcome[END_REF]. 25 subjects with chronic HIV infections than in those with acute infections. This result agrees 1 with the significant correlation between KSHV DNA and low CD4+ cell count and seems to 2 confirm that it is the immunological deficiency (which is more pronounced in advanced HIV 3 infection) rather than the level of HIV replication that favours KSHV viraemia. The relevance 4 of the immune response to the control of KSHV replication is also demonstrated by the results 5 of a study in which KS risk was found to be associated positively with a reduced number of 6 lymphocytes, including CD4-positive cells (<457 cells/mL;) [START_REF] Brown | Virologic, hematologic,and 23 immunologic risk factors for classic Kaposi sarcoma[END_REF]. 7

Almost 60% of patients with KSHV DNA-positive PBMCs also showed the presence of 8 HHV8 in plasma. Absolute CD4+ cell counts and percentages were significantly lower in 9 patients who were KSHV DNA-positive in both plasma and PBMCs compared to patients 10 positive only in PBMCs. Again, the active replication of KSHV was associated significantly 11 with a low number and low percentage of CD4+ cells, and this confirms the more relevant 12 role of immune response in controlling KSHV replication. 13

Previous studies have indicated a correlation between KSHV replication and KS pathogenesis 14 men who seroconverted to KSHV antigens, KSHV viraemia persisted for up to 6 months 2 [START_REF] Goudsmit | Human herpesvirus 8 infections in the Amsterdam 11 Cohort Studies (1984-1997): analysis of seroconversions to ORF65 and ORF73[END_REF]. This issue was addressed in a subgroup of patients from the cohort of 3 this study. KSHV viraemia persisted in the majority of patients who were untreated or treated 4 unsuccessfully with HAART. By contrast, all of the patients on successful HAART cleared 5 KSHV from their blood after a mean period of 17 months. 6

Before drawing any conclusions, some potential biases of the study must be addressed. The 7 cross-sectional design of the study does not allow conclusive explanation of the correlation 8 between KSHV viraemia and low CD4+ cell count. However, although the small number of 9 patients enrolled in this longitudinal study may limit its conclusiveness, the low CD4+ cell 10 counts in patients on HAART was associated significantly with active replication of KSHV. 11 Studies in a larger population are required to confirm this result. Finally, the lack of risk 12 factors for HIV infection for less than 10% of the patients may have caused misclassification, 13 potentially biasing the correlation between unprotected homosexual relationships and KSHV 14 viraemia. 15

In conclusion, KSHV DNA-positive results were observed in approximately 15% of an HIV-16 positive cohort of Italian male subjects. The active replication of KSHV was associated 17 significantly with a low number of CD4+ cells, and it was more frequent in patients with 18 chronic than acute HIV infections. A longitudinal study of a limited number of patients 19

  Moreover, correlations between KSHV viraemia and the virological and immunological 18 parameters of HIV infection were evaluated. hundred and eighty one men infected with HIV-1 were enrolled at six Infectious 4 Diseases Units located in Veneto, Italy. The patients were recruited consecutively from July 5 1, 2004 to December 31, 2008 after obtaining their written informed consent to participate in 6 the study. The ethics committee of the hospitals gave approval for the study. 7

  Biosystems, Foster City, CA) of open reading frame 26 (ORF26), as described 1

1 13

 1 analyses were performed on an intention-to-treat basis using SPSS for Windows

  in PBMCs in comparison to patients with chronic infections. No significant 19 differences in HIV RNA or HIV DNA levels were found between KSHV DNA-positive and 20 KSHV DNA-negative patients. 21 By multivariate analysis adjusted for different epidemiological and virological parameters, 22 having a homosexual relationship (OR: 3.25; 95% CI: 1.1-9.7; p=0.035 ) was the only 23 parameter that was associated independently with the presence of KSHV DNA in PBMCs.24

  the 14 patients treated with HAART, the 11 patients who were negative for 1 KSHV DNA had a mean increase in their CD4+ cell counts of 249 cells/µl (range 57-603), 2 whereas the 3 treated patients with detectable levels of KSHV DNA had CD4+ cell count 3 increases of 57, 97, and 74 cells/µl. 4

( 22 (

 22 [START_REF] Whitby | Detection of Kaposi sarcoma associated 20 herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi's 21 sarcoma[END_REF][START_REF] Cannon | Risk factors for Kaposi's sarcoma 10 in men seropositive for both human herpesvirus 8 and human immunodeficiency virus[END_REF][START_REF] Lorenzen | HHV-8 DNA in blood and the development of 15 HIV-associated Kaposi's sarcoma in the era of HAART--a prospective evaluation[END_REF][START_REF] Smith | Detection of 1 human herpesvirus 8 DNA in Kaposi's sarcoma lesions and peripheral blood of human 2 immunodeficiency virus-positive patients and correlation with serologic measurements[END_REF] Broccolo 15 et al., 2002;[START_REF] Engels | 1 Detection and quantification of Kaposi's sarcoma-associated herpesvirus to predict AIDS-2 associated Kaposi's sarcoma[END_REF]. In individuals infected with HIV the use of HAART has a 16 role in reducing the frequency of KSHV viraemia, mostly because of the immuno-restorative 17 properties of HIV treatment[START_REF] Gill | Prospective study of the effects of antiretroviral therapy on 6 Kaposi Sarcoma-associated Herpesvirus infection in patients with and without Kaposi 7 Sarcoma[END_REF][START_REF] Rizzieri | Clearance of HHV-8 from peripheral 15 blood mononuclear cells with a protease inhibitor [letter[END_REF] Lebbe et al., 1998).[START_REF] Chang | Identification of Herpesvirus-like DNA sequences in AIDS-associated Kaposi's 20 sarcoma[END_REF] Treatment with a regimen that includes a protease inhibitor was associated with the clearance 19 of KSHV DNA in KS lesions and PBMCs, and with the regression of KS lesions in AIDS 20 patients (Lebbe et al., 1998;[START_REF] Blum | Complete 11 remission of AIDS-related Kaposi's sarcoma associated with undetectable human herpesvirus 12 8 sequences during anti-HIV protease therapy[END_REF]. Furthermore, a number of reports have linked 21 tumour regression after the initiation of HAART to the restoration of immune function[START_REF] Dupont | Long-term efficacy on Kaposi's sarcoma of highly active antiretroviral 24 therapy in a cohort of HIV-positive patients[END_REF][START_REF] Marcelin | Quantification of Kaposi's sarcoma-associated herpesvirus in blood, oral mucosa, and 20 saliva in patients with Kaposi's sarcoma[END_REF], and it has been reported that KSHV DNA amount 23 in PBMCs rebounded in patients with KS after a short interruption of efficient antiretroviral 24 therapy(Parisi et al., 2002). 25 KSHV viraemia are limited. In a large cohort of homosexual 1

  demonstrated a direct correlation between immune recovery during HAART and the 20 disappearance of KSHV DNA. 21

Table 1 .

 1 Demographic, virological, and immunological characteristics of untreated HIV-1 seropositive patients according to the presence of KSHV DNA in PBMCs.

	2
	3
				All patients	KSHV neg	KSHV pos	p value
				(n=481)	(n=407)	(n=74)
		Mean age in years (± SD)		40.3 (+11.2) 41.7 (+11.7)	40.0 (+11.1)	0.26
		Risk factor for HIV infection:
		*unprotected homosexual relationship, (%)	282 (64.4)	226 (61.4)	56 (80.0)	0.003
		F Patients with primary HIV infection, n. (%) o r **Patients with B subtype (%)	102 (21.2) 401 (88.5)	92 (22.6) 339 (88.1)	10 (13.7) 62 (91.2)	0.09 0.45
		P Mean CD4+ cell count /µl (± SD)	449 (+288)	461 (+291)	389 (+264)	0.06
		Patient CD4+ cell count, n (%)	e			0.08
		<200 cells 200-350 cells	e r	87 (18.1) 112 (23.3)	70 (17.2) 89 (21.9)	18 (24.3) 22 (29.7)
		>350 cells Percentage of CD4+ cells (±SD) HIV RNA cp/ml (±SD) HIV DNA cp x 10 6 PBMCs (+SD)	282 (58.6) R 22.1 (+10.6) 22.3 (+10.5) 248 (60.9) e 268061 280623 v (+872183) (+940158) i e 2893 (+4429) 2799 (+4483) 3340 (+4162) 34 (46.0) 21.2 (+10.6) 200150 (+304285)	0.025 0.44 0.50 0.38
	4 5	* % of 438 evaluable patients ** % of 453 evaluable data		w
	6
	7
						1

Table 2 1

 2 Univariate analysis of 60 untreated HIV-seropositive patients with KSHV DNA

	2
	3	in PBMCs and for whom plasma samples were available/analysed
	4
			All patients	KSHV pl pos	KSHV pl neg	P
			(n=60)	(n=33)	(n=27)	value
		Mean age in years (±SD)	42.1 (+11.5)	44.1 (+12.8)	39.2 (+8.8)	0.10
		Risk factor for HIV infection:
		unprotected homosexual relationship (%)	47 (78)	29 (87.9)	18(66.6)	0.37
		Patients with primary HIV infection, n. (%)	7 (11.6)	5 (15.1)	2 (7.4)	0.46
		Patients with B subtype (%)	55 (91.6)	29 (87.8)	26 (96.3)	0.27
		CD4+ cell count/µl (±SD)	404 (+267)	325 (+258)	513 (+244)	0.006
		Patient CD4+ cell count, n. (%)				0.002
		<200 cells	13 (21.7)	13 (39.3)	0 (0)
		200-350 cells	19 (31.7)	9 (27.2)	10 (37.0)
		>350 cells	28 (46.6)	11 (33.3)	17 (63.0)	0.01
		Percentage of CD4+ cells (±SD)	22.1 (+10.4)	19.2 (+11.4)	25.8 (+7.7)	0.019
		HIV RNA cp/ml (±SD)	197691	224658	162095	0.45
			(+311073)	(+313325)	(+310812)
		HIV DNA cp x 10 6 PBMCs (+SD)	3446 (+4362) 3252 (+3786) 3786 (+5407) 0.67

Table 3

 3 Multivariate analysis of untreated HIV-seropositive patients with KSHV DNA in 1

	2	PBMCs according to the presence of KSHV DNA in plasma
	3
	4
			p	OR	95% CI
		Patients with age > median age	0.70	0.06	-0.24-0.35
		Risk factor for HIV infection
		unprotected homosexual relationship	0.55	0.09	-0.22-0.40
		CD4+ cell count/ml	0.03	0.001	<0.001-0.001
		Patients with >median HIV RNA cp	0.28	-0.17	-0.48-0.14
	5


Fabrizio Racca

Giacomo Berta

Marco Sequi

Elisabetta Bignamini

Elena Capello

Renato Cutrera

Giancarlo Ottonello

Marco V Ranieri

Ida Salvo

Raffaele Testa

San Giovanni

V M Ranieri

Andrea Wolfler

MD Maurizio Bonati

Anna Costantino

Raffaella Pagni

Anna Maria Miorelli

Paolo Castaldi

Mariano Cabras

Bruno Dengo

Augusto Biasini

Francesco Morandi

Carlo Minetti

Paolo Banfi

Edoardo Calderini

Edi Prandi

Marco Zucconi

Giuseppe Fiorentino

Antonio Maddalena

Grazia Crescimanno

Anna Guddo

Livio Carnevale

Andrea Vianello

Giancarlo Piccinini

Valter Bottari

Reggio Emilia

Maurizio Corbari

Martino Pavone

Lorenzo Del Sorbo

Cesare Gregoretti

Maria Maspoli

Antonio Peratoner

Monica Benedetti

Castelfranco Veneto

Long-term Home Ventilation of Children in Italy: a National Survey

Keywords: Chronic respiratory failure, home respiratory care, epidemiology, disability, long term ventilation, home mechanical ventilation Chronic respiratory failure, home respiratory care, long term ventilation, home mechanical ventilation, epidemiology, artificial ventilation, disability Chronic respiratory failure, home respiratory care, long term ventilation, home mechanical ventilation, epidemiology, artificial ventilation, disability

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

INTRODUCTION

Continuous advances in neonatal and pediatric intensive care are likely to result in more infants, children, and adolescents with critical, chronic conditions, who survive and who are discharged home, but require long-term technological support. [1][2][3] A growing population of children exists who has chronic respiratory failure due to conditions such as neuromuscular diseases, obstructive sleep apnea, craniofacial abnormalities, or spinal cord injuries, and who require either tracheostomy or mask ventilation.

Pressure to reduce the hospital stay of these children and awareness of the effects of long termhospitalization on a child's development have, during the last decade -especially, in a few countries such as UK, France, and US, -increased the numbers of ventilated children discharged home. However, the availability and practical details of complex respiratory support at home involve a lot of variables such as appropriate home environment, healthcare support, and funding, that cannot always be guaranteed. Findings of the Eurovent study, a wide cross sectional survey including adults as well as children, highlighted how the collection of information about home mechanical ventilation (HMV) could facilitate national planning for HMV. In particular, it could aid the development of HMV services in order to ensure equality of provision and access. 3 Surveys in Canada, 4,5 Chile, 6 Germany, 7 France, 8,9 Italy, 10,11 Japan, 12 New Zealand, 13 Switzerland, 14 The Netherlands, 15 Turkey, 16 UK, [17][18][19] and USA 20-- 22 have defined the size, main features, and needs of the pediatric population requiring home mechanical ventilation. However there are many differences between these observational studies: the setting (single referring center, state, or country), the time period (one year or trend over time), the data collected, and how it was collected. Thus, estimates of the national prevalence of home ventilator-dependent children range from 0.7/100,000 children in 1993 in Japan 12 to 3.4/100,000 in 2000 in Switzerland. 14 Because information on long-term home ventilation for children in Italy is scant and erratic (no regional or national population-based data exist), this survey was conducted to describe the size and

METHODS

Study Population

A questionnaire was sent to all 27 Italian pediatric intensive care and pulmonary units to get data on children up to 17 years old receiving HMV in January 2007. To identify as many children as possible the questionnaire was sent also to another 275 hospital-based units (eg. pulmonary services, and critical care units) of the National Health Service (NHS) that are potentially involved in the care of the target population. Information referred to children discharged home from hospital whorequired tracheostomy or mask ventilation.

Home respiratory care

HMV was defined as invasive mechanical ventilation (IMV) or non invasive mechanical ventilation (NIV), for a period of at least 3 months on a daily basis, carried out mostly in the user's home or other long-term care facility (not a hospital). 3 Basic demographic data (age, sex, place of residence), causes of respiratory failure, hospitalization history, duration of ventilatory support, type of ventilation and management-related description and home care provider information were collected.

Data analysis

Data were entered into a SAS/STAT database (Statistical Analysis Software, Cary, NC) for recording and analysis. The prevalence with 95% confidence intervals (CI) of Italian children ventilated at home on the long term was calculated using the Istat 2007 annual report (Italian National Institute of Statistics) for children up to 17 years old, resident in the provinces in which patients were identified as the denominator. Age was calculated on the basis of the date of birth and the date of onset of ventilation was recorded. Results are presented as frequencies, or median and interquartile range; P < 0.05 was considered significant.

All data collected on centres and patients were kept strictly confidential. Data on patients were anonymous. The San Giovanni Battista-Molinette Hospital, University of Turin, Committee on Clinical Investigation reviewed and approved the study protocol. Because this was an observational study without an intervention and without the presentation of any identifying data, the committee ruled that informed consent was not necessary.

RESULTS

A total of 362 children were identified by 1).

The most frequent diagnostic category was neuromuscular disorders (49%), followed by lung and upper respiratory diseases (18%), hypoxic (ischemic) encephalopathy (13%), and abnormal ventilation control (12%) ( 23 Health inequalities in childhood can lead to distress for the patient and the family, as well as financial waste. Thus, for this group of patients the burden of care is higher for southern residents.

The median length of home ventilation was one year, indicating that hospital discharge of these patients is a recent practice in Italy. In line with surveys from other countries 9,12,16,21 neuromuscular disorders were the most frequent indication for long-term home ventilation, as the established supportive therapy reduces the morbidity and mortality of these children. [24][25][26][27][28] Non-invasive support was used in the majority of the investigated population (59%) and was more prevalent in later infancy and adolescence. This finding is consistent with the literature, 29 invasive mechanical ventilation, the prevalence of non-invasive ventilation in late childhood and adolescence probably reflects the better cooperation by older children. 24,25 However, the paucity of non-invasive interfaces suitable for small children continues to be an important consideration that guides the choice of approach.

Invasive ventilation was significantly related not only to young age, but also to residence in the south, longer time spent under mechanical ventilation, and neuromuscular disorders or hypoxic (ischemic) encephalopathy. Inability to cooperate, to clear airway secretions, and to swallow are more common in neurological disorders and in younger children, and are contraindications to using non-invasive ventilation. 29,30 The survey showed that children affected by neurological disorders provide the vast majority of patients receiving ventilation via tracheotomy. This finding is consistent with the results of Eurovent study. 3 According to criteria proposed by Australasian consensus statement children requiring invasive ventilation spent longer time per day under mechanical ventilation. 31 Regional differences in the type of ventilation reflect the health, social and organizational conditions and the medical staff expertise in the different settings. In fact, in the South of Italy, the vast majority of patients was discharged from pediatric intensive care units.

While NIV is generally accepted to support patients affected by Duchenne muscular dystrophy and SMA type 2, 27,32 the use of NIV for children with SMA type 1 is less clear and respiratory management differs around the world. 33 The benefits and the burdens of both NIV and IV in children affected by SMA type 1 have to be again adequately evaluate. 34 Different views and attitudes have the clinicians recommending/offering the type of ventilation to the parents of children with SMA type 1. 35,36 Findings suggest that in Italy both NIV and IV are used in this population at a similar rate, and further efforts are needs defining appropriate decisions.

One of the most surprising findings of the survey is the relatively high prevalence of children affected by encephalopathy supported with HMV. This finding is consistent with the results of an other Italian study. 10 However, recent surveys documented that in other countries these patients don't represent one of the most important disorder categories of HMV children. 9,18,21,22 Moreover, this study showed differences also in the type of interface used to ventilate these children. While in the United Kingdom NIV is usually preferred, 37 in Italy the majority of children affected by encephalopathy is invasively ventilated. Both these differences are probably due to a different ethical approach around the world.

The main people involved in home ventilation surveys in UK 18 and Massachusetts 22 are pediatric pulmonologists and general pediatricians, and pediatric intensive care physicians are seldom involved (11% and 1% of children respectively). In the present survey the majority of patients (59%) were discharged from pediatric intensive care units or pediatric pulmonary units, suggesting a different attitude and involvement of Italian general pediatricians in the management of these patients compared to other countries.

As expected, 22,38 pulse oximetry was the preferred monitoring modality at home, during noninvasive and invasive ventilation, particularly for children with neuromuscular disorders. This approach is in agreement with the regimen for managing acute and chronic neuromuscular respiratory failure at home described by Bach and colleagues. 24 Similarly to French survey 9 follow up was performed during outpatient visits in 60% of the patients.

Records of arterial blood gases, nocturnal pulse oximetry and sleep study were the most common applied tests during routine follow up. This finding is consistent with the French study. 9 In our study population uncuffed tracheotomy tubes are preferred over cuffed tubes. This finding is consistent with the literature. In fact, the indication for cuffed tracheotomy tubes are limited (i.e., patients with chronic translaryngeal aspiration, patients requiring ventilation with high pressures). 39 There is no consensus regarding the frequency of tracheotomy tube changes. While recommended tube change frequency ranges from daily to monthly, the most common frequency is weekly. 39 According to the literature, the frequency of tracheotomy tube changes in our population varies widely. However, the majority of patients required a tube change every 16-30 days. According to the literature, in most cases the tube change is performed by the caregiver. In fact, before the home discharge, caregivers should be trained in the replacement of tracheotomy tubes. 39 The majority of patients that required home health care need invasive ventilation for more than 12 hours. This finding is consistent with criteria recommended by Australasian consensus statement. 31 In fact, these children require high level of care and close supervision, which families are unable to sustain without assistance.

Heavy workload, loss of personal time, financial, and emotional resources are the main burden of care related to home care ventilation, falling on parents who, in at least half the cases met all their child's medical needs themselves. Both the hospital discharge and home care need adequate and appropriate approaches. Children with chronic ventilation at home need a coordinated multidisciplinary approach by trained and accountable professionals, parents, and lay people, based on interactions between different teams from the hospital, community, family, and carers. 18,[40][41][42] In our country specific state programs for pediatric home-care are devoted only to children affected by HIV or cystic fibrosis, while a program of respiratory home care for children with chronic respiratory failure have not yet been realized. Specific health care plan is already present only in selected regions. 10,11 There are several limitations to the present survey. Although some children may have been Pressure to reduce the hospital stay of these children and awareness of the effects of long termhospitalization on a child's development have, during the last decade -especially, in a few countries such as UK, France, and US, -increased the numbers of ventilated children discharged home. However, the availability and practical details of complex respiratory support at home involve a lot of variables such as appropriate home environment, healthcare support, and funding, that cannot always be guaranteed. Findings of the Eurovent study, a wide cross sectional survey including adults as well as children, highlighted how the collection of information about home mechanical ventilation (HMV) could facilitate national planning for HMV. In particular, it could aid the development of HMV services in order to ensure equality of provision and access. 3 Surveys in Canada, 4,5 Chile, 6 Germany, 7 France, 8,9 Italy, 10,11 Japan, 12 New Zealand, 13 Switzerland, 14 The Netherlands, 15 Turkey, 16 UK, [17][18][19] and USA 20-- 22 have defined the size, main features, and needs of the pediatric population requiring home mechanical ventilation. However there are many differences between these observational studies: the setting (single referring center, state, or country), the time period (one year or trend over time), the data collected, and how it was collected.

Thus, estimates of the national prevalence of home ventilator-dependent children range from 0.7/100,000 children in 1993 in Japan 12 to 3.4/100,000 in 2000 in Switzerland. 14 Because information on long-term home ventilation for children in Italy is scant and erratic (no regional or national population-based data exist), this survey was conducted to describe the size and

METHODS

Study Population

A questionnaire was sent to all 27 Italian pediatric intensive care and pulmonary units to get data on children up to 17 years old receiving HMV in January 2007. To identify as many children as possible the questionnaire was sent also to another 275 hospital-based units (eg. pulmonary services, and critical care units) of the National Health Service (NHS) that are potentially involved in the care of the target population. Information referred to children discharged home from hospital whorequired tracheostomy or mask ventilation.

Home respiratory care

HMV was defined as invasive mechanical ventilation (IMV) or non invasive mechanical ventilation (NIV), for a period of at least 3 months on a daily basis, carried out mostly in the user's home or other long-term care facility (not a hospital). 3 Basic demographic data (age, sex, place of residence), causes of respiratory failure, hospitalization history, duration of ventilatory support, type of ventilation and management-related description and home care provider information were collected.

Data analysis

Data were entered into a SAS/STAT database (Statistical Analysis Software, Cary, NC) for recording and analysis. The prevalence with 95% confidence intervals (CI) of Italian children ventilated at home on the long term was calculated using the Istat 2007 annual report (Italian National Institute of Statistics) for children up to 17 years old, resident in the provinces in which patients were identified as the denominator. Age was calculated on the basis of the date of birth and the date of onset of ventilation was recorded. Results are presented as frequencies, or median and interquartile range; P < 0.05 was considered significant.

All data collected on centres and patients were kept strictly confidential. Data on patients were anonymous. The San Giovanni Battista-Molinette Hospital, University of Turin, Committee on Clinical Investigation reviewed and approved the study protocol. Because this was an observational study without an intervention and without the presentation of any identifying data, the committee ruled that informed consent was not necessary.

RESULTS

A total of 362 children were identified by 30 1).

The most frequent diagnostic category was neuromuscular disorders (49%), followed by lung and upper respiratory diseases (18%), hypoxic (ischemic) encephalopathy (13%), and abnormal ventilation control (12%) (Table 1). The most represented neuromuscular disorders were spinal muscular atrophy (SMA) (78 patients), Duchenne muscular dystrophy (31 patients) and Congenital muscular dystrophy (29 patients). In particular, 25 children were affected by SMA type 1 ( 16receiving NIV, 64%) and 53 children by SMA type 2 (30 receiving NIV, 57%).

There were wide difference between the type of ventilation and diagnoses ( F = 5.94, P < 0.016).

Time per day spent under mechanical ventilation was longer for children requiring invasive ventilation (χ 2 = 148; P << 0.001) (Table 1), particularly for those with neuromuscular disorders (χ 2 = 73; P << 0.001), hypoxic (ischemic) encephalopathy (χ 2 = 37; P << 0.001), or chronic lung and upper respiratory diseases ( χ 2 = 28; P << 0.001) .

Nurses (for 62 % of children), physiotherapists (20 %), social workers (7 %), psychologists ( The majority of children with a tracheotomy had uncuffed tube (64,2%). The at home tracheotomy tube changes were performed by parents (39% of tracheotomised patients), by intensivists (25%), by nurses (19%), by pulmonologists (10%), or by others (7%). The majority of patients with tracheotomy (63%) required a tube change every 16-30 days.

The medical professionals at the reference center were named as the primary provider for home ventilation management. However, only 48% of children required home health care, mostly those needing invasive ventilation for ≥12 hours daily (χ 2 MH = 46; P << 0.001) (Table 1). Parents were identified as the first care giver for all the children, and in 171 cases (47%), the sole care giver.

Other care givers were different professionals (40% of children), family friends (8%), and non professional volunteers (3%).

Pulse oximetry (saturation) was used as monitoring modality. Cardiovascular/respiratory lead monitoring was done in 10% of patients, particularly younger children (≤ 5 year age), receiving non-invasive ventilation, and with neuromuscular disorders.

Multivariate logistic regression

DISCUSSION

This is the first report about children receiving long-term home ventilation in Italy. Approximately 4 per 100.000 children up to 17 years old had chronic mechanical respiratory support at home in 2007, in good agreement with estimates from other countries. 12,14,21 If the figures are generalized to the whole country, a total 404 children in Italy would be found, supporting the reliability of the present survey involving 362 children. Thus, even if the reported findings do not include all possible cases, they do provide the best available information for this group of children. Patients were resident in 75 of the 107 Italian provinces (70%), and were mainly discharged from centers in the North of Italy, which may be attributed to fewer pediatric services in the South, in terms of quality and quantity. 23 Health inequalities in childhood can lead to distress for the patient and the family, as well as financial waste. Thus, for this group of patients the burden of care is higher for southern residents.

The median length of home ventilation was one year, indicating that hospital discharge of these patients is a recent practice in Italy. In line with surveys from other countries 9,12,16,21 neuromuscular disorders were the most frequent indication for long-term home ventilation, as the established supportive therapy reduces the morbidity and mortality of these children. [24][25][26][27][28] Non-invasive support was used in the majority of the investigated population (59%) and was more prevalent in later infancy and adolescence. This finding is consistent with the literature, 29 invasive mechanical ventilation, the prevalence of non-invasive ventilation in late childhood and adolescence probably reflects the better cooperation by older children. 24,25 However, the paucity of non-invasive interfaces suitable for small children continues to be an important consideration that guides the choice of approach.

Invasive ventilation was significantly related not only to young age, but also to residence in the south, longer time spent under mechanical ventilation, and neuromuscular disorders or hypoxic (ischemic) encephalopathy. Inability to cooperate, to clear airway secretions, and to swallow are more common in neurological disorders and in younger children, and are contraindications to using non-invasive ventilation. 29,30 The survey showed that children affected by neurological disorders provide the vast majority of patients receiving ventilation via tracheotomy. This finding is consistent with the results of Eurovent study. 3 According to criteria proposed by Australasian consensus statement children requiring invasive ventilation spent longer time per day under mechanical ventilation. 31 Regional differences in the type of ventilation reflect the health, social and organizational conditions and the medical staff expertise in the different settings. In fact, in the South of Italy, the vast majority of patients was discharged from pediatric intensive care units.

While NIV is generally accepted to support patients affected by Duchenne muscular dystrophy and SMA type 2, 27,32 the use of NIV for children with SMA type 1 is less clear and respiratory management differs around the world. 33 The benefits and the burdens of both NIV and IV in children affected by SMA type 1 have to be again adequately evaluate. 34 Different views and attitudes have the clinicians recommending/offering the type of ventilation to the parents of children with SMA type 1. 35,36 Findings suggest that in Italy both NIV and IV are used in this population at a similar rate, and further efforts are needs defining appropriate decisions.

One of the most surprising findings of the survey is the relatively high prevalence of children affected by encephalopathy supported with HMV. This finding is consistent with the results of an other Italian study. 10 However, recent surveys documented that in other countries these patients don't represent one of the most important disorder categories of HMV children. 9,18,21,22 Moreover, this study showed differences also in the type of interface used to ventilate these children. While in the United Kingdom NIV is usually preferred, 37 in Italy the majority of children affected by encephalopathy is invasively ventilated. Both these differences are probably due to a different ethical approach around the world.

The main people involved in home ventilation surveys in UK 18 and Massachusetts 22 are pediatric pulmonologists and general pediatricians, and pediatric intensive care physicians are seldom involved (11% and 1% of children respectively). In the present survey the majority of patients (59%) were discharged from pediatric intensive care units or pediatric pulmonary units, suggesting a different attitude and involvement of Italian general pediatricians in the management of these patients compared to other countries.

As expected, 22,38 pulse oximetry was the preferred monitoring modality at home, during noninvasive and invasive ventilation, particularly for children with neuromuscular disorders. This approach is in agreement with the regimen for managing acute and chronic neuromuscular respiratory failure at home described by Bach and colleagues. 24 Similarly to French survey 9 follow up was performed during outpatient visits in 60% of the patients.

Records of arterial blood gases, nocturnal pulse oximetry and sleep study were the most common applied tests during routine follow up. This finding is consistent with the French study. 9 In our study population uncuffed tracheotomy tubes are preferred over cuffed tubes. This finding is consistent with the literature. In fact, the indication for cuffed tracheotomy tubes are limited (i.e., patients with chronic translaryngeal aspiration, patients requiring ventilation with high pressures). 39 There is no consensus regarding the frequency of tracheotomy tube changes. While recommended tube change frequency ranges from daily to monthly, the most common frequency is weekly. 39 According to the literature, the frequency of tracheotomy tube changes in our population varies widely. However, the majority of patients required a tube change every 16-30 days. According to The majority of patients that required home health care need invasive ventilation for more than 12 hours. This finding is consistent with criteria recommended by Australasian consensus statement. 31 In fact, these children require high level of care and close supervision, which families are unable to sustain without assistance.

Heavy workload, loss of personal time, financial, and emotional resources are the main burden of care related to home care ventilation, falling on parents who, in at least half the cases met all their child's medical needs themselves. Both the hospital discharge and home care need adequate and appropriate approaches. Children with chronic ventilation at home need a coordinated multidisciplinary approach by trained and accountable professionals, parents, and lay people, based on interactions between different teams from the hospital, community, family, and carers. 18,[40][41][42] In our country specific state programs for pediatric home-care are devoted only to children affected by HIV or cystic fibrosis, while a program of respiratory home care for children with chronic respiratory failure have not yet been realized. Specific health care plan is already present only in selected regions. 10,11 There are several limitations to the present survey. Although some children may have been

  pediatric Italian population receiving chronic mechanical ventilation at home and the profile of home respiratory care.

6

 6 Descriptive statistics were computed for the entire study population and subgroups. Student's t test was used to compare the type of ventilation and patient's age (continuous variable). χ 2 tests were used to compare the type of ventilation and categorical variables, such as sex and home health care.The Mann-Whitney U test was used to compare the type of ventilation in each class of time/day spent under mechanical ventilation. ANOVA was used to check the association between the type of ventilation and class of diagnosis. Comparisons of the type of ventilation in each region were tested by χ 2 trend analysis. Multivariate logistic regression was used to look at the association between the type of ventilation and all monitored factors, simultaneously.

5 characteristics

 5 of the pediatric Italian population receiving chronic mechanical ventilation at home and the profile of home respiratory care.

6

 6 Descriptive statistics were computed for the entire study population and subgroups. Student's t test was used to compare the type of ventilation and patient's age (continuous variable). χ 2 tests were used to compare the type of ventilation and categorical variables, such as sex and home health care.The Mann-Whitney U test was used to compare the type of ventilation in each class of time/day spent under mechanical ventilation. ANOVA was used to check the association between the type of ventilation and class of diagnosis. Comparisons of the type of ventilation in each region were tested by χ 2 trend analysis. Multivariate logistic regression was used to look at the association between the type of ventilation and all monitored factors, simultaneously.

  SuperscriptDeleted: Interestingly, the results of our study suggest that both NIV and IV are possible option for patients affected by SMA type 1.

Deleted: 4 Deleted: 5 Deleted: 6 Deleted: 6 Page

 4566 in most cases the tube change is performed by the caregiver. In fact, before the home discharge, caregivers should be trained in the replacement of tracheotomy tubes.39

Table 1

 1 hospital units and wards (i.e., adult intensive care units, adult pulmonary units, pediatric neurology units, pediatric palliative care units). The median number of children followed by each center was seven (interquartile range3-19).

	The median age was 8 years (interquartile range 4-14) and was significantly different for children
	receiving invasive or non-invasive ventilation (P << 0.001), as well as for the median age at the
	start of mechanical ventilation (P << 0.001) (
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

). The majority of patients (59%) were discharged from 12 of 27 NHS pediatric intensive care units or pediatric pulmonary units that were following mechanically ventilated children at home, and the others came from 18 other

  ). All children received positive pressure ventilation. The majority of children receives NIV (Table1), which is delivered mostly by nasal mask (85% of patients receiving NIV). Non-invasive ventilator support was the most used and

skewed toward late infancy and adolescence.. Overall, 41% of the survey population had ventilation via tracheotomy with the highest percentage of children affected by neurological disorders. The median duration of home mechanical ventilation was one year (Table

Table

  home-ventilated children could help in monitoring and improving the care of these children (and their families) throughout the country.In conclusion, the present survey provides, for the first time, reliable estimates of the prevalence of children receiving home mechanical ventilation in Italy. The majority of children are noninvasively ventilated. Invasive ventilation is significantly related to younger age, regional residence, more hours on ventilation and diagnosis (i.e., neurological disorders). Further work is needed to promote health program to monitor and improve the wellbeing of these children. However, this is a recognized challenge and technical and political commitments are necessary to provide the needed resources for patients and their families.McCormick MC, Escobar GJ, Richardson DK, Zheng Z, Coleman-Phox K, Roberts R, Zupancic JAF. Neonatal Intensive Care Unit Census Influences Discharge of Moderately Preterm Infants. Pediatrics. 2007; 119:314-319. 39. Sherman JM, Davis S, Albamonte-Petrick S, Chatburn RL, Fitton C, Green C et al. Care of the child with chronic tracheostomy. Am J Respir Crit Care Med. 2000;161(1):297-308. 40. Jardine E, Wallis C. Core guidelines for the discharge home of the child on long-term assisted ventilation in the United Kingdom. UK Working Party on Paediatric Long Term Ventilation. Thorax. 1998;53(9):762-7. 41. Perrin J, Erenberg G, LaCamera Rea. Guidelines for home care of infants, children, and adolescents with chronic disease. American Academy of Pediatrics Committee on Children with Disabilities. Pediatrics.1995;96(1Pt1):161-4. 42. Wang KW, Barnard A. Caregivers' experiences at home with a ventilator-dependent child.

	F o r 38. Profit J, Qual Health Res. 2008;18(4):501-508. national registry of F o r F o r F o r F o r
	P P P P P
	e e e e e
	e r e r e r e r e r
	R R R R R
	e e e e e
	v i e v i e v i e v i e v i e
	w w w w w
	13 16 1

excluded, the data from many different hospital providers give a comprehensive figure of the use of home mechanical ventilation in pediatric patients in Italy. No attempt was made to collect data retrospectively to describe the number of children previously ventilated at home or who either died or were weaned from the ventilator, or switched from invasive to non-invasive ventilation. This will be one of the topics of future investigations. Families' desires and acceptance of home mechanical ventilation may be different, and can be influenced by several reasons and factors that have to be evaluated to guide and support the discharge home. An Italian national ventilator program, and a

TABLE 1 Children's characteristics and type of ventilation Characteristics Invasive ventilation Noninvasive ventilation Total

 1 Continuous advances in neonatal and pediatric intensive care are likely to result in more infants, children, and adolescents with critical, chronic conditions, who survive and who are discharged home, but require long-term technological support.[1][2][3] A growing population of children exists who has chronic respiratory failure due to conditions such as neuromuscular diseases, obstructive sleep apnea, craniofacial abnormalities, or spinal cord injuries, and who require either tracheostomy or mask ventilation.

	No. of subjects	149	213	362
	Male/female, n	87/62	116/97	203/159 a1
	Area of residence b
	Northern Italy	84	115	199
	Central Italy	21	35	56
	Southern Italy	44	63	107
	Age, median (interquartile range), y	6 (3-9)	11 (5-15)	8 (4-14) c1
	Age at start of mechanical ventilation
	median (interquartile rage), y	1 (0-4)	8 (3-13)	4 (1-11) c2
	Time on ventilation support
	median (interquartile range), y	3 (1 -5)	1 (0 -2)	1 (0 -4) c3
	Major diagnostic category d , n
	Neuromuscular disorders	66	112	178
	Chronic lung and upper respiratory diseases	12	52	64
	Hypoxic (ischemic) encephalopathy	31	17	48
	Abnormal ventilation control	25	19	44
	Spinal cord injury	10	1	11
	Chest anomalies	4	12	16
	Other	1	0	1
	Hours/day spent under mechanical ventilation
	<12 / ≥ 12, n	29/120	178/35	207/155 a2
	Ventilation monitoring modality, n
	Pulse oximetry (saturation)	136	144	280
	Cardiovascular/respiratory leads	8	29	37

  hospital units and wards (i.e., adult intensive care units, adult pulmonary units, pediatric neurology units, pediatric palliative care units). The median number of children followed by each center was seven (interquartile range3-19).The median age was 8 years (interquartile range 4-14) and was significantly different for children receiving invasive or non-invasive ventilation (P << 0.001), as well as for the median age at the start of mechanical ventilation (P << 0.001) (Table1). All children received positive pressure ventilation. The majority of children receives NIV (Table1), which is delivered mostly by nasal mask (85% of patients receiving NIV). Non-invasive ventilator support was the most used and skewed toward late infancy and adolescence.. Overall, 41% of the survey population had ventilation via tracheotomy with the highest percentage of children affected by neurological disorders. The median duration of home mechanical ventilation was one year (Table

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	were discharged from 12 of 27 NHS pediatric intensive care units or pediatric pulmonary units that
	were following mechanically ventilated children at home, and the others came from 18 other
	7

centers (67% in the North of Italy) as receiving home mechanical ventilation in January 2007, with an estimated prevalence of 4.2 per 100,000 ≤17 year old children (95% CI 3.8-4.6). Patients were resident in 75 of the 107 Italian provinces (70%); 200 (55%) were resident in the North, 55 (15%) in the Center, and 107 (30%) in the South, in good agreement with the national distribution of children up to 17 years old. No difference was observed between the type of ventilation and the area of residence (Table

1

). The majority of patients (59%)

  6% during outpatient visits) and in a regular monitoring program. At home visits were planned in 60% of patients (36% of patients only during planned hospitalization, 30% only during outpatient visits, 29% with both modalities, 5 and % only at home). The mean number of hospital visits was 2.8/year, while the mean number of at home visits was 27/year. The at home visit

	inpatient visits; program involves general practitioners (63% of patients), hospital physicians (65% of patients),
	nurses (49% of patients), physical therapists (22% of patients). Arterial blood gases (59% of
	patients), sleep study (41% of patients), and nocturnal pulse oximetry (33% of patients), were the
	principal investigations during monitoring program.
	F o r
	P	Deleted: 80
	e e r	Deleted: ) Deleted: 32 Deleted: 31 Deleted: 24 Deleted: 56
	R
	e
	v i e
	w	7 %),
	pediatricians (5%), and other professionals (4%) participated in the home discharge process with
	hospital physicians. All children were involved in a patient ventilator training program (94% during
		8

  which identifies NIV as the preferred ventilation strategy and recommended IV only if required (i.e., inability to swallow, to cooperate, to clear airway secretions). Although, improvements in the technical features of mechanical ventilators and non-invasive interfaces have made a larger proportion of the pediatric population, including very young or small children, suitable for non-

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	10

Table Formatted :

 Formatted excluded, the data from many different hospital providers give a comprehensive figure of the use of home mechanical ventilation in pediatric patients in Italy. No attempt was made to collect data retrospectively to describe the number of children previously ventilated at home or who either died or were weaned from the ventilator, or switched from invasive to non-invasive ventilation. This will be one of the topics of future investigations. Families' desires and acceptance of home mechanical ventilation may be different, and can be influenced by several reasons and factors that have to be evaluated to guide and support the discharge home. An Italian national ventilator program, and aIn conclusion, the present survey provides, for the first time, reliable estimates of the prevalence of children receiving home mechanical ventilation in Italy. The majority of children are noninvasively ventilated. Invasive ventilation is significantly related to younger age, regional residence, more hours on ventilation and diagnosis (i.e., neurological disorders). Further work is needed to promote health program to monitor and improve the wellbeing of these children. However, this is a recognized challenge and technical and political commitments are necessary to provide the needed resources for patients and their families.McCormick MC, Escobar GJ, Richardson DK, Zheng Z, Coleman-Phox K, Roberts R, Zupancic JAF. Neonatal Intensive Care Unit Census Influences Discharge of Moderately Preterm Infants. Pediatrics. 2007; 119:314-319. 39. Sherman JM, Davis S, Albamonte-Petrick S, Chatburn RL, Fitton C, Green C et al. Care of the child with chronic tracheostomy. Am J Respir Crit Care Med. 2000;161(1):297-308. 40. Jardine E, Wallis C. Core guidelines for the discharge home of the child on long-term assisted ventilation in the United Kingdom. UK Working Party on Paediatric Long Term Ventilation. Thorax. 1998;53(9):762-7. 41. Perrin J, Erenberg G, LaCamera Rea. Guidelines for home care of infants, children, and adolescents with chronic disease. American Academy of Pediatrics Committee on Children with Disabilities. Pediatrics.1995;96(1Pt1):161-4. 42. Wang KW, Barnard A. Caregivers' experiences at home with a ventilator-dependent child. Qual Health Res. 2008;18(4):501-508. Indent: Left: 12.6 pt, Tabs: 12.6 pt, Left Indent: Left: 12.6 pt, Tabs: 12.6 pt, Left Formatted: Indent: Left: 12.6 pt, Tabs: 12.6 pt, Left Formatted: Indent: Left: 12.6 pt

	Deleted: 6 Deleted: 37-39 Formatted: Font: Not Bold Formatted: Font: Italic Formatted: Indent: Left: 12.6 pt, Tabs: 12.6 pt, Left Page 38 of 41 Deleted: TABLE 2 Ventilation monitoring modality according to the type of ventilation used ¶ ¶ Modality ... [1] Deleted: FIGURE 1 Age of children receiving home mechanical ventilation (A) and at start of mechanical ventilation (B) ¶ ¶ ¶ ¶ Formatted Page 37 of 41 F o r P e e r R e v i e w national registry of home-ventilated children could help in monitoring and improving the care of these children (and their families) throughout the country. 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 38. Profit J, F o r P e e r R e v i e w TABLE 1 Children's characteristics and type of ventilation Characteristics Invasive ventilation Noninvasive ventilation Total No. of subjects 149 213 362 Male/female, n 87/62 116/97 203/159 a1 Area of residence b Northern Italy 84 115 199 Central Italy 21 35 56 Southern Italy 44 63 107 Age, median (interquartile range), y 6 (3-9) 11 (5-15) 8 (4-14) c1 Age at start of mechanical ventilation median (interquartile rage), y 1 (0-4) 8 (3-13) 4 (1-11) c2 Time on ventilation support median (interquartile range), y 3 (1 -5) 1 (0 -2) 1 (0 -4) c3 Major diagnostic category d , n Neuromuscular disorders 66 112 178 Chronic lung and upper respiratory diseases 12 52 64 Hypoxic (ischemic) encephalopathy 31 17 48 Abnormal ventilation control 25 19 44 Spinal cord injury 10 1 11 Chest anomalies 4 12 16 Other 1 0 1 Hours/day spent under mechanical ventilation <12 / ≥ 12, n 29/120 178/35 207/155 a2 Ventilation monitoring modality, n Pulse oximetry (saturation) 136 144 280 Cardiovascular/respiratory leads 8 29 37 Pediatric Pulmonology F o r P e e r R e v i e w End tidal CO 2 11 0 11 None 3 38 41 Other 4 3 7 Home health care, yes/no, n 121/28 51/162 172/190 a3 a1 : 2 χ =0.55; P > 0.459; a2 : 2 χ = 148; P << 0.001; a3 : 2 χ = 115; P << 0.001 b : 2 trend χ = 0.07; P > 0.80 c Mann-Whitney U test: c1 = -6.147; P << 0.001; c2 : = -8.733; P << 0.001; c3 = 5.873; P << 0.001 d : F= 5.94; P <0.016 Pediatric Pulmonology F o r P e e r R e v i e w Page 39 of 41 Pediatric Pulmonology F o r P e e r R e v i e w <sp><sp>
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Formatted:

TABLE 2

 2 Ventilation monitoring modality according to the type of ventilation usedThe figures do not add up to 100% because some patients used more than one monitoring modality.

	Modality	Invasive	Noninvasive	Total
		ventilation	ventilation
		n (%)	n (%)	n (%)
	Pulse oximetry (saturation)	136 (84)	144 (67)	280 (74)
	Cardiovascular/respiratory leads	8 (5)	29 (14)	37 (10)
	End tidal CO 2	11(7)	0	11 (3)
	None	3 (2)	38 (18)	41 (11)
	Other	4 (2)	3 (1)	7 (2)

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