Daniela Toniolo
email: daniela.toniolo@hsr.it

Rossella Sorice

Silvia Bione

Serena Sansanelli

Sheila Ulivi

Emmanouil Athanasakis

Carmen Lanzara

Teresa Nutile

Cinzia Sala

Carmela Lanzara

Clara Camaschella

Pio D' Adamo

Paolo Gasparini

Marina Ciullo

Association of a variant in the CHRNA5-A3-B4 gene cluster region to

Keywords: Nicotine addiction, CHRNA5-A3-B4 cluster, genetic association

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

Introduction

Cigarette consumption is a major health problem worldwide. It is the cause of about 5 million deaths each year and this number is estimated to double by 2020 1,2 . Tobacco use is a strong risk factor for leading causes of death in the world: a significant proportion of ischemic heart disease, cerebro-vascular disease, respiratory infections, tuberculosis, chronic obstructive pulmonary disease and cancers of the lung, trachea and bronchus are attributable to smoking behavior. Despite its recognized deleterious impact on health, smoking prevalence is very high in many countries with about 22% of the worldwide adult population currently consuming tobacco. Many studies have revealed that genetic factors, together with environmental factors, have a strong impact on smoking-related behavior. A meta-analysis of different twin studies estimated a mean heritability of 0.5 for smoking initiation and of 0.59 for nicotine dependence 3 .. Identification of genetic factors affecting smoking-related behavior is consequently a major interest of medicine today, to assess individual vulnerability and to develop more effective intervention.

Recently, several genome-wide association studies have established the association of one locus on the chromosome 15q25 with nicotine dependence and smoking quantity. This locus implicates a cluster of three genes, CHRNA5, CHRNA3 and CHRNB4, which encode neuronal nicotinic acetylcholine receptor subunits (alpha5, alpha3 and beta4), acting as risk-factors for nicotine dependence or heavy smoking [3][4][5][6][7][8][9][10] . The same variants were also found to be associated with lung cancer and peripheral arterial diseases 5,11-12 . However, it is still debated whether there is a direct effect on disease etiology or an indirect effect through smoking. So far, different variants in the 15q25.1 region, strongly correlated by linkage disequilibrium, were reported as associated with smoking behavior. One of them caused an amino acid change in the CHRNA5 encoded protein but its functional role is still unclear. One study also showed evidence for the presence of at least two independent variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster region contributing to nicotine dependence 6 . In another study, this nicotine dependence locus has been tagged by the single nucleotide polymorphism (SNP) rs1051730, located in the CHRNA3 gene, which has been reported to have an effect on the number of cigarettes smoked per day 5 . This variant has already been associated with nicotine dependence [START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF] in a previous genome-wide study that used the low-quantity smokers as controls.

The main focus of our analysis was to evaluate the influence of the rs1051730 variant on smoking behavior, smoking quantity within current and former smokers and related diseases in three Italian isolated populations, belonging to the Italian Network on Genetic Isolates (http://www.netgene.it/ita/ingi_6.asp)

Materials and Methods

Study participants and data collection

Individuals enrolled in the study belong to three isolated populations from different parts of Italy; the basic characteristics of the three populations have been previously described [START_REF] Sala | Variation of hemoglobin levels in normal Italian populations from genetic isolates[END_REF] . All individuals recruited gave their written informed consent and the study protocol was approved by the ethical committees of the institutions involved. All participants provided a detailed medical and lifestyle history collected through structured questionnaires. The information about the smoking behaviour and smoking quantity was acquired using a specific questionnaire. With regard to the smoking habit, three categories were available: never, former and current smokers. According to the number of cigarettes smoked per day (CPD) the individuals were clustered in two categories: low-dose smokers (10 or fewer cigarettes per day) or high-dose smokers (more than 10 cigarettes per day). A dichotomous trait was derived from the CPD categories and it was used for association analysis: the subjects belonging to the lowdose category were classified as controls and those from the high-dose categories as cases.

For the Val Borbera population information was available for 1007 never smokers, 424 former smokers and 283 current smokers. In this sample all smoking individuals (current and former smokers) were used for clustering in the two CPD categories: 304 individuals were classified as low-dose smokers and 402 people as high-dose smokers.

In the Cilento population we had 861 never smokers, 211 former smokers and 254 current smokers. However, for this sample the CPD information was available only for current smokers generating two CPD categories with 89 light and 165 heavy smokers.

In the Carlantino population, no information was available to distinguish between current and former smokers (=304) then the CPD categories included 191 heavy smokers and 113 light smokers, the total number of never smokers was 806,

Genotyping

Genotypes for SNP rs1051730 were determined by TaqMan genotyping (Applied Biosystems, cat. n. 4351379, assay C___9510307_20). Genotype calls were done with the SDS 1.08 software (Applied Biosystems). Call rates were 98.4, 93.8 and 89.4% respectively for the Val Borbera (1713/1741), Cilento (1326/1414) and Carlantino (1110/1241) populations. SNP genotypes used for LD estimation were obtained from Illumina 370K chip data available for each population study.

Association Analysis

Association analyses were performed using a χ 2 test implemented in PLINK software (http://pngu.mgh.harvard.edu/~purcell/plink/) and an additive genetic model was tested.

The analyses were carried out considering both each population separately and combining the three population all together.

Relatedness between individuals was considered in the association testing taking advantage of genealogical information available for each population. A corrected statistics was obtained by the estimation of a correction factor λ through simulations following the procedure proposed by Thorgeirsson et al . 5 In detail, the genotype of the rs1051730 variant was randomly assigned to the founders of each genealogy using the allele frequency distribution estimated with the BLUE estimator on the data and assuming the Hardy-Weinberg equilibrium for the marker. Genotypes of all nonfounder individuals were then randomly drawn conditional on their parent genotypes Cilento/ValBorbera) and applies a simulation procedure to assess the type I error. For each simulation, the individuals are randomly assigned to the two populations and an empirical statistics is recomputed. In our study 100.000 simulations were applied. smokers definition was increased to 15 CPD, the difference in the A allele frequency among cases and controls resulted more marked (+3.2%) but still not statistically significant.

A comparison of the LD pattern in the variant region across the three population samples was performed to investigate about the lack of association in Carlantino. The LD between 18 SNPs, located approximately 100 kb upstream and downstream the rs1051730 variant, was analyzed. Interesting, LD pattern was significantly different between Carlantino and the other two populations: Val Borbera (pval < 10 -6 ) and Cilento (pvalue= 9 10 -4 ). As expected, no significant difference in the LD pattern was found between Val Borbera and Cilento (pvalue=0.514) (see Supplementary Figure 1). This result suggests that LD difference in Carlantino could be a possible explanation of the association result observed in this population .

In order to increase the power of the analysis the case/control association was also carried out on the combined sample (Val Borbera-Cilento-Carlantino). The Cochran-Mantel-Haenszel was applied to avoid the confounding effects due to the population origin and an OR of 1.26 ( 95% C.I 1.07-1.49, p-value=0.0057) was obtained confirming the association of the variant A with the heavy smoking. This result was strengthened by the Breslow-Day test that supported the hypothesis of the homogeneity of odds ratio giving a p-value of 0.285, data shown in the figure 1.

The effect of the rs1051730 variant in the etiology of some diseases for which smoking is a strong risk-factor was examined. 138 cases of malignant tumors (about one third being breast cancers) were assessed in the three populations and were compared to 3185 not affected individuals. No association was found taking into account sex, age and smoking as covariates in logistic regression analysis. Similarly, no association was found comparing 160 individuals presenting cardiovascular disorders (stroke and myocardial infarction) with 3979 control individuals (data not shown).

Discussion

Genetic factors have been suggested to contribute to individual variations in smoking behavior and several have been identified in recent years through GWA studies and linkage analysis 5-6,8-9, [START_REF] Li | Identifying susceptibility loci for nicotine dependence: 2008 update based on recent genome-wide linkage analyses[END_REF] . Many have been replicated in different populations and genetic factors for smoking cessation, number of cigarettes smoked per day, smoking initiation and nicotine addiction have been confirmed 5-6,8-9, 19, 20 . Here, we report a replication of the rs1051730 variant in the CHRNA5-A3-B4 gene cluster, associated with smoking quantity, in isolated populations from Italy. As shown by recent studies of the European population [START_REF] Novembre | Genes mirror geography within Europe[END_REF] , the Italian population is rather heterogeneous from the genetic point of view, possibly due to its geographical position and its history, characterized by multiple population admixtures. The study of isolated populations from different parts of the country seems therefore a useful tool to define the complexity of the population structure and to highlight genetic differences that may lead to discovery of less common genetic variations 22 .

The A allele of the rs1051730 variant, in all three isolated populations, showed a frequency similar to those described in European and Middle Eastern population [START_REF] Hung | A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25[END_REF] , even if in Cilento sample this variant was less frequent. The association was replicated in two isolates, Val Borbera (in the North) and Cilento (in the South) thus, confirming the association of this variant to smoking quantity also in our country. Interestingly, Carlantino did not show any significant association but only a trend that was stronger if the cut-off of smokers was set at 15 CPD. The lack of association observed in Carlantino could be attributed to a reduced power in this sample. However, the different LD pattern detected in Carlantino in the variant region, suggests that the rs1051730 variant is not the functional variant but is in LD with the causal variant in Cilento and Legend to Fig, 1 Odds ratios and confidence intervals from all studies A forest plot for the three studies with odds ratios (OR) and 95% confidence intervals (95% IC) is shown. A log scale is used on the x-axis. The width of the boxes is proportional to the precision of the study and the ends of the horizontal lines represent lower and upper 95% confidence limits. The left vertical line marks an odds ratio of 1.0, corresponding to any risk. For the Val Borbera and Cilento populations the reported odds ratios are greater than unity indicating an increase in the risk of becoming a heavy smoker. The central diamond corresponds to the odd ratio for the combined sample and it is obtained applying the Cochran-Mantel-Haenszel test. The homogeneity of effects between the three groups is tested by Breslow-Day statistic test.

Table 1 . Association analysis of the rs1051730 variant * >10 cpd/die ^ <= 10 cpd/die ° 10.000 simulations using GeneDrop tool (http://www.stat.washington.edu/thompson/Genepi/). § As lambda lower than 1, no correction was applied to the statistics.

 1

		Sample Size	f A	Chi-square p-value	Λ°	Corrected chi-square p-value
	Val Borbera	Cases* (402) Controls^ (304)	0.423 0.365	6.21 p=0.012	1.05	5.91 p=0.0151
	Cilento	Cases* (165) Controls^ (89)	0.394 0.292	5.21 p=0.022	0.984	5.21 § p=0.022
	Carlantino	Cases* (191) Controls^ (113)	0.421 0.411	1.52 p=0.22	1.01	1.51 p=0.22

Acknowledgments

We are indebted to the inhabitants of the three isolates, Val Borbera, Carlantino and Cilento (Gioi/Cardile and Campora) who, participating to the projects, have made this study possible. We also thank the local administrations, the MD, and the numerous people from each village who helped with data collection. The work was supported by Compagnia di San Paolo, Torino, Cariplo Fundation, Milano, Italian Ministry of Health (Grants Ricerca Corrente and Grants Ricerca Finalizzata, to PG and DT), Telethon Fundation, the Italian Ministry of Universities (FIRB -RBIN064YAT) to MC. RS and TN were supported by a fellowship of Regione Campania, Italy.

22 Kristiansson K, Naukkarinen J, Peltonen L: Isolated populations and complex disease gene identification. Genome Biol 2008; 9:109.

and independently from their phenotype. Only genotypes of individuals available in the real data were considered, all others were set to missing. A number of 100,000 simulations were performed using this gene-dropping method implemented in the Genedrop program from the MORGAN package (http://www.stat.washington.edu/thompson/Genepi/).

For each simulation, a χ 2 test was carried out and the mean of χ 2 distribution was considered as λ. Moreover, for the Cilento sample, the λ was also estimated from genetic data (1073 microsatellite markers distributed in the genome) by applying the genomic control approach [START_REF] Bacanu | Association Studies for Quantitative Traits in Structured Populations[END_REF] .

For the combined sample (Val Borbera-Cilento-Carlantino) the Cochran-Mantel-Haenszel (CMH) and Breslow-Day tests (both implemented in PLINK) were performed.

The CMH provides a test based on an "average" odds ratio adjusted for the possible confounding effects due to population stratification; whereas the Breslow-Day statistic tests for homogeneity of effects between the three groups taking into account the population of origin.

Linkage disequilibrium and HW estimation

The Hardy-Weinberg equilibrium were assessed using the Haploview v. 4.1 program.

For each population the LD pattern and the HW equilibrium were evaluated on a subset of poor-correlated individuals. The LD pattern in the variant region was estimated using 18 SNPs located approximately 200 kb around the rs1051703 variant. The method proposed by Zaykin et al [START_REF] Zaykin | Contrasting linkage-disequilibrium patterns between cases and controls as a novel association-mapping method[END_REF] was applied to compare the LD matrices between each couple of samples (Cilento/ValBorbera, Cilento/Cardile, Valborebera/Cardile). Briefly, this method tests the difference of pair-wise ∆-prime matrix between two groups ( i.e.

Results

Allele frequency of the rs1051730 variant was calculated in each population sample.

Interestingly, the minor allele frequency (f A ) in the Cilento population (f A =0. A comparison of the rs1051730 variant allele frequency between the current and never smokers and between the former and the current smokers was performed and no significant difference was observed (not shown).

A significant difference was evident between low-dose smokers and high-dose smokers.

In Val Borbera and Cilento populations a strong increase of the A allele (+ 5.8 and + 10.2% respectively) was observed among the heavy smokers (Table I). In both cases the enrichment reached the threshold of statistical significance also after correction for relatedness among individuals (Val Borbera pval=0.0151 Cilento pval=0.022 ).

However, as in the Cilento sample the value of λ was lower than 1, it did not affect the original statistics. The λ was estimated by a genealogy-based approach in the three populations. For the Cilento population a genomic-based approach was also used, as described in Materials and Methods. Comparable values of the λ were obtained from the two approaches (0.984 versus 1 from genealogy and genome respectively).

The A allele of rs1051730 resulted only slightly enriched (+1%) among high-dose smokers in the Carlantino population. Interestingly, if the threshold for low/high-dose

Conflict of interest.

The authors declare no conflict of interest.


MD MSc PhD Irene C Joziasse

PhD Kelly A Smith

BSc Sonja Chocron

BSc Maarten Van Dinther

Guryev Victor

Jasper Van Der Smagt

PhD Edwin Cuppen

PhD Peter Ten Dijke

MD PhD Barbara Jm Mulder

PhD Cheryl L Maslen

PhD Victor Guryev

MD Jasper J Van De Smagt

Benjamin Reshey

Bsc

MD PhD Pieter A Doevendans

PhD Jeroen Bakkers
email: j.bakkers@hubrecht.eu

ALK2 mutation in a patient with Down syndrome and a congenital heart defect

Keywords: Down Syndrome, Congenital Heart defects, gene mutation

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

Introduction

Down syndrome (DS) is the most frequent cause of mental retardation, most often caused by trisomy 21 (in approximately 95% of cases). DS occurs in about one in 700-1000 live births [START_REF] Bell | Down's syndrome: occurrence and outcome in the north of England, 1985-99[END_REF][START_REF] Mikkelsen | Incidence study of Down's syndrome in Copenhagen, 1960-1971; with chromosome investigation[END_REF][START_REF] Owens | The incidence of Down's syndrome over a 19-year period with special reference to maternal age[END_REF] . The syndrome is characterized by well-defined and distinctive phenotypic features including characteristic facies, minor limb anomalies, hypotonia and growth retardation [START_REF] Korenberg | Down syndrome phenotypes: the consequences of chromosomal imbalance[END_REF] . Whilst all individuals with DS manifest these phenotypes, there is significant phenotypic variability between DS individuals [START_REF] Wiseman | Down syndrome-recent progress and future prospects[END_REF] . In conjunction with these phenotypic traits, DS individuals suffer a higher incidence of several conditions, including congenital heart defects (CHDs) [START_REF] Wiseman | Down syndrome-recent progress and future prospects[END_REF] .

The (birth) prevalence of congenital heart defects in DS is about 40-60%, most frequently atrioventricular septal defects (AVSD) and ventricular septal defects (VSD) [START_REF] Loffredo | Atrioventricular septal defects: possible etiologic differences between complete and partial defects[END_REF][START_REF] Paladini | The association between congenital heart disease and Down syndrome in prenatal life[END_REF][START_REF] Vis | Down syndrome: a cardiovascular perspective[END_REF] . Efforts have been made to determine the critical chromosomal region for specific phenotypic features of DS by deletion mapping and characterization of patients with partial trisomy 21 [START_REF] Korenberg | Down syndrome phenotypes: the consequences of chromosomal imbalance[END_REF][START_REF] Korenberg | Molecular definition of a region of chromosome 21 that causes features of the Down syndrome phenotype[END_REF][START_REF] Kerstann | Linkage disequilibrium mapping in trisomic populations: analytical approaches and an application to congenital heart defects in Down syndrome[END_REF] . One potential candidate gene for these trisomy 21-related CHD is collagen Type VI. However, the evidence for the involvement of collagen Type VI has, to date, been restricted to correlative expression pattern analysis [START_REF] Klewer | Expression of type VI collagen in the developing mouse heart[END_REF][START_REF] Gittenberger-De Groot | Collagen type VI expression during cardiac development and in human fetuses with trisomy 21[END_REF] .

While this susceptibility and variability of the DS phenotype is widely accepted, the factors contributing to this variability have not been established. It is postulated that variations in gene dosage of chromosome 21, environmental factors and genetic modifications not linked to chromosome 21 account for this variability; and it is likely that contributions from each of these elements are involved. Genetic imbalance caused by the presence of an extra copy of chromosome 21 will seriously disrupt one or more developmental pathways. In addition the presence of non-chromosome 21 loci variation may predispose for a heart defect. To date, CRELD1 (3p25.1) is the only non-chromosome 21 gene that has been reported to associate with DS-related CHDs [START_REF] Maslen | CRELD1 mutations contribute to the occurrence of cardiac atrioventricular septal defects in Down syndrome[END_REF] .

We have previously reported mutations in ALK2, a type I receptor for bone morphogenetic proteins (BMPs), in patients with endocardial cushion-associated CHDs. One of these variants identified has a dominant-interfering effect on BMP signaling and is associated with primum type atrial septal defect (ASD type I) [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] . Here we report an additional ALK2 variant that was identified in a patient with trisomy 21 and a primum type atrial septal defect (ASD type I). In addition to the ALK2 variant, two other variations were detected in another type I BMP receptor gene, ALK3, and in the epidermal growth factor receptor family, ErbB3. Using both in vitro and in vivo assays, we show that the ALK2 variant has reduced BMP-inductive capacity and a mild dominant-interfering effect on BMP signaling. This effect on BMP signaling from the ALK2 variation in conjunction with the additional variations identified in this patient is likely to contribute to the manifestation of a CHD in this individual.

Material and Methods

This study was prospectively reviewed and approved by the Local Research Ethics

Committee of the University Medical Center Utrecht and the Academic Medical Center, Amsterdam, The Netherlands.

Genetic material and clinical evaluation

The patient described in this study was identified in a large-scale candidate gene sequencing screen as previously described [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] . In total 9 candidate genes were sequenced in this patient (ALK2, ALK3, EGFR, GATA4, HAS2, UGDH, ERBB2, ERBB3, CRELD1). Patient material was acquired from the CONCOR (CONgenital CORvitia) database [START_REF] Van Der Velde | CONCOR, an initiative towards a national registry and DNA-bank of patients with congenital heart disease in the Netherlands: rationale, design, and first results[END_REF]

Detection of variants

PCR primers (sequence available upon request) for amplification were designed using an in-house developed management system (http://limstill.niob.knaw.nl), which was also used for the semi-automated identification and classification of mutations [START_REF] Prandini | Natural gene-expression variation in Down syndrome modulates the outcome of gene-dosage imbalance[END_REF] .

Selected genes were analysed by dideoxy sequencing of PCR-amplified exonic fragments.

Cell lines and transfections.

Bovine aortic endothelial cells [START_REF] Hammerschmidt | Dorsoventral patterning in the zebrafish: bone morphogenetic proteins and beyond[END_REF] were used for luciferase assays and cos7 cells were used for construct expression. All transfections were performed using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions.

Constructs, luciferase assay and kinase activity assays.

Expression constructs encoding full-length human ALK2 have been cloned into the pcDNA3 vector as previously described [START_REF] Ten Dijke | Characterization of type I receptors for transforming growth factor-beta and activin[END_REF] and mutant variants were generated by sitedirected mutagenesis using a QuikChange kit (Stratagene, La Jolla, CA, USA). Inserts were fully sequenced and recloned into the original parental vector or the pCS2+ vector (for mRNA synthesis) to ensure against additional mutations. The luciferase assay was performed as previously described [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF] . This included an injection with vector only or in combination with a short hairpin RNA (shRNA), the latter to knockdown endogenous bovine ALK2 but not vector-based human ALK2, as described previously [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] .

Zebrafish lines, mRNA synthesis and injections

Wild type fish were kept under standard conditions as previously described [START_REF] Westerfield | The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)[END_REF] . pCS2+ ALK2 constructs were linearised with NotI enzyme and capped mRNA was prepared with the Message Machine kit (Ambion, Austin, TX, USA). mRNA was diluted to appropriate concentration in nuclease-free water and 1 nl [START_REF] Westerfield | The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)[END_REF] per embryo injected at the 1-2 cell stage. Embryos were imaged live and mounted in methylcellulose.

Statistical analysis

Results are expressed as mean ± SEM. Statistical significance was determined by oneway analysis of variance, followed by a Bonferroni t test using the program SigmaStat version 2.03 (Jandel Scientific, San Rafael, CA).

Results

We recently performed a screen for the detection of cSNPs (coding single nucleotide polymorphisms) in patients with congenital heart defects arising from improper endocardial cushion formation [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] . During this screen we identified a patient with Down syndrome in possession of compound cSNPs in ALK2 resulted in a His286Asp substitution (ALK2 p.His286Asp) (Figure 1b); the ALK3 cSNP caused a Glu414Lys substitution (ALK3 p.Glu414Lys); whilst the cSNP in ERBB3 resulted in a Thr1169Ile substitution (ERBB3 p.Thr1169Ile) (Supplement figure 1). These cSNPs were analyzed with the prediction tools PolyPhen [START_REF] Sunyaev | Towards a structural basis of human nonsynonymous single nucleotide polymorphisms[END_REF][START_REF] Sunyaev | Prediction of deleterious human alleles[END_REF] and SIFT [START_REF] Ng | SIFT: Predicting amino acid changes that affect protein function[END_REF] (Table 1). Only ALK2 p.His286Asp was predicted to damage protein function in both models and was not detected in 350 controls of the Dutch population. The coding regions of a total of 9 genes were sequenced in this individual, including CRELD1, and no other cSNPs were identified. This patient with DS was diagnosed with a primum type atrial septal defect with a cleft mitral valve and the coronary sinus draining into the left atrium (Figure 1e).

Sequencing of DNA from peripheral blood lymphocytes confirmed the 3 cSNPs in this individual (figure 1d). To determine heritability and segregation of the cSNPs, we screened all first degree relatives of the proband for the presence of each substitution (Figure 1d). The father of the proband was found to possess both the ALK2 p.His286Asp and the ALK3 p.Glu414Lys variants whereas the mother was a carrier of the ERBB3 p.Thr1169Ile variant. Of the proband's two siblings, only the sister carried the ALK3 p.Glu414Lys and the ERBB3 p.Thr1169Ile variants whereas the brother was negative for the three cSNPs. None of these relatives were found to have a CHD by cardiac echocardiography.

We utilised our previous experience in functionally characterising ALK2 variants to investigate the ALK2 p.His286Asp variant. Structural modelling of the cytoplasmic domain of the ALK2 receptor demonstrated that H286 resides within the ATP binding pocket of the kinase domain, indicating that a missense substitution at this residue may impair receptor activity (Figure 1a,c). To test this hypothesis, ALK2 p.His286Asp was examined for its capacity to induce BMP-specific transcriptional reporter activity [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF] . In this reporter assay the ALK2 p.His286Asp variant showed significantly lower luciferase activity with and without BMP6 induction compared with wtALK2 (p < 0.05) (Figure 2a). Next, endogenous ALK2 expression was knocked down by an RNAi construct as previously described [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] . Transfection with the ALK2 p.His286Asp variant demonstrated significantly lower levels of luciferase activity in the uninduced state (p< 0.05) but a difference from wtALK2 was not observed upon stimulation with BMP6 (Figure 2b). These results indicate that the ALK2 p.His286Asp receptor is capable of propagating a signal when stimulated with a BMP ligand, but this activity is impaired when the ALK2 p.His286Asp variant is in the presence of the wtALK2 form of the receptor.

To investigate the effect of the ALK2 p.His286Asp variant on receptor function in vivo, synthetic wtALK2 and ALK2 p.His286Asp mRNAs was injected into zebrafish embryos at the single cell stage. Injection of the ALK2 p.His286Asp variant resulted in mild dorsalisation of the zebrafish (C1 in 17% versus 3% of injection with wtALK2; p<0.05) [START_REF] Mullins | Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes[END_REF] , which demonstrates a mild dominant-interfering effect of the ALK2 p.His286Asp variant on BMP signalling in the gastrulating embryo (Figure 3a). As a further measure of the reduction of ALK2 signalling for the ALK2 p.His286Asp variant, rescue experiments were performed in the zebrafish ALK2/8 mutant, lost-a-fin (laf), which is identifiable for its C1 dorsalisation phenotype (Figure 3c). While both wtALK2 and variant ALK2 mRNA were able to rescue the C1 dorsalization phenotype in laf/alk2 embryos, the ALK2 p.His286Asp did so less effectively than wt (51% versus 65%, respectively; p < 0.05; Figure 3a).

Discussion

We recently reported a dominant-negative ALK2 allele in a patient with a congenital heart defect [START_REF] Smith | Identification of a dominant-negative ALK2 allele in a family with congenital heart defects[END_REF] . In the current study we report a patient with Down syndrome and a primum type atrial septal defect who harbours three cSNPs in genes associated with endocardial cushion development. The cSNPs result in missense substitutions in the type I BMP receptors genes, ALK2 and ALK3 resulting in the genetic variants p.His286Asp and p.Glu414Lys, respectively, and in the epidermal growth factor receptor family gene, ERRB3, resulting in the p.Thr1169Ile variant We show that, whilst subtle, there is a statistically significant difference in BMP signalling activity in ALK2 p.His286Asp compared with the wtALK2 allele by both in vitro and in vivo analysis. Additionally, a genetic variant in ALK3 was identified. How these compound substitutions in two distinct type I BMP receptors affect overall BMP signalling was not tested here but could affect BMP signalling to a greater extent when present in a compound manner, since ALK2 and ALK3 can form a heterodimeric complex [START_REF] Little | Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis[END_REF] . Furthermore, it is difficult to predict what adverse effects may arise when occurring in concert with the non-conservative ERBB3 p.Thr1169Ile substitution. What can be determined from the family data is that none of these variants are sufficient to cause a CHD when present in isolation in the absence of trisomy 21.

The three variants were detected in the parents of the probands demonstrating that these variants were not acquired de novo. The father of the proband carries the ALK2 p.His286Asp and ALK3 p.Glu414Lys variants and the sister of the proband harbours the ALK3 p.Glu414Lys and the ERBB3 p.Thr1169Ile variants. Neither of these individuals presented with a CHD, demonstrating that the possession of either of these two combinations of cSNPs is either not sufficient to cause the heart defect or the variants are not completely penetrant.

Our data are consistent with the notion that ALK2 is a susceptibility gene for primum type atrial septal defects, in combination with variants at other loci, or in combination with trisomy 21. This case is also suggestive that additional predisposing genetic factors exist for heart defects in DS patients thus underlining the polygenic nature of these defects. These genetic factors can be identified by large scale candidate gene screens.

  . DNA material was obtained from the patient and first degree relatives by buccal swabs. Family members were clinically evaluated and individuals testing positive for the ALK2, ERBB3 and ALK3 variant were examined by echocardiography. DNA material from patients with non-cardiac-related diseases (Fragile X [n=170], male infertility [n=90] and Rett Syndrome [n=90]) was used for controls. Controls were sourced from the Dutch population which comprises 80% of individuals of European ancestry (according to the Central Bureau of Statistics, The Netherlands, for 2008).

Table 1 .

 1 Predictions of ALK3 and ErbB3 variant using PolyPhen and SIFT.

	PolyPhen	SIFT
	ALK3 p.Glu415Lys Benign variant	Affect protein function
	ErbB3 p.Thr1169Ile Benign variant	Alter protein function (low confidence)

Acknowledgements

We kindly acknowledge the cooperation of the index patient and his family members.

Conflict of interest

All authors state that they have no conflicting interests to declare.


B W M Van Bon

J Balciuniene

G Fruhman

S C S Nagamani

D L Broome

E Cameron

D Martinet

E Roulet

S Jacquemont

J S Beckmann

M Irons

L Potocki

B Lee

S W Cheung

A Patel

M Bellina

A Selicorni

M Silengo

Annalisa Vetro

N V Knoers

N De Leeuw

R Pfundt

B Wolf

P Jira

S Aradhya

P Stankiewicz

H G Brunner

O Zuffardi

S B Selleck

J R Lupski

MD Bert B A De Vries
email: b.devries@antrg.umcn.nl

The phenotype of recurrent 10q22q23 deletions and duplications

Keywords: 10q22.3q23.2, NRG3, BMPR1A, PTEN, GRID1, cerebellum, breast development, cardiac

The genomic architecture of the 10q22q23 region is characterized by two low copy repeats (LCR3 and LCR4) and deletions in this region appear to be rare.

We report the clinical and molecular characterisation of eight novel deletions and six duplications within the 10q22.3q23.3 region. Five deletions and three duplications occur between LCRs 3-4, whereas three deletions and three duplications have unique breakpoints.

Most of the individuals with the LCRs3-4 deletion had developmental delay, mainly affecting speech. In addition, macrocephaly, mild facial dysmorphisms, cerebellar anomalies, cardiac defects and congenital breast aplasia were observed. For congenital breast aplasia the NRG3 gene, known to be involved in early mammary gland development in mice, is a putative candidate gene. For the cardiac defects, BMPR1A and GRID1 are putative candidate genes because of their association with cardiac structure and function.

Duplications between LCRs3-4 are associated with variable phenotypic penetrance.

Probands had speech and/or motor delays and dysmorphisms including a broad forehead, deep-set eyes, upslanting palpebral fissures, a smooth philtrum and a thin upper lip.

In conclusion, duplications between LCRs 3-4 on 10q22.3q23.2 may lead to a distinct facial appearance and delays in speech and motor development. However, the phenotypic spectrum is broad and duplications have also been found in healthy family members of a proband. Reciprocal deletions lead to speech and language delay, mild facial dysmorphisms and, in some individuals, to cerebellar, breast developmental and cardiac defects.

Introduction

Patients with submicroscopic aberration syndromes have traditionally been recognized by a specific combination of clinical features. [START_REF] Schmickel | Contiguous gene syndromes: a component of recognizable syndromes[END_REF] When chromosomal banding techniques became available in the 1970s the cytogenetic basis of many of these syndromes was revealed. [START_REF] Ewart | Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome[END_REF][START_REF] Driscoll | A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11[END_REF][START_REF] Bowen | The critical segment for the Langer-Giedion syndrome: 8q24.11----q24[END_REF][START_REF] Ledbetter | Deletions of chromosome 15 as a cause of the Prader-Willi syndrome[END_REF] The development of subtelomeric fluorescent in situ hybridization (FISH), targeting all telomeres in a single assay, led to a shift from the original 'phenotype first' approach, to a 'genotypefirst' approach. In the absence of a recognisable phenotype, individuals were screened by FISH for novel submicroscopic chromosomal abnormalities. Based on similar genomic aberrations in various patients, the clinical presentation was delineated. A process designated 'reverse phenotypics'. [START_REF] De Vries | Telomeres: a diagnosis at the end of the chromosomes[END_REF] Using new molecular karyotyping techniques such as subtelomeric MLPA and micro-array analysis, reverse phenotypics has proven successful, by the constantly increasing list of microdeletion/microduplication syndromes. [START_REF] Slavotinek | Novel microdeletion syndromes detected by chromosome microarrays[END_REF] Some of these have a recognisable phenotype, such as the 17q21.31 microdeletion syndrome. [START_REF] Shaw-Smith | Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability[END_REF][START_REF] Koolen | Clinical and molecular delineation of the 17q21.31 microdeletion syndrome[END_REF][START_REF] Sharp | Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome[END_REF] Others, such as deletions of chromosome band 1q21.1, 15q13.3 or 16p13.11, give rise to less consistent phenotypes. [START_REF] Brunetti-Pierri | Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities[END_REF][START_REF] Mefford | Recurrent Rearrangements of Chromosome 1q21.1 and Variable Pediatric Phenotypes[END_REF][START_REF] Ullmann | Array CGH identifies reciprocal 16p13.1 duplications and deletions that predispose to autism and/or mental retardation[END_REF][START_REF] Sharp | A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures[END_REF][START_REF] Stefansson | Large recurrent microdeletions associated with schizophrenia[END_REF][START_REF] Stone | Rare chromosomal deletions and duplications increase risk of schizophrenia[END_REF][START_REF] Ben-Shachar | Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders[END_REF][START_REF] Dibbens | Familial and sporadic 15q13.3 microdeletions in Idiopathic Generalized Epilepsy: Precedent for Disorders with Complex Inheritance[END_REF][START_REF] Helbig | 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy[END_REF][START_REF] Hannes | Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant[END_REF][START_REF] Van Bon | Further delineation of the 15q13 microdeletion and duplication syndromes: A clinical spectrum varying from non-pathogenic to a severe outcome[END_REF] These aberrations show incomplete penetrance, as demonstrated by their presence in clinically unaffected relatives. Several of these aberrations are associated with an increased risk of neuropsychiatric disorders, such as schizophrenia and autism. 15;16;22 Chromosomal anomalies, with an unpredictable and inconsistent phenotypic outcome, represent a difficult counselling situation for clinicians. Micro-array analysis during pregnancy is now offered in several countries and prenatal detection of these submicroscopic changes is a particularly challenging counselling situation. [START_REF] Van Den Veyver | Clinical use of array comparative genomic hybridization (aCGH) for prenatal diagnosis in 300 cases[END_REF] Therefore, the need for extensive information about phenotypic outcomes of such recurrent aberrations is essential.

Recurrent deletions of 10q22.3q23.2 have been characterised by cognitive and behavioural abnormalities. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] The 10q22.3q23.2 region is characterised by a complex set of low-copy repeats (LCRs), which can give rise to various genomic changes mediated by non-allelic homologous recombination (NAHR). [START_REF] Lupski | Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes[END_REF] Balciuniene et al. described three probands with interstitial deletions in this region. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] The breakpoints in two of these mapped within these LCRs (LCR3 and LCR4) and inheritance was reported in one. This deletion segregated with a wide range of cognitive and behavioural phenotypes within a large family. The third proband had a complex non-contiguous rearrangement consisting of two deletions with breakpoints in unique genomic sequence, with the most proximal breakpoint between LCR3 -LCR4 and a telomeric breakpoint ~630 kb distal of LCR4. A considerable overlap of cognitive and behavioural phenotypes was noted among probands and affected family members. The presence of LCRs in this region suggests that this locus has an increased susceptibility to chromosomal rearrangements. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] In contrast to expectations based on the chromosomal architecture defined by LCR3 and LCR4, only six novel deletions and no duplications comprising this region have been reported. [START_REF] Alliman | Clinical and molecular characterization of individuals with recurrent genomic disorder at 10q22.3q23.2[END_REF] Of these, four were clinically assessed.

In this report we describe the clinical and molecular characterisation of eight novel deletions and six duplications within the 10q22.3q23.3 region. Five deletions and three duplications occur between LCRs 3 and 4, whereas three deletions and three duplications have unique breakpoints.

Methods

Patients and DNA samples

In this study, 14 individuals with an aberration in 10q22q23 were studied. DNA was isolated according to standard procedures. Except for individual 4, who had a 47,XYY karyotype, all others had normal karyotypes by G-banded chromosome studies. Clinical information was obtained from the respective physicians.

The submicroscopic aberrations were detected by array analysis using different array platforms.

All deletions were fine mapped using high-resolution, custom-tiled oligonucleotide CGH arrays. These were designed, manufactured, processed and analyzed by Roche NimbleGen Systems, Inc (Madison, WI). Except for patient 3, all patients were analyzed using arrays (design ID 2794) that contained 385,000 isothermal oligonucleotide probes fine-tiled across the human chromosome 10 region from 44 to 92 Mb (human genome reference sequence version hg18). Data analysis included 371,000 "unique" probes providing an average coverage of one probe every 130 nucleotides. A probe was considered "unique" if it differed by at least five nucleotides from homologous hits retrieved by a Blastn search against the entire human genome. Less stringent criteria for "uniqueness" were applied for probes in LCR sequences, and probes showing only one nucleotide mismatch to paralogous sequences were included for the analysis. Patient 3 was analyzed using a different design array (design ID 5621). The chromosome 10 region from 77 Mb to 92 Mb (human genome reference sequence version hg18) was finely tiled with 68,291 probes which yielded a mean coverage of one probe per 210 nucleotides. All probes on this array were "unique" based on the aforementioned criteria. Each sample was subjected to one hybridization experiment, except for patient 2 whose DNA was subjected to two independent hybridization arrays. Test DNA samples were labelled with Cy3 and paired with a reference DNA sample labelled with Cy5.

We used a human DNA reference sample (Promega) provided by Roche NimbleGen Systems which is a pool of genomic DNA from six anonymous males. Probe hybridization signals were expressed as the log2 ratio of signal intensities of a test sample versus signal intensities of a reference sample. Array data were analyzed with the segMNT or DNACopy algorithms. More detailed description of probe design, array construction, DNA labelling, hybridization and data analysis can be found on Roche Nimblegen Systems webpage (http://www.nimblegen.com/products/lit/index.html).

Copy Number variations (CNVs) with breakpoints mapping within LCRs, the automated segmentation algorithm performed poorly. The breakpoints assigned by analyses of different averaging windows were inconsistent and fell within large genomic intervals up to 5 Mb in length. Instead, we visualized the data using our previously published colour binning method and compared the data of our patients with three previously obtained control samples. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] For Patient 2, log 2 ratios from two independent experiments were averaged and the averaged values were used for colour binning analysis. Breakpoint genomic coordinates provided in the result section represent our best estimate and are in reference to the hg18 version of the Human Genome assembly (http://genome.ucsc.edu/).

Results

Molecular findings

The breakpoints of the deletions and duplications are shown in figure 1 and figure 2, respectively. In addition to the aberration in the 10q22q23 region, patient 3 had a de novo 722 kb gain in 2q36.3q36.

Clinical data

The clinical findings of the patients are described in detail in the supplementary information.

The clinical features of probands 1-5 with an LCRs3-4 deletion are summarized in table 1 and of probands 10-12 with an LCRs3-4 duplication in table 2. The latter table also includes clinical features of patient 9, who had a largely overlapping duplication with breakpoints located in unique sequences, outside LCRs 3 and 4. The clinical features of probands 6-8 and of probands 13-14 are displayed in tables 3 and 4 respectively. Photographs of six patients (cases 1, 3, 4, 9, 10 and 11), for whom the parents consented for publication, are shown in figure 3.

Discussion

In recent years, many recurrent rearrangements have been added to the increasing list of genomic disorders [START_REF] Lupski | Genomic disorders ten years on[END_REF] including deletions in the 10q22.3q23.2 region. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] The breakpoints of the this deletion are flanked by low copy repeats (LCRs). LCR3 is flanking the proximal breakpoint and harbors two large (>300 kb) highly homologous (99.8% identity) segmental duplications.

They are composed of smaller modules with different orientations that are dispersed elsewhere on chromosome 10 and on other chromosomes. LCR4 is flanking the distal breakpoint and contains ~170kb of sequence homologous to LCR3, as well as >100 kb of sequence homologous to LCRs located near the chromosome 10 centromere. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] Based on this genomic architecture, an increased susceptibility of chromosomal rearrangements between LCR 3 and 4 can be expected in this region. However, since the description of two deletions by Balciuniene et al. in 2007 only six additional cases have recently been reported. 24;26 The additional five new cases with the LCR3-4 deletion in the current study were detected within a cohort of 30,991 individuals with MR and/or multiple congenital anomalies, leading to a frequency of 0.016%. This is a much lower frequency than reported for other genomic disorders, such as Williams syndrome (0.31 %) [START_REF] Stevenson | Genetic syndromes among individuals with mental retardation[END_REF] , proximal 16p11.2 deletions (0.6 %) [START_REF] Walters | A new highly penetrant form of obesity due to deletions on chromosome 16p11.2[END_REF] , 17q21.31 deletions (0.64 %) [START_REF] Koolen | Clinical and molecular delineation of the 17q21.31 microdeletion syndrome[END_REF] and 15q13.3 deletions (0.24 %). [START_REF] Van Bon | Further delineation of the 15q13 microdeletion and duplication syndromes: A clinical spectrum varying from non-pathogenic to a severe outcome[END_REF] Based on genomic architecture, the lower frequency might be explained by the ~7 Mb unique sequence between the LCRs in the 10q22.3q23.2 region, which is much larger than the distance between segmental duplications in other LCR-mediated recurrent rearrangements reported thus far. The distance between two LCRs is known to be one of the genomic architectural features that influences the efficiency of NAHR. [START_REF] Lupski | Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits[END_REF] In fact, based on the 18 new genomic disorders described since 2005, recently reviewed by Mefford and Eichler, the distance between LCR3 and LCR4 on 10q22.3q23.2 is the largest in size (Figure 4). [START_REF] Mefford | Duplication hotspots, rare genomic disorders, and common disease[END_REF] Except for the 16p11.2p12.2 deletion, having a size of 7 Mb, all aberrations are less than 2.5 Mb. However, the latter study did not include previously described LCR-mediated syndromes delineated through a 'phenotype first approach'. Some of these also include intermediate size deletions, such as the Williams (MIM # 19450), Velo-Cardio-Facial (MIM # 192430), Smith-Magenis (MIM # 182290), Prader-Willi (MIM # 176270) and Angelman (MIM # 105830) syndromes (Figure 5). [START_REF] Mefford | Duplication hotspots, rare genomic disorders, and common disease[END_REF][START_REF] Shaffer | The identification of microdeletion syndromes and other chromosome abnormalities: cytogenetic methods of the past, new technologies for the future[END_REF][START_REF] Bayes | Mutational mechanisms of Williams-Beuren syndrome deletions[END_REF][START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF][START_REF] Shaw | Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2[END_REF][START_REF] Makoff | Detailed analysis of 15q11-q14 sequence corrects errors and gaps in the public access sequence to fully reveal large segmental duplications at breakpoints for Prader-Willi, Angelman, and inv dup(15) syndromes[END_REF] In addition, it has been observed that larger-sized genomic rearrangements, using LCRs positioned further apart, are regularly associated with larger LCRs. [START_REF] Lupski | Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits[END_REF] Although the aberration on 10q22.3q23.2 is largest in size, its LCRs are intermediate in size compared to LCRs in other genomic rearrangements.

The low frequency of 10q22.3q23.2 deletions among MR/MCA individuals may reflect lack of ascertainment because of the mild phenotype in some individuals with this deletion. Balciuniene et al. reported multiple family members having this deletion, which segregated with a wide range of cognitive and behavioural phenotypes, including learning difficulties, speech-and language delay, ADHD and autism. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] Individuals with a similar mild phenotype will often not be tested for submicroscopic aberrations. Similar phenotypic variability has been described for other rearrangements, such as on chromosomes 1q21.1, 15q13.3 and 16p13.11. All aberrations are enriched in affected persons compared to control individuals. 12-16;18-21 A less likely explanation for the low frequency might be that 10q22.3q23.2 deletions in foetuses often lead to miscarriages. Although miscarriages were not reported in our families, the family reported by Balciuniene et al. showed spontaneous abortions and a stillbirth. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] In addition to the six clinically reported 10q22.3q23.2 cases, we present the clinical features of five novel patients with this deletion. Of these 11 deletions, seven had arisen de novo, two were inherited and of two the inheritance remained unknown. Two patients had additional chromosome abnormalities. The phenotype of patient 2 might have been influenced by the additional de novo duplication of chromosome 2q36.3. However, this aberration has also been reported in a healthy father of an affected individual (DECIPHER database ID 248304) and several partially overlapping duplications in this region have been reported in the Database of Genomic Variants. The phenotype of patient 4 might have been influenced by the 47,XYY karyotype. In general, individuals with an XYY karyotype have lower IQ scores, mainly verbally, expected for their social background, but not impaired in relation to general population norms. In addition, problems with anger control and attention problems have been described. [START_REF] Leggett | Neurocognitive outcomes of individuals with a sex chromosome trisomy: XXX, XYY, or XXY: a systematic review[END_REF] Cognitive development was impaired in all 11 probands varying from mild to moderate impairment. All patients had apparent speech and language problems. Three patients also had motor developmental delay, but their speech was more severely affected than their motor development.

Patients were mildly dysmorphic and low set ears, hypertelorism and a flat nasal bridge were frequently noted. Head circumference ranged from the 3 rd centile to above the 97 th centile, but the majority of patients were macrocephalic.

Behavioural problems were present in some but non-overlapping in type; Two patients had autism 24;26 , two patients showed hyperactivity [START_REF] Alliman | Clinical and molecular characterization of individuals with recurrent genomic disorder at 10q22.3q23.2[END_REF] and patient 4 had aggressive behaviour. In the latter this may have been due his 47,XYY karyotype.

Two patients had cerebellar anomalies; low-lying cerebellar tonsils in patient 3 and a retrocerebellar cyst with a small cerebellum in patient JHU10qDel-01 reported by Balciuniene et al. [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF] Patient 1 had unilateral congenital breast aplasia. Except for two patients, all other LCR3-LCR4 deletion probands were male. Patient 2 and 4 and case 1 of Alliman et al. had a cardiac defect (AVSD,tricuspid and pulmonic regurgitation and persistent ductus arteriosus respectively) and patient 3 had epilepsy. An AVSD was also noted in patient 7 and epilepsy in patient 8. However, these two latter patients had smaller intragenic deletions, located in unique sequences.

The breakpoints of the deletion in proband 6 were also located in unique sequences, including LCR4 and the PTEN gene. This patient had macrocephaly and juvenile colonic polyps, common features of contiguous gene deletions including tumor suppression genes BMPR1A and PTEN. [START_REF] Delnatte | Contiguous gene deletion within chromosome arm 10q is associated with juvenile polyposis of infancy, reflecting cooperation between the BMPR1A and PTEN tumor-suppressor genes[END_REF] Germline loss-of-function heterozygous mutations in PTEN have been described, causing an overlapping phenotype including macrocephaly and gastrointestinal polyps in patients with Cowden and Banayan-Riley-Ruvalcaba syndrome. [START_REF] Eng | PTEN: one gene, many syndromes[END_REF] Furthermore, heterozygous germ line loss-of-function mutations in BMPR1A cause juvenile polyposis. [START_REF] Howe | Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis[END_REF] Three currently reported patients (2,4 and 7) had a cardiac defect, indicating that a dosagesensitive gene or genes involved in the pathogenesis of cardiac defects may be located in the 10q22.3q23.2 region. There are two interesting candidate genes located in this region, BMPR1A and GRID1. BMPR1A is deleted in patients 3 and 4 and in all individuals who have been reported with overlapping deletions and cardiac septal defects. [START_REF] Shapiro | Deletions of the long arm of chromosome 10[END_REF][START_REF] Menko | Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes[END_REF][START_REF] Salviati | Deletion of PTEN and BMPR1A on chromosome 10q23 is not always associated with juvenile polyposis of infancy[END_REF][START_REF] Zigman | Localization of the Bannayan-Riley-Ruvalcaba syndrome gene to chromosome 10q23[END_REF][START_REF] Sweet | Molecular classification of patients with unexplained hamartomatous and hyperplastic polyposis[END_REF] Of these deletions, at least one did not include the GRID1 gene. [START_REF] Menko | Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes[END_REF] Cardiac-specific deletion of BMPR1A disrupts cardiac morphogenesis in mice, showing ventricular septum, trabeculae, compact myocardium and endocardial cushion defects. [START_REF] Gaussin | Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3[END_REF] BMPR1A was not deleted in patient 7 with a cardiac defect, who had an intragenic deletion of GRID1, which is located only about 500 kb upstream of BMPR1A. The inheritance of this deletion was unknown, hampering conclusions on pathogenicity. Similar deletions have not been reported in the Database of Genomic Variants nor in the chromosomal databases DECIPHER and ECARUCA. GRID1 encodes for a subunit of glutamate receptor channels, which play a role in mediating excitatory synaptic transmission in the central nervous system. [START_REF] Yamazaki | Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family[END_REF] The deletion in patient 7 removes exons 5-8 of the GRID1 gene. The removal of these exons would lead to an in-frame protein that would partly lack the extracellular ligandbinding domain and therefore, may represent a dominant-negative mutation. A recent meta-analysis of genome wide association data, aimed to identify common genetic variants associated with cardiac structure and function, proposed GRID1 as a candidate gene for left ventricle wall thickness. [START_REF] Vasan | Genetic variants associated with cardiac structure and function: a meta-analysis and replication of genome-wide association data[END_REF] Remarkably, this glutamatergic gene has also been associated with entirely different pathology, namely schizophrenia. [START_REF] Treutlein | Dissection of phenotype reveals possible association between schizophrenia and Glutamate Receptor Delta 1 (GRID1) gene promoter[END_REF][START_REF] Guo | A case-control association study between the GRID1 gene and schizophrenia in the Chinese Northern Han population[END_REF][START_REF] Zhu | A MicroRNA gene is hosted in an intron of a schizophrenia-susceptibility gene[END_REF][START_REF] Fallin | Bipolar I disorder and schizophrenia: a 440single-nucleotide polymorphism screen of 64 candidate genes among Ashkenazi Jewish caseparent trios[END_REF] The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system [START_REF] Monaghan | The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system[END_REF] and changes in this system are the basis of the 'glutamate hypothesis' aiming to explain symptoms of schizophrenia. 54;55 After finding linkage evidence for schizophrenia in 10q22 in different populations, [START_REF] Chen | Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia[END_REF][START_REF] Fallin | Genomewide linkage scan for schizophrenia susceptibility loci among Ashkenazi Jewish families shows evidence of linkage on chromosome 10q22[END_REF][START_REF] Faraone | Genome scan of Han Chinese schizophrenia families from Taiwan: confirmation of linkage to 10q22.3[END_REF][START_REF] Liu | Evidence for a putative bipolar disorder locus on 2p13-16 and other potential loci on 4q31, 7q34, 8q13, 9q31, 10q21-24, 13q32, 14q21 and 17q11-12[END_REF] GRID1 was implicated as being the causative gene in several association studies. [START_REF] Treutlein | Dissection of phenotype reveals possible association between schizophrenia and Glutamate Receptor Delta 1 (GRID1) gene promoter[END_REF][START_REF] Guo | A case-control association study between the GRID1 gene and schizophrenia in the Chinese Northern Han population[END_REF][START_REF] Zhu | A MicroRNA gene is hosted in an intron of a schizophrenia-susceptibility gene[END_REF][START_REF] Fallin | Bipolar I disorder and schizophrenia: a 440single-nucleotide polymorphism screen of 64 candidate genes among Ashkenazi Jewish caseparent trios[END_REF] Another gene in the 10q22 region, associated with schizophrenia risk and presentation, is [START_REF] Chen | Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia[END_REF] This gene is located 2.6 Mb proximal of GRID1 and affected in copy number variation in twelve patients in this study. Neuregulins are signalling proteins that mediate cell-cell interactions in the nervous system, heart, breast and other organ systems. [START_REF] Falls | Neuregulins: functions, forms, and signaling strategies[END_REF] Although several family members of proband 8 had a psychiatric disorder, the intragenic deletion found in the proband and his father was not segregating in the rest of the family. In addition, this intragenic deletion minimally overlaps a CNV (loss) described in two healthy individuals [START_REF] Shaikh | High-resolution mapping and analysis of copy number variations in the human genome: a data resource for clinical and research applications[END_REF] and in our patient 7; however, it extends beyond the CNV telomeric boundary. Therefore, the pathogenic significance of this CNV remains uncertain.

NRG3 (Neuregulin 3).
Taking into account that most probands in this study are still young, definitive evidence linking haploinsufficiency of NRG3 or GRID1 to psychiatric disease could not be established.

NRG3 is also a candidate gene for congenital breast aplasia, which was present in patient 1.

NRG3 has been implicated to play a role in early mammary gland development, and Nrg3 signalling in mice promotes differentiation of mammary epithelial cells at the initial stages of organ formation. Mice harbouring a hypomorphic allele of Nrg3, exhibit defects in mammary gland formation, such as hypoplastic and supernumerary mammary gland formation. 62;63 NRG3 was also involved in two patients with an intragenic NRG3 duplication and congenital anomalies. Patient 13 had vision problems, most likely due to prematurity whereas patient 14 had Tetralogy of Fallot, hypospadias and ambiguous genitalia. Both patients inherited the duplication from an unaffected parent. Although a possible role in organ development cannot be excluded, these duplications did not influence neurocognitive functioning and are most likely non-pathogenic variants.

Three patients (10-12) had a duplication between LCR3 and LCR4. Patient 9 had a duplication that did not involve LCR4, but shared a 5 Mb overlap with the three LCR3-4 duplications.

Sibpair patients 10 and 11 inherited this duplication from their healthy mother. The duplication in patient 12 occurred de novo. This patient also had a de novo 16p13.11 deletion which has been reported as a risk factor, strongly predisposing for, but not suffice to cause autism, epilepsy and mental retardation. 13;20 Duplications involving the 10q22-q23 region are rare. To date, only four cases overlapping this region have been reported. [START_REF] Erdogan | Fine mapping of a de novo interstitial 10q22-q23 duplication in a patient with congenital heart disease and microcephaly[END_REF][START_REF] Dufke | De novostructural chromosomal imbalances: molecular cytogenetic characterization of partial trisomies[END_REF][START_REF] Han | Partial trisomy of chromosome 10(q22-q24) due to maternal insertional translocation (15;10)[END_REF][START_REF] Goss | Duplication 10q22.1-q25.1 due to intrachromosomal insertion: a second case[END_REF] Two of these patients had microcephaly and three had cardiac defects. Precise genotypephenotype correlations are hampered because these aberrations have often been detected by conventional cytogenetic techniques. In addition, except for one case reported by Dufke and colleagues, [START_REF] Dufke | De novostructural chromosomal imbalances: molecular cytogenetic characterization of partial trisomies[END_REF] As could be noted in the healthy mother of sibpair patients 10 and 11, duplication of the LCRs3-4 region does not always lead to an abnormal phenotype. Incomplete penetrance of chromosomal aberrations is a well-known phenomenon, which has been described for several abnormalities such as 1q21.1, 15q13.3, 16p13.11 and 22q11.2 aberrations, 11-14;20;21;68- 71 with reciprocal duplications of the same interval often less penetrant. 72;73 To determine the full spectrum and penetrance of the LCRs3-4 duplication, more information about probands carrying this duplication and especially information about other carriers in their families are needed.

In conclusion, duplications of the LCR3-LCR4 region on 10q22.3q23.2 are associated with variable phenotypic penetrance. In affected individuals these aberrations may lead to a distinct facial appearance and developmental speech and/or motor function delays.

Deletions between LCRs 3-4 are clinically penetrant in all cases reported thus far. These deletions are associated with cognitive impairment, mainly affecting speech development, and cardiac defects. In addition, these deletions may also be associated with cerebellar and breast developmental defects.

Database of Genomic Variants: http://projects.tcag.ca/variation/?source=hg18 European Cytogeneticists Association Registered Unbalanced Chromosome Aberrations www.ecaruca.net Frequency of LCR mediated micro-deletions in cohorts of individuals with MR, multiple congenital anomalies and/or autism (Y-axis) 9;12;20;21;29;32;74;75 compared to the size of unique sequence between LCRs (x-axis). [START_REF] Mefford | Duplication hotspots, rare genomic disorders, and common disease[END_REF][START_REF] Shaffer | The identification of microdeletion syndromes and other chromosome abnormalities: cytogenetic methods of the past, new technologies for the future[END_REF][START_REF] Bayes | Mutational mechanisms of Williams-Beuren syndrome deletions[END_REF][START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF][START_REF] Shaw | Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2[END_REF][START_REF] Makoff | Detailed analysis of 15q11-q14 sequence corrects errors and gaps in the public access sequence to fully reveal large segmental duplications at breakpoints for Prader-Willi, Angelman, and inv dup(15) syndromes[END_REF] q 1 1 . 2 1 q 1 1 . 2 2 q 1 1 . 2 3 q 2 1 . 1 q 2 1 . 2 q 2 1 . 3 q 2 2 . 1 q 2 2 . 2 q 2 2 . 3 q 2 3 . 1 q 2 3 . 2 q 2 3 . 3 1 q 2 3 . 3 3 q 2 4 . 1 q 2 4 . 2 q 2 5 . 1 q 2 5 . 2 q 2 5 . 3 q 2 6 . 1 1 q 2 6 . 1 3 q 2 6 . 2 q 2 6 . 3 q 2 4 .

Legends to the figures and tables

  these duplications were much larger in size. Patients 9 and 12 displayed speech delay and impaired social interaction. Sibpair patients 10 and 11 were mentally retarded. Probands 9-11 and the patient described by Dufke et al. had a strikingly similar facial appearance including a broad forehead, deep-set eyes, upslanting palpebral fissures, a smooth philtrum and a thin upper lip.

Figure 1 .

 1 Figure 1. Schematic representation of 10q genomic rearrangements identified in the probands. A. Structure of LCRs located in 10q22.3-q23.2 is shown. 24 Blocks of the same color and/or pattern denote paralogous sequences. Degree of sequence identity between paralogous sequences ranges from 90.8% to 99.8%. Genomic position (in Mb) is shown on a scale below the LCRs and corresponds to human reference human genome reference sequence version hg18. B. Map of deletions predicted by oligonucleotide CGH arrays in patients 1-8 and of previously published individuals JHU10qDel-01, JHU10qDel-02, UM10qDel-01. 24 Grey bar indicates intact DNA sequence. Red dashed line indicates hemizygous deletion. Grey dashed line denotes genomic area that contains a breakpoint. Genes located in this genomic region are shown below the diagram of the rearrangements.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

  WBS = Williams-Beuren Syndrome, PWS = Prader Willi Syndrome, AS = Angelman syndrome, SMS = Smith-Magenis syndrome, VCF = Velo-Cardio-Facial syndrome. Frequencies of PW/AS and VCF syndromes were corrected for the most common deletion causing these syndromes.

Table 1 . clinical features in literature cases and probands with a LCRs3-4 10q22.3q23.3 deletion. UM10qDel-01 and JHU10qDel-01 from Balciuniene et al.²² Case 1-4 from Alliman et al. ²⁶ AVSD=atrial ventricular septal defect, PDA=persistent ductus arteriosus, upsl/downsl palp fissures= upward/downslanting slanting palpebral fissures u = unknown

 1

		1/89.2	81.6-89.1/89.3	81.6-88.9	81.6-88.9	81.6-88.9	81.6-88.9	81.6-88.9/89.1	81.6-88.9/89.1	81.4-89.1/89.3	81.6-88.6/89.1	81.6-89.1
	LCR involvement	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4
	Inheritance	inherited mat	u	de novo	de novo	de novo	de novo	de novo	u	de novo	de novo	inherited mat
	Additional aberrations									gain 2q36.3q36.3	47,XYY
	Developmental delay	+	+	+	borderline		+	+	+	+	+	+
	Autism	+	-	+				-	-	-		-
	Speech delay	+	NA	+	+		+	-	+	+	+	+
	Hyperactivity				+			-	-	+		-
	Seizures	-	-					-	-	+	-	-
	OFC	P97	P97 at birth				>P97	P10	P3	P10	>P97	P84
	Height	u	P10-P90				P50	P10	<P3		>P97	P30
	Cardiac evaluation	u	u	PDA				normal	AVSD	normal	tricuspid regurgitation normal
											pulmonic regurgitation
	Brain anomalies	u	retrocerebellar cyst	u			-	u	u	Chiari I malformation	-	u
			small cerebellum
			wide ventricles
	Dysmorphisms &	minor features	white forelock	micrognathia	mild facial asymmetry	hypertelorism	frontal bossing	ptosis	telecanthus	dolichocephaly	long face	low set ears
	congenital anomalies			thin upper lip	prognathia	mild epicanthal folds	narrow philtrum	low set small ears	low set ears	low-set prominent ears hypertelorism	hypertelorism
				mild hypertelorism	downsl palp fissures	low-set ears	retrognathia	hypotelorism	hypertelorism	hypertelorism	radioulnar synostosis	broad base of nose
				upsl palp fissures	mild arachnodactyly	narrow ear canals	small mouth	upsl palp fissures	anteverted nares	epicanthal folds	scoliosis	flat nasal bridge
				earlobe creases	joint hyperextensibility bilateral club feet	downsl palp fissures	broad thumbs	flat nasal bridge	flat nasal bridge	kyphosis	shawl scrotum
				overfolding lateral pinna		bilateral hearing loss	hypertelorism	broad halluxes	large mouth	midface retraction	pectus excavatum	club feet
				arachnodactyly			small ears	breast aplasia		high arched palate	café-au-lait spots
				joint hyperextensibility						maligned teeth
										retroplaced thumbs

Table 1 . clinical features in literature cases and probands with a LCRs3-4 10q22.3q23.3 deletion AVSD=atrial ventricular septal defect, PDA=persistent ductus arteriosus, upsl/downsl palp fissures= upward/downslanting slant u = unknown, *brain anomalies in the same person Patient 2

 1

		Patient 3	Patient 4	Patient 5
	81.6-88.9/89.1	81.4-89.1/89.3	81.6-88.6/89.1	81.6-89.1
	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4
	u	de novo	de novo	inherited mat
		gain 2q36.3q36.3	47,XYY
	+	+	+	+
	-	-		-
	+	+	+	+
	-	+		-
	-	+	-	-
	P3	P10	>P97	P84
	<P3		>P97	P30
	AVSD	normal	tricuspid regurgitation	normal
			pulmonic regurgitation
	u	Chiari I malformation	-	u
	telecanthus	dolichocephaly	long face	low set ears
	low set ears	low-set prominent ears hypertelorism	hypertelorism
	hypertelorism	hypertelorism	radioulnar synostosis	broad base of nose
	anteverted nares	epicanthal folds	scoliosis	flat nasal bridge
	flat nasal bridge	flat nasal bridge	kyphosis	shawl scrotum
	large mouth	midface retraction	pectus excavatum	club feet
		high arched palate	café-au-lait spots
		maligned teeth
		retroplaced thumbs
	10q22.3q23

.3 deletion upsl/downsl palp fissures= upward/downslanting slanting palpebral fissures Patient 1

			Patient 2	Patient 3
	Deletion position (Mb) 81.6-88.9/89.1	81.6-88.9/89.1	81.4-89.1/89.3
	LCR involvement	LCR3 -LCR4	LCR3 -LCR4	LCR3 -LCR4
	Inheritance	de novo	u	de novo
	Additional aberrations			gain 2q36.3q36.3
	Developmental delay	+	+	+
	Autism	-	-	-
	Speech delay	-	+	+
	Hyperactivity	-	-	+
	Seizures	-	-	+
	OFC	P10	P3	P10
	Height	P10	<P3
	Cardiac evaluation	normal	AVSD	normal
	Brain anomalies	u	u	Chiari I malformation
	Dysmorphisms &	ptosis	telecanthus	dolichocephaly
	congenital anomalies	low set small ears	low set ears	low-set prominent ears
		hypotelorism	hypertelorism	hypertelorism
		upsl palp fissures	anteverted nares	epicanthal folds
		broad thumbs	flat nasal bridge	flat nasal bridge
		broad halluxes	large mouth	midface retraction
		breast aplasia		high arched palate
				maligned teeth
				retroplaced thumbs

Table 1 . clinical features in literature cases and probands with a LCRs3-4 10q22.3q23.3 deletion AVSD=atrial ventricular septal defect, PDA=persistent ductus arteriosus, upsl/downsl palp fissures= upward/downslanting u = unknown Patient 4

 1

		Patient 5
	81.6-88.6/89.1	81.6-89.1
	LCR3 -LCR4	LCR3 -LCR4
	de novo	inherited mat
	47,XYY
	+	+
		-
	+	+
		-
	-	-
	>P97	P84
	>P97	P30
	tricuspid regurgitation	normal
	pulmonic regurgitation
	-	u
	long face	low set ears
	hypertelorism	hypertelorism
	radioulnar synostosis	broad base of nose
	scoliosis	flat nasal bridge
	kyphosis	shawl scrotum
	pectus excavatum	club feet
	café-au-lait spots

ssures= upward/downslanting slanting palpebral fissures

Acknowledgments

We thank all the parents and children who have participated in this study. This work was supported by a grant from the European commission: AnEUploidy project (LSHG-CT-2006-037627) under FP6 (B.v.B., H.B., B.B.A.d.V.), supplemental grants from the Netherlands Organisation for Health Research and Development (ZonMW 917-86-319 to B.B.A.d.V), Hersenstichting Nederland (B.B.A.d.V.), Cure Autism Now (SBS), the Harrison Endowment and Autism Initiative Funds (JB, SBS).

Conflict of interest.

The authors declare no conflict of interest.

Web Resources

Human Genome Browser: http://genome.ucsc.edu/cgi-bin/hgGateway (