A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms

Lisa S Mathew
Manuel Spannagl
†
Ameena Al-Malki
Binu George
Maria F Torres
Eman K Al-Dous
Eman K Al-Azwani
Emad Hussein
Sweety Mathew
Klaus Fx Mayer
Yasmin Ali Mohamoud
Karsten Suhre
Joel A Malek
A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms
R E S E A R C H A R T I C L E Open Access
Background:The date palm is one of the oldest cultivated fruit trees. It is critical in many ways to cultures in arid lands by providing highly nutritious fruit while surviving extreme heat and environmental conditions. Despite its importance from antiquity, few genetic resources are available for improving the productivity and development of the dioecious date palm. To date there has been no genetic map and no sex chromosome has been identified.Results: Here we present the first genetic map for date palm and identify the putative date palm sex chromosome. We placed~4000 markers on the map using nearly 1200 framework markers spanning a total of 1293 cM. We have integrated the genetic map, derived from the Khalas cultivar, with the draft genome and placed up to 19% of the draft genome sequence scaffolds onto linkage groups for the first time. This analysis revealed approximately~1.9 cM/Mb on the map. Comparison of the date palm linkage groups revealed significant long-range synteny to oil palm. Analysis of the date palm sex-determination region suggests it is telomeric on linkage group 12 and recombination is not suppressed in the full chromosome. Conclusions: Based on a modified gentoyping-by-sequencing approach we have overcome challenges due to lack of genetic resources and provide the first genetic map for date palm. Combined with the recent draft genome sequence of the same cultivar, this resource offers a critical new tool for date palm biotechnology, palm comparative genomics and a better understanding of sex chromosome development in the palms.

Background

The date palm (Phoenix dactylifera L.) is a critical crop tree in a large section of North Africa, the Middle East and South Asia. It is one of 3 commercially important palms (Arecaceae) that include the monoecious oil and coconut palms. The date palm is dioecious with separate male and female trees. Only the female bears the commercially important date fruit. Trees grown from seed require approximately 6-8 years to flower before gender can be determined and no sex chromosome has been identified [1]. Sex determination in plants is highly heterogeneous and sex chromosomes in the palms appear to have developed more than once offering an excellent model for studying this process [2,3].

Despite the clear importance of the date palm, few genetic resources exist mainly due to a long generation time of approximately 6 years. A breeding program began in the United States in the 1940's resulted in backcrossed varieties that exhibited depressed breeding ability and other physiological problems [4]. Previous research into the number of chromosomes in date palm suggests it has 18 chromosome pairs (2n = 36) [5] though some evidence for alternate numbers have been presented [6,7]. Technological advances allowed us to generate a draft sequence of a commercial cultivar of date palm [8] that was recently improved on [9] and revealed a genome size of approximately 670 Mb. The draft sequence, while critical to understanding gene content and polymorphism, does not provide structural information of the genome. We were able to use the draft sequence to identify scaffolds containing polymorphisms segregating with date palm gender but other studies such as a search for selective sweeps and quantitative trait loci (QTL) would benefit from a genetic map.

Construction of a genetic map in the absence of defined genetic resources is challenging. Specifically, outcrossed pedigrees require the identification of sufficient polymorphic markers between the male and female parents to allow map construction [10]. While date palm females, which bears the fruit, are well defined by cultivar, the males are much less so as they are simply used for pollination. The introduction of genotyping-by-sequencing (GBS) was important in overcoming these challenges and provides a method for genotyping thousands of new markers in potentially hundreds of offspring [11,12]. Accuracy in the GBS data is critical in our case as errors would result in lack of linkage or highly inflated map distances. We have adapted the method to our requirements by using a reduced representation method of GBS. We employed this adapted approach as each marker needed to be accurately genotyped by high depth sequencing rather than summed over a region as by Andolfatto and colleagues [13,14]. Here we present a first genetic map for date palm and its comparison to the other commercially important palms.

Results and discussion

Genotyping

Using various genotype quality requirements (see Methods below) such as minimum coverage and alignment scores, a total of 64,783 Single Nucleotide Polymorphisms (SNPs) were considered as genotyped at high quality. Using this method, comparison of 'genotypes between two Khalas control libraries carried separately through the entire process revealed a genotype accuracy rate of 99.4%. Our previous genome sequence and SNP data from the Khalas cultivar allowed detection of male parent specific SNPs where Khalas was known to be homozygous. These male parent-specific SNPs were used to segregate progeny by which male parent they derived from. Four major populations derived from the same female parent but different male parents were selected for use in the genetic map and contained 29, 24, 17, and 15 individuals.

We obtained 5873 markers that fit our quality requirements, showed similar segregation in the populations and were called in at least 53 of the 85 individuals genotyped. Markers that shared the same segregation pattern across all progeny were grouped and a single representative marker was selected from each group. This collapsing resulted in 1235 representative markers of which 1199 were placed on the framework map. Markers placed on the map represented as few as one marker (especially male/female segregating markers) and as many as 28 markers. The 1199 representative markers represent 3976 total markers and these derive from 2115 unique segregating parent/scaffold combinations. That is, a scaffold could share two markers but this was only allowed if the markers segregated in different parents. The mapped marker set derived from 1823 scaffolds and these scaffolds span a total of 74,079,588 bp or~20% of the 380 Mb of sequence in the assembly.

Genetic map features

The total genetic map length was 1293 centimorgans (cM) (Figure 1). Assuming a genome size of approximately 670 Mb leads to a genetic to physical distance conversion of approximately 1.93 cM/Mb of sequence or 520 kb/cM. Inter-marker distances were evenly distributed ( Figure 2) though some biases were observed. The largest gap between any two markers was 9.7 cM on linkage group (LG) 6. Both LG5 and LG10 had an excess of gaps greater than 3 cM with a total of 9 and 7 respectively. Distribution of marker types among the linkage groups showed pronounced marker type bias in LG5, 6, 9, and 10. As an example, LG9 shows essentially no markers heterozygous in both parents or heterozygous in the male parent for 50 cM between cM 20 and the end of the linkage group. Long stretches on the map without certain marker types are likely due to not enough heterozygous markers genotyped in both parents. Markers heterozygous in both parents are necessary to integrate markers homozygous in only one parent into the map. Markers heterozygous in both parents were least likely to be captured given the requirement that they had to be heterozygous in all males used to create the mapping populations. Additionally, as the cross used for the map was taken from commercial cultivar, it is possible that there are indeed large stretches of homozygosity that decrease the chance for capturing those marker types. This both highlights the challenges of genetic mapping with outcrossed resources and the necessity of next-generation sequencing approaches to achieve high enough density to allow construction of a genetic map. The markers in the map derived from 1823 unique sequence scaffolds in the draft genome sequence. If each scaffold is considered to provide a single marker, this provides a unique marker every~210 kb of the genome sequence.

The exact number of date palm chromosomes has been uncertain with some publications reporting 14, 16, or 18 pairs [6,7] with most evidence pointing to 18 pairs [5]. Our results agree well with Al Salih and Al-Rawi's findings of both chromosome number and predicted length [7]. In their study of the Ashgar cultivar they detected 18 chromosome pairs and measured them with microscopy. To compare our genetic map we scaled lengths of our date palm linkage groups by adjusting the largest linkage group's centimorgan length to their longest chromosome measured in micrometers ( Figure 3). This revealed very good consistency between our genetic linkage group lengths to the physical lengths of the chromosomes. Indeed, only LG10 and 11 were significantly outside the standard deviation of physical measurements for the chromosomes. This discrepancy may be due to unplaced markers on the end of our linkage groups.

Sex chromosome features

Previous karyotypic analysis of the date palm genome has not identified a specific sex chromosome [1]. We observed that markers from scaffolds previously segregating with gender were all localized to the lower half of LG12 suggesting this may be the sex chromosome. We checked for the density of SNPs segregating with gender in all scaffolds and indeed the highest density was found in LG12 ( Figure 4). This region segregating with gender is approximately 26 cM in length indicating the total physical length may be as long as 13 Mb or 2% of the genome. This is larger than we originally predicted [8] and may be due to the fact we utilized all markers in this region rather than collapsing to high-quality filtered representative markers as for the rest of the genome (see Methods). The use of non-quality filtered markers could lead to inclusion of genotyping errors and an inflation of the region's size through a false increase in recombination frequency. Using markers from LG12 mapped to the more complete Saudi date palm genome (discussed below) scaffolds spanning 4.3 Mb were obtained. If the recent draft sequence is indeed~82% complete, this would suggest the region is approximately 5.2 Mb. Additional work will be required to determine the exact non-recombining boundaries and would benefit from large clone sequencing such as bacterial artificial chromosome libraries. Comparison of the gender segregating region to the oil palm genome suggests that it may be rearranged in date palm. Indeed, markers from LG12 of date palm mostly mapped to Chr10 of oil palm though the sex determination region showed additional shorter regions of consecutive markers matching to oil palm chromosomes 1, 2 and 5 ( Figures 5 and 6b). More detailed analysis at the DNA sequence level should shed light on why the date palm is dioecious despite sharing such high synteny and sequence similarity with the monoecious oil palm.

Comparison to the recently updated draft sequence

Markers were mapped to the Saudi date palm genome [9] by BLAST [15] alignment of~1000 bp surrounding the SNP. To be considered, BLAST alignments were required to be unique in the genome. 3884 (98%) of the marker regions uniquely aligned to 702 scaffolds in the Saudi date palm sequence. These 702 scaffolds span~304 Mb (54%) of the 563.5 Mb assembled genome.

High synteny in palm genome structure

To study palm genome structure, sequence surrounding markers on the genetic map were aligned to the recently The distance between adjacent markers on the genetic map were plotted as a histogram. A majority of markers are within 1 cM of each other, however, a tail of markers extends to 9.7 cM. This is likely due to bias of the method for certain gene dense regions as it relies on alignment to a reference sequence assembled from short read alignments. Figure 3 Comparison of centimorgan lengths of linkage groups to physically measured date palm chromosomes. Al-Salih and Al-Rawi [7] reported the chromosome lengths (μm) of the date palm Ashgar cultivar utilizing microscopy. We plotted their reported lengths and the centimorgan based lengths of linkage groups reported in this study for the Khalas cultivar. The scaled lengths from the two methods show very good correlation over all. Differences in lengths may be due to differences in cultivar type or incomplete extension of the linkage group from unincorporated markers. released oil palm genome [16]. 2000 bp of sequence surrounding the markers were aligned to the oil palm scaffolds and oil palm chromosome locations were documented. It is clear from this comparison ( Figure 5) that the two genomes maintain high levels of synteny. Indeed most of the 18 date palm linkage group were syntenic with one of the 16 oil palm chromosomes ( Figure 6). How the two genomes diverged from 16 to 18 chromosomes is of interest. Based on telomere repeats detected in putative oil palm chromosome centromeres, it has been suggested that a robertsonian fusion caused the collapse of chromosomes into 2 and 14 in oil palm [16]. Our data supports this theory for oil palm chromosome 2. Synteny determined between the date palm genetic map and the oil palm chromosomes suggests that oil palm chromosome 2 constitutes a fusion of date palm chromosomes 1 and 10 whereas date palm LG11 shows blocks shared with oil palm chromosomes 1, 6 and 10. In addition, longer stretches on oil palm chromosome 1 correspond to regions on date palm chromosomes 4 and 16.

The same analysis approach was used to compare available sequence markers from the coconut genetic map [17,18] to the oil palm genome. While only 45 markers from the coconut map could be unambiguously aligned to the oil palm genome, a similar pattern appears with most markers from each coconut linkage group aligning to the same oil palm genome (Figure 7).

Despite the fact that the oil palm genome is more than double the size (1.535 Gb) of the date palm genome (0.67 Gb), long-range synteny is maintained and overall chromosome structure is very well preserved. It will be of interest to understand if the oil palm has added nearly double the sequence to the genome while maintaining chromosome structure or the date palm has lost ancestral DNA.

Conclusions

We provide a first genetic map for the commercially important date palm tree and strong evidence for identifying the sex chromosome. Using next-generation sequencing we have overcome challenges due to lack of genetic resources for this tree. While markers on our map are not guaranteed to be polymorphic in other mapping populations, they nevertheless provide ordering of scaffolds for improved genome analysis. Additionally, by using SNPs from sequence scaffolds, other polymorphic markers within the same scaffolds could be selected for other populations. The high density of the markers on the map sets a target for future date palm genome sequence projects. Future research should aim for greater than 500 kb average scaffold length to ensure that most of the genome sequences will be properly ordered on the genetic map. To resolve more markers onto the map, it will be critical to expand these approaches with a larger pedigree (such as 200 offspring) derived from a single male and female parent. Extending the methods presented here will allow for an even denser genetic map of date palm and an improved resource for quantitative trait locus mapping.

For the first time we have localized the gender segregating region in date palm to LG12 suggesting this region may contain the sex determination genetic alteration. Using the genetic map we estimate the size of the region to be approximately 5 to 13 Mb and more work will be needed to ensure the true recombination boundaries. Identification of the gender segregating region will facilitate future studies on dioecy and the development of sex chromosomes. Indeed, our comparison of the region to the monoecious oil palm revealed shorter blocks of synteny in this region than the rest of the genome.

Analysis of the genetic map reveals important features in the date, oil and coconut palm genomes including maintenance of long range synteny despite dramatic genome size differences. Analysis of the genetic maps combined with genomic data will assist in the comparison and improvements of these 3 commercially important palms.

Methods

Genotyping-by-Sequencing

Date palm seeds were collected from a single Khalas cultivar individual at the agricultural affairs department farm in Rawdat Al-Faras, Al-Ghuwayriyah, Qatar. Seeds were washed and germinated at 29 C for 1 week and then transferred to potting soil. They were grown with minimal watering until primary leaves were~15 cm. Leaf samples of 10 cm were collected for DNA extraction. Leaf material from each plant was arrayed into individual wells of 96-well plates, crushed with the TissueLyser II (Qiagen), and DNA extracted with the DNeasy 96 Plant Kit (Qiagen) according to the manufacturer's protocol. As control for the full process, Khalas (female parent) leaves replaced two individuals in the 96 well plate. DNA was quantified and normalized using a Nanodrop (Ther-moScientific). Genotyping-by-sequencing (GBS) libraries were constructed according to a modified protocol of Elshire and colleagues [11]. Samples were processed in batches of 24 to match barcodes available. Briefly, 150 ng of DNA per sample were digested with 1.25 units of ApeKI (New Engand Biolabs, USA) and then ligated to 10pmol of a universal PCR adapter and one of 24 possible barcoded adapters. DNA from the 24 samples, each with unique barcodes, was then pooled and cleaned with 1.2X Agencourt AMPure XP beads (Beckman Coulter, USA). To increase coverage of fewer SNPs and allow high quality calling of heterozygous alleles, a reduced-representation approach was used. Specifically, after pooling of 24 samples, the libraries were size selected on a 1.5% agarose gel from 350-550 bp using the automated Pippin Prep system (Sage Science, USA). Size selected samples were then amplified by PCR for 16 cycles and cleaned with a 1.2X Agencourt AMPure XP (Beckman Coulter, USA). Pools containing 24 libraries were sequenced on separate lanes of the HiSeq2000 (Illumina, USA) according to the manufacturer's paired-end sequencing protocol. Five lanes of high-quality data representing 120 individuals were collected. Libraries were 'spiked' with 20% of a balanced nucleotide library to improve base-calling within the barcode.

DNA sequences were separated for each barcoded library, matched to the Date Palm V3 genome [8] using Figure 7 Synteny between date palm genetic map linkage group 18, coconut sequence markers and oil palm chromosome sequences. Similar to Figure 5 using coconut genetic map to compare to the oil palm. Coconut genetic map markers were mapped to scaffolds in the date palm genetic map. Linkage groups (LG) were colored by the most likely orthologous chromosome (chr) with orthology based on frequency of shared markers. Date palm LG1 is added for reference.

BOWTIE2 [19]. SNPs were then called with SAMTOOLS [20] to generate a VCF file. To ensure high-quality genotype calling, we required a SNP contain at least 15X coverage to be called. We required a SNP to be called in at least 80% of individuals to be considered. Individuals were then clustered in the Partek Genomics Suite (Partek, USA) using~4000 SNPs that segregated in the male parent to determine their respective population. To detect the sex chromosome markers in the clustered data with similar inheritance patterns as markers in scaffolds previously shown to segregate with sex [8] were collected and added to the set of markers used to construct the map. That is, even single occurrence markers were used. While this may increase error rates in the region it provides higher numbers of mapped gender related scaffolds for future analysis.

Genetic map construction

Utilizing the collected female parent genotype data we inferred male parent data by looking for 1:1 or 1:2:1 segregation patterns in the 4 populations. SNPs that shared segregation patterns in all populations were selected for use in the genetic map. Different markers having the same genotypes in all individuals were collapsed and the marker with the highest call-rate was selected as a representative. In the case of markers known to segregate male and female [8,21], we used all markers that clustered with markers on scaffolds known to segregate with sex (see above).

Genotypes were phased separately for each population and all populations merged using TMAP [22]. Twopoints recombination data and LOD scores were calculated using CarthaGene [23]. Markers were grouped and ordered using JoinMap 4.0 [24]. Grouping was conducted with a LOD score of 10, the regression algorithm was used for marker ordering and the Kosambi function was used in marker distance calculation.

To study the sex control region, we utilized 5 genomes sequenced in our original study [8] including Khalas, Deglet Noor, Deglet Noor Backcross 5 male, Medjool, and Medjool Backcross 4 male. We documented SNPs that were homozygous in all females while heterozygous in both males. We then plotted the counts of these SNPs per 100 kb of scaffolds within a 5 cM sliding window and step size of 2.5 cM (Figure 4).

Syntenic relationships between date palm genetic map and oil palm finished genome sequence

To determine syntenic relationships between the genetic map of Phoenix dactylifera (date palm) [8] and its close relative Elaeis guineensis (pisifera fruit form; oilpalm) we first downloaded the latest genome sequence release of oilpalm [16] from http://genomsawit.mpob.gov.my (16 EG5 chromosome sequences, without unanchored scaffolds) and extracted the position of 4850 distinct genetic markers from the datepalm genetic map (Additional file 1). From their position on a date palm sequence scaffold 1,000 bp upstream and downstream sequences were extracted for each marker where possible. In case of conflicts with a scaffold end or start we extracted sequence from the SNP until the beginning or end of the scaffold. BLASTN with an e-value cutoff of 10e-05 was used to identify homologous regions on the oil palm chromosomes for the 2000 bp marker-flanking date palm sequences. For a total of 4513 (out of 4850) date palm markers one or more homologous regions were found on the oil palm chromosome sequences; in case of multiple hits, only best blast hits (BBH) were considered for downstream analyses (in case of identical BBH bitscores all hits with this bitscore were considered).

The syntenic relationships established between date palm genetic map and oil palm chromosome sequences were plotted using the CIRCOS software [25] and are illustrated in Figure 5. Chromosomes with strong syntenic relations between date palm and oil palm were assigned the same color.

Syntenic relationships between date palm genetic map, oil palm genome sequence and coconut genetic markers

To determine syntenic relationships between date palm, oil palm and coconut, markers from coconut were downloaded from GENBANK (http://www.ncbi.nlm.nih.gov) selecting only microsatellite DNA markers with significant (~500 bp) surrounding sequence. Location of the downloaded markers on the coconut genetic map was obtained from http://tropgenedb.cirad.fr/.

Fifty six coconut markers were associated with genomic sequence. BLASTN with an e-value cutoff of 10e-05 was used to identify homologous regions on the oil palm chromosomes for the coconut marker sequences. For a total of 45 (out of 56) coconut markers one or more homologous regions were found on the oil palm chromosome sequences; in case of multiple hits, only Best Blast Hits (BBH) were considered for downstream analyses (in case of identical BBH bitscores all hits with this bitscore were considered).

The syntenic relationships established between date palm genetic map, coconut markers and oil palm chromosome sequences were plotted individually for all 18 date palm linkage groups using the CIRCOS software [25] in Figure 7. Chromosomes with strong syntenic relations between date palm and coconut palm were assigned the same color.

Availability of supporting data

The draft genome sequence used in this research is available at: http://qatar-weill.cornell.edu/research/date palmGenome/download.html.

Figure 1
1A genetic map of date palm. The consensus genetic map of a cross between Khalas female and 4 different male parents. Linkage groups were sorted by size and marker types were assigned colors. Red: heterozygous in female parent and homozygous in male parent; Green: heterozygous in both parents; Blue: heterozygous in the male parent and homozygous in the female parent; Purple: markers selected as linked to gender.

Figure 2
2Date palm genetic map Inter-marker distance plot.

Figure 4
4Male/Female segregating single nucleotide polymorphism (SNP) density in selected linkage groups. SNPs segregating with gender in 3 female and 2 male genomes were plotted. A 5 cM sliding window was used to determine the density of segregating SNPs per 100 kb and this was plotted as a red line next to the linkage group.LG12 contains the only region of high density segregating SNPs in the genome, suggesting it is indeed the sex chromosome. Localization of the sex determination region to a portion of the chromosome suggests the rest of the chromosome is still freely recombining.

Figure 5
5Synteny between date palm genetic map and oil palm chromosomes. Sequence surrounding markers from the date palm genetic map were aligned to oil palm chromosomes and locations reported. Linkage groups (LG) were colored by the most likely orthologous chromosome (chr) with orthology based on frequency of shared markers.

Figure 6
6Schematic overview of syntenically conserved blocks between the date palm genetic map and oil palm chromosome sequences as identified by CIRCOS plot infigure 5. Sequence surrounding markers from the date palm genetic map were aligned to oil palm chromosomes and locations reported on the (a) oil palm chromosomes (chr) or (b) date palm linkage groups (LG). Different color codes indicate syntenic blocks between the individual date palm linkage groups and corresponding regions on oil palm chromosomes as identified in CIRCOSFigures 5 and 7. In case of regions on oil palm chromosomes with indication of significant syntenic relationships to more than one distinct oil palm linkage group, split color bars were used. Areas on oil palm chromosomes with no support for any syntenic relation to date palm or showing promiscuous relations are colored in white.
http://www.biomedcentral.com/1471-2164/15/285
AcknowledgementsFunding for this research was provided by Qatar Foundation's National Priorities Research Program -Exceptional Award (NPRP-EP) NPRPX-014-4-001.And at http://www.ncbi.nlm.nih.gov/genbank under the accession number: ACYX00000000.Additional fileAdditional file 1: The date palm genetic map, representative markers and SNP marker locations. Date Palm Genetic Map and Marker information. A spreadsheet containing all representative marker locations on the genetic map, the corresponding date palm sequence scaffold and location the SNP marker was derived from and all markers included in the representative group.Competing interestThe authors declare no competing interests.
Towards sex determination of date palm. S A Bekheet, M S Hanafy, Date palm biotechnology, XVIII. Jain SM, Al-Khayri JM, Johnson DVNew York, New York, USASpringerBekheet SA, Hanafy MS: Towards sex determination of date palm. In Date palm biotechnology, XVIII, 551-566. Edited by Jain SM, Al-Khayri JM, Johnson DV. New York, New York, USA: Springer; 2011.

Phylogenetic Analysis of Dioecy in Monocotyledons. G Weiblen, R Oyama, M Donoghue, Am Nat. 155Weiblen G, Oyama R, Donoghue M: Phylogenetic Analysis of Dioecy in Monocotyledons. Am Nat 2000, 155:46-58.

Sex chromosomes in land plants. R Ming, A Bendahmane, S S Renner, Annu Rev Plant Biol. 62Ming R, Bendahmane A, Renner SS: Sex chromosomes in land plants. Annu Rev Plant Biol 2011, 62:485-514.

The Date Palm (Phoenix dactylifera L.): Overview of Biology, Uses, and Cultivation. C T Chao, R R Krueger, HortScience. 425Chao CT, Krueger RR: The Date Palm (Phoenix dactylifera L.): Overview of Biology, Uses, and Cultivation. HortScience 2007, 42(5):1077-1082.

Cytological Studies in the Genus Phoenix. J M Beal, Bot Gaz. 99Beal JM: Cytological Studies in the Genus Phoenix. Bot Gaz 1937, 99:400-407.

. On, Status, Chromosomes, The, Palm (phoenix Dactylifera L, ON THE STATUS OF CHROMOSOMES OF THE DATE PALM (PHOENIX DACTYLIFERA L). http://www.actahort.org/books/882/882_28.htm.

A study of the cytology of two female cultivars of date palm. A A Al-Salih, Al-Rawi Ama, Date Palm J. 52Al-Salih AA, Al-Rawi AMA: A study of the cytology of two female cultivars of date palm. Date Palm J 1987, 5(2):123-142.

De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). E K Al-Dous, B George, Al-Mahmoud Me, Al-Jaber , M Y Wang, H Salameh, Y M Al-Azwani, E K Chaluvadi, S Pontaroli, A C Debarry, J Arondel, V Ohlrogge, J Saie, I J Suliman-Elmeer, K M Bennetzen, J L Kruegger, R R Malek, J A , Nat Biotechnol. 29Al-Dous EK, George B, Al-Mahmoud ME, Al-Jaber MY, Wang H, Salameh YM, Al-Azwani EK, Chaluvadi S, Pontaroli AC, Debarry J, Arondel V, Ohlrogge J, Saie IJ, Suliman-Elmeer KM, Bennetzen JL, Kruegger RR, Malek JA: De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat Biotechnol 2011, 29:521-527.

Genome sequence of the date palm Phoenix dactylifera L. Al-Mssallem Is Hu, S Zhang, X Lin, Q Liu, W Tan, J Yu, X Liu, J Pan, L Zhang, T Yin, Y Xin, C Wu, H Zhang, G , Ba Abdullah, M M Huang, D Fang, Y Alnakhli, Y O Jia, S Yin, A Alhuzimi, E M Alsaihati, B A Al-Owayyed, S A Zhao, D Zhang, S Al-Otaibi, N A Sun, G Majrashi, M A Li, F Tala, Nat Commun. 42274Al-Mssallem IS, Hu S, Zhang X, Lin Q, Liu W, Tan J, Yu X, Liu J, Pan L, Zhang T, Yin Y, Xin C, Wu H, Zhang G, Ba Abdullah MM, Huang D, Fang Y, Alnakhli YO, Jia S, Yin A, Alhuzimi EM, Alsaihati BA, Al-Owayyed SA, Zhao D, Zhang S, Al-Otaibi NA, Sun G, Majrashi MA, Li F, Tala, et al: Genome sequence of the date palm Phoenix dactylifera L. Nat Commun 2013, 4:2274.

Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. D Grattapaglia, R Sederoff, Genetics. 137Grattapaglia D, Sederoff R: Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics 1994, 137:1121-1137.

A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. R J Elshire, J C Glaubitz, Q Sun, J A Poland, K Kawamoto, E S Buckler, S E Mitchell, PLoS One. 619379Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE: A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 2011, 6:e19379.

Genome-wide genetic marker discovery and genotyping using next-generation sequencing. J W Davey, P A Hohenlohe, P D Etter, J Q Boone, J M Catchen, M L Blaxter, Nat Rev Genet. 12Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML: Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet 2011, 12:499-510.

Marker density and read depth for genotyping populations using genotyping-by-sequencing. T M Beissinger, C N Hirsch, R S Sekhon, J M Foerster, J M Johnson, G Muttoni, B Vaillancourt, C R Buell, S M Kaeppler, N De Leon, Genetics. 193Beissinger TM, Hirsch CN, Sekhon RS, Foerster JM, Johnson JM, Muttoni G, Vaillancourt B, Buell CR, Kaeppler SM, de Leon N: Marker density and read depth for genotyping populations using genotyping-by-sequencing. Genetics 2013, 193:1073-1081.

Multiplexed shotgun genotyping for rapid and efficient genetic mapping. P Andolfatto, D Davison, D Erezyilmaz, T T Hu, J Mast, T Sunayama-Morita, D L Stern, Genome Res. 21Andolfatto P, Davison D, Erezyilmaz D, Hu TT, Mast J, Sunayama-Morita T, Stern DL: Multiplexed shotgun genotyping for rapid and efficient genetic mapping. Genome Res 2011, 21:610-617.

Basic local alignment search tool. S F Altschul, W Gish, W Miller, E W Myers, D J Lipman, J Mol Biol. 215Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.

Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds. R Singh, M Ong-Abdullah, E-Tl Low, Maa Manaf, R Rosli, R Nookiah, Lc-L Ooi, S-E Ooi, K-L Chan, M A Halim, N Azizi, J Nagappan, B Bacher, N Lakey, S W Smith, D He, M Hogan, M A Budiman, E K Lee, R Desalle, D Kudrna, J L Goicoechea, R A Wing, R K Wilson, R S Fulton, J M Ordway, R A Martienssen, R Sambanthamurthi, Nature. 500Singh R, Ong-Abdullah M, Low E-TL, Manaf MAA, Rosli R, Nookiah R, Ooi LC-L, Ooi S-E, Chan K-L, Halim MA, Azizi N, Nagappan J, Bacher B, Lakey N, Smith SW, He D, Hogan M, Budiman MA, Lee EK, DeSalle R, Kudrna D, Goicoechea JL, Wing RA, Wilson RK, Fulton RS, Ordway JM, Martienssen RA, Sambanthamurthi R: Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds. Nature 2013, 500:335-339.

Construction of a linkage map of the Rennell Island Tall coconut type (Cocos nucifera L.) and QTL analysis for yield characters. P Lebrun, L Baudouin, R Bourdeix, J L Konan, J H Barker, C Aldam, A Herrán, E Ritter, Genome. 44Lebrun P, Baudouin L, Bourdeix R, Konan JL, Barker JH, Aldam C, Herrán A, Ritter E: Construction of a linkage map of the Rennell Island Tall coconut type (Cocos nucifera L.) and QTL analysis for yield characters. Genome 2001, 44:962-970.

Microsatellite-based high density linkage map in oil palm. N Billotte, N Marseillac, A-M Risterucci, B Adon, P Brottier, F-C Baurens, R Singh, A Herrán, H Asmady, C Billot, P Amblard, T Durand-Gasselin, B Courtois, D Asmono, S C Cheah, W Rohde, E Ritter, A Charrier, Elaeis guineensis Jacq.). Theor Appl Genet. 110Billotte N, Marseillac N, Risterucci A-M, Adon B, Brottier P, Baurens F-C, Singh R, Herrán A, Asmady H, Billot C, Amblard P, Durand-Gasselin T, Courtois B, Asmono D, Cheah SC, Rohde W, Ritter E, Charrier A: Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.). Theor Appl Genet 2005, 110:754-765.

Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. B Langmead, C Trapnell, M Pop, S L Salzberg, Genome Biol. 1025Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009, 10:R25.

The Sequence Alignment/Map format and SAMtools. H Li, B Handsaker, A Wysoker, T Fennell, J Ruan, N Homer, G Marth, G Abecasis, R Durbin, Bioinformatics. 25Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R: The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25:2078-2079.

Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. E Cherif, S Zehdi, K Castillo, N Chabrillange, S Abdoulkader, J-C Pintaud, S Santoni, A Salhi-Hannachi, S Glémin, F Aberlenc-Bertossi, New Phytol. 197Cherif E, Zehdi S, Castillo K, Chabrillange N, Abdoulkader S, Pintaud J-C, Santoni S, Salhi-Hannachi A, Glémin S, Aberlenc-Bertossi F: Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm. New Phytol 2013, 197:409-415.

Genetic mapping in the presence of genotyping errors. D A Cartwright, M Troggio, R Velasco, A Gutin, Genetics. 176Cartwright DA, Troggio M, Velasco R, Gutin A: Genetic mapping in the presence of genotyping errors. Genetics 2007, 176:2521-2527.

CARHTA GENE: multipopulation integrated genetic and radiation hybrid mapping. S De Givry, M Bouchez, P Chabrier, D Milan, T Schiex, Bioinformatics. 21De Givry S, Bouchez M, Chabrier P, Milan D, Schiex T: CARHTA GENE: multipopulation integrated genetic and radiation hybrid mapping. Bioinformatics 2005, 21:1703-1704.

Multipoint maximum likelihood mapping in a full-sib family of an outbreeding species. J W Ooijen, Van, Genet Res (Camb). 93Ooijen JW VAN: Multipoint maximum likelihood mapping in a full-sib family of an outbreeding species. Genet Res (Camb) 2011, 93:343-349.

M Krzywinski, J Schein, I Birol, J Connors, R Gascoyne, D Horsman, S J Jones, M A Marra, Circos: an information aesthetic for comparative genomics. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA: Circos: an information aesthetic for comparative genomics.

. 10.1186/1471-2164-15-285Genome Res. 19Genome Res 2009, 19:1639-1645. doi:10.1186/1471-2164-15-285

A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. Mathew, BMC Genomics. 15285Cite this article asCite this article as: Mathew et al.: A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms. BMC Genomics 2014 15:285.


Influence of socioeconomic status on antipsychotic prescriptions among youth in France
July 1, 2013-June 30, 2014

Olivier Bonnot
Mélanie Dufresne
Paula Herrera
Emmanuelle Michaud
Jacques Pivette
Anicet Chaslerie
Anne Sauvaget
Caroline Vigneau
Influence of socioeconomic status on antipsychotic prescriptions among youth in France
July 1, 2013-June 30, 201410.1186/s12888-017-1232-3R E S E A R C H A R T I C L E Open Access
Background: Recent studies analysing the trends in antipsychotic (AP) prescriptions for children and adolescents have raised concerns regarding the influence of socioeconomic status. Previous findings have also shown variable prescription rates for first-generation (FG) and second-generation (SG) APs. Method: Our objectives were to assess the proportion of patients from low-income families receiving APs and the most commonly prescribed APs in France. We conducted a descriptive analysis of AP drugs dispensed during a 1-year period () in a northwestern region of France with 941,857 subjects less than 18 years old. All data were extracted from an exhaustive, individual and anonymous social security database. We obtained each subject's socioeconomic status (by identifying their affiliation with a specific social security program) and also collected sociodemographic data, drug type, prescribing and dispensing dates and amount, and prescriber type (e.g., hospital physician, general practitioner, psychiatrist, paediatrician). Results: There were two main novel findings. First, we found that the proportion of patients with AP prescriptions was nearly ten times higher in low-income families than in the general population: 35.9% of CMU-C patients compared to 3.7% in all of Pays de la Loire (X 2 = 7875.1, p < 0.001). Additionally, we found a higher rate of FGAP than SGAP prescriptions (65% vs. 57%). Conclusions: Our study suggests two types of AP misuse that could provide interesting targets for public healthcare interventions. First, our results strongly suggest an over-representation of patients from low-income families. Low-income families primarily resided in areas with low physician density and appeared to receive drugs to treat their conditions more frequently than other individuals. This increased prescription rate is a public health issue, potentially requiring political action. Second, the use of FGAPs did not adhere to the latest recommendations for drug use in this population, and this discrepancy should be addressed with informational campaigns targeted to medical practitioners.

Background

Antipsychotic (AP) drugs are recommended for the treatment for schizophrenia [1] and bipolar disorders [2] in children and adolescents. These drugs are effective in managing the core symptoms of these two diseases [1,2]. APs are also partly effective and thus prescribed to manage attention deficit hyperactivity disorders, disruptive behaviour disorders, Tourette's syndrome, post-traumatic stress disorder, anorexia nervosa and autism spectrum disorders [3,4]. The recent literature regarding the prescription of APs raises two main issues:

The first pertains to the use of first-and secondgeneration APs. Considering the data on their efficacy and side effects, the literature recommends a first-line prescription of a second-generation AP (SGAP) and then switching to another SGAP if necessary before considering a first-generation AP (FGAP) [1]. The choice of prescription should balance the benefits and risks of the drug, particularly in younger populations, who experience more side effects than adults [5,6]. A recent study showed that AP treatment is associated with increased body weight and cardiometabolic side effects as well as different side effect patterns [5]. The use of SGAPs is now recommended, and longitudinal studies have shown an increase in AP prescriptions in the last two decades with a switch from FGAPs to SGAPs [7][8][9]. However, some European studies, including those in France, have found that FGAPs are still much prescribed in this population, whereas in the U.S., the rate of FGAP prescriptions among youth is now very low [4,10].

The second issue is related to the patients' socioeconomic status (SES). Very few studies have explored the influence of socioeconomic/residential status on AP prescription rates. Existing research has focused on rural vs. urban inhabitants [11,12] and has found no correlations between these parameters and AP prescriptions. Two recent studies on this topic yielded conflicting results. One study reported that AP prescriptions were more common among people from low-income areas in the UK [13], whereas the second study failed to find an association between AP consumption by youth and the rate of welfare benefits [14]. These inconsistent findings may have occurred because the authors correlated the overall rate of AP prescriptions with welfare benefits of geographic locations rather than individual welfare data. The effect of SES might be more related to individual factors than to the geographic location.

To study these two issues, we conducted a descriptive analysis of drugs dispensed during a 1-year period (July 1, 2013-June 30, 2014) in an exhaustive study of a northwestern region of France with 3,658,000 inhabitants, including 941,857 subjects younger than 18 years old whose socioeconomic conditions were known.

Methods

Data source and population sample

We conducted an observational study of a retrospective cohort using a healthcare database that included information on subjects living in one area (Pays de la Loire) with five different counties (Loire Atlantique, Maine et Loire, Mayenne, Sarthe, and Vendée).

The data were extracted from one database (datamart de Consommation Inter Regimes, DCIR) that was part of the French healthcare insurance database (Système National d'Information Inter Régimes de l' Assurance Maladie, SNIIR-AM), which contains all of the information from the main insurance schemes in France; the SNIIR-AM covers salaried workers, farm workers, non-salaried workers (i.e., craftsmen and tradesmen), and a few other special groups (i.e., teachers, policemen, and armed forces). Affiliation with one of these schemes is mandatory (i.e., persons cannot be outside of the database). The DCIR database is an exhaustive, individual and anonymous healthcare database that includes all information on refunded healthcare claims. Individuals cannot purchase any of the included drugs without a pharmacist's report of this prescription being documented in the DCIR. Therefore, it is the main point of access for information on drug consumption by a large group of individuals. As with all subjects, individuals younger than 18 years have their own anonymous health service code, which is a lifetime code that uniquely identifies each resident.

Our population sample was composed of all children and adolescents younger than 18 years old at the end of the study period (July 1, 2013, to June 30, 2014) who were living in the Pays de la Loire area (northwestern France, 3.658 million inhabitants in 2013 [15]). All characteristics of the youth who received at least one dispensation of psychotropic drugs were obtained (n = 17.198).

Psychotropic drug selection process in the database

Drugs were identified using the International Nonproprietary Name (INN) and categorized using the Anatomical Therapeutic Chemical (ATC) classification system [16], which is commonly applied in epidemiologic studies. We selected APs, which are a subcategory of N05, the N05A group. We also added valproic acid (N03AG01) and valpromide (N03AG02). The total list of drugs included was as follows: Chlorpromazine, Levomepromazine, Cyamemazine, Valproic acid, Valpromide, Periciazine, Pipamperone, Haloperidol, Flupentixol, Zuclopenthixol, Pimozide, Loxapine, Clozapine, Olanzapine, Quetiapine, Sulpiride, Tiapride, Amisulpride, Risperidone, Aripiprazole, and Paliperidone.

Valproic acid and valpromide are commonly used for their AP effects by prescribers in general practice in France (even if they are off-label), and we chose to include them after a consensus meeting with all the authors of this article (i.e., OB, CC, PH, CV, PJ).

Study measures

We collected information on participants' sociodemographic data (gender, county, affiliated healthcare insurance scheme, age, and CMU-C status). Couverture Maladie Universelle Complémentaire (CMU-C) is a supplemental universal public insurance coverage scheme for low-income persons. The criteria for accessing the CMU-C include an income less than 721 euros/month for a single individual, adding 250 euros for each member of the family. Affiliation with CMU-C is considered a poverty threshold in France. Therefore, CMU-C status defines the group of people under the poverty threshold in France.

Regarding medications, we collected the type of drug dispensed (INN and ATC code (5th level)), the date of dispensation, the date of prescription, the number of dispensations, and the prescriber type (e.g., hospital physician, general practitioner, psychiatrist, paediatrician).

Our database granted access to all drugs dispensed and reimbursed in a pharmacy (and thus all prescriptions). We were able to determine how many different dispensations each patient had received during the study period. In France, drugs are provided to patients in complete blister cards (with a pre-defined number of slots), not by pills. Patients are usually given 1 month of treatment per dispensation (with the number of complete blister cards calculated accordingly). Therefore, patients typically receive extra pills, which may represent more than a 1-month supply. We determined the treatment duration for each subject by accessing the number of different months of dispensation in the follow-up for each drug. The month of dispensation was obtained to determine whether treatment was chronic (continuous variable), and we considered subjects receiving at least 6 months of dispensations as chronic users, which was consistent with the methodology of a previous study using this database [14].

Finally, it was difficult to determine the type of hospital practitioner prescribing APs. All prescriptions from physicians working in a hospital are dispensed with the unique identification number of the hospital. Currently, the only way to access information on the type of hospital practitioner is to access the actual prescription, which would have provided the name and medical speciality of the physician; however, this approach was not possible in our study for ethical reasons (lack of anonymity).

We also searched for national data regarding the general population. We focused on the number of inhabitants [15], proportion of CMU-C patients [17] and medical density in the study region [18].

Statistical analysis

Statistical analyses were performed using SAS Enterprise software version 4.3, and the detailed data were extracted and analysed by individuals who manage the healthcare insurance database according to the national commission for liberty and informatics (CNIL) laws.

A descriptive analysis was performed for sociodemographic and drug prescription data. We also calculated the proportion of AP prescriptions in comparison with general population data.

A proportions test based on chi2 was performed to compare the prevalence of CMU-C patients in our sample with that of the general population.

Results

Prescription overview

Among all patients in the Pays de la Loire region, 0.33% (2,897 children and adolescents under 18 years old) had an AP prescribed and dispensed during the observation period (see Table 1). Half of these prescriptions were for patients aged 13 years or younger (48%). Prescriptions increased with age and were higher in boys than in girls (0.47 in boys and 0.18 in girls).

Our results also showed that the overall AP prescription rate differed by county. Two counties were above the average prescription rate (0.33%): Mayenne (0.72%) and Maine et Loire (0.40%). Some rates, such as AP prescriptions for boys 5 to 9 years old in Mayenne vs. all areas (0.98 compared to 0.30), were of particular importance. The results in Mayenne were higher than the average for all sex and age subgroups; for example: 0.52 vs. 0.24% for girls aged 10-14 years and 1.75 vs 0.74% for boys aged 10-14 years. We present AP prescription rates by county with medical density in Table 2 (using data from the Conseil National de l'Ordre des Medecins) [18].

CMU-C-affiliated population

The proportion of individuals affiliated with CMU-C in our study was approximately 1/3 of our population (35.9%). When compared to the rates in the general population in Pays de la Loire, Chi 2 comparison tests showed a higher proportion of CMU-C patients in our sample (X 2 = 7875.1, p < 0.001). The results were similar in all counties (see Table 3). CMU-C affiliation was higher in families with children over 5 years: 22.7% for ages 0-4, 36.3% for ages 5-9, 35.5% for ages 10-14 and 36.7% for ages 15-17 years.

AP-specific medication data

Regarding single prescriptions, FGAPs were prescribed more frequently than SGAPs (65% vs. 57%). The ratio of FGAPs to SGAPs decreased with age, at 86% vs. 32% for subjects 0-4 years old and 63% vs. 61% for subjects 15-17 years old. In our study, 23% of subjects received both FGAPs and SGAPs. The overall data for all prescriptions (single and combination) are presented in Table 4.

The most commonly prescribed APs were cyamemazine and risperidone, totalling 1,379 and 1,303 prescriptions, respectively. Aripiprazole was the third-most prescribed AP, with only 207 prescriptions.

Regarding the duration of cyamemazine treatment, 33% of children/adolescents (463 of 1,379) had only a 1month dispensation, and 27% (366 of 1 379) had 6 separate months with one or more dispensations per year. For risperidone, 18% of children/adolescents (230 of 1,303) had only one month of dispensed drugs, and 41% (532 of 1,303) had 6 separate months with one or more dispensations per year.

Regarding the type of prescribers, hospital physicians comprised 73.2% of the total. For 46.3% of the patients, these doctors were their only prescriber. General practitioners prescribed 11.4% of the patients' prescriptions during the study period (results are in Table 5).

Discussion

This study presented two main novel results. First, we found that the proportion of patients with APs was almost ten times higher in low-income families than in the general population: 35.9% of low SES patients compared to 3.7% in all of Pays de la Loire (X 2 = 7875.1, p < 0.001). In addition, we found a higher rate of FGAP than SGAP prescriptions (65% vs. 57%).

In general, our sample is comparable to those of other recent French studies, as our overall AP prescription rate was 0.33% (0.47 in boys and 0.18 in girls) for patients under 18 years old. Three major studies on the use of AP medications have been conducted using the French healthcare insurance database. In 2004, a study was conducted in two different regions, showing that 0.3% of patients used APs and that the rates increased with age [19,20]. Using a different methodology and a random sample (1%) from the 2010 national database, another study reported an overall AP prescription rate of approximately 0.27-0.33% (0.35-0.45% for boys) [21]. A recent study examined a random representative sample of the French population (1%) extracted from the healthcare insurance database from 2006 to 2013 [14] and found an overall AP prescription rate of 0.49% for patients under 25 years old, 0.47% for patients between 11 and 15 years old, and 0.26% for patients between 6 and 10 years old.

These results are also consistent with recent studies in Europe. For example, a study in Germany used a similar method involving an exhaustive data source from a large area and found that the rate of AP prescriptions in 2012 was 0.33% and increased with age [13,22]. Compared to older studies and those with different methodologies (smaller, non-exhaustive samples or random partial extractions of a large population), our results are within the mean range of data from Western countries, which show prevalences of APs ranging from 0.08% in Italy [23] to 0.76% in the U.S. [24]. Our rates in France are lower than those in the U.S., with the most recent French data reporting AP use in 2006 and 2010 by 0.14% and 0.11% of younger children, 0.85% and 0.80% of older children, 1.10% and 1.19% of adolescents, and 0.69% and 0.84% of young adults [21].

Socioeconomic status

The results regarding SES are important and similar in all counties (see Table 3). To our knowledge, this type of data has not been presented in previous studies. Although these results differ from comparable French [14] and Canadian [12] studies, previous authors compared the global rate of  low SES patients in an area to the AP rate, whereas our study included each parent's CMU-C affiliation. Our approach provides a different perspective and suggests, by incorporating individual status, that being from a low-income family instead of simply living in a low-income area may be associated with increased prescription of APs.

In the same vein, we also found that the geographical distribution of AP users was not homogeneous and was greater in counties with the lowest medical density compared to those with a higher medical density (see Table 2). The ratio of medical facilities in the entire region was 272.7/100,000 inhabitants, which is slightly lower than the national density of 299.7/100,000 [18]. Moreover, the county with the lowest rate of AP prescriptions was Loire Atlantique, which also has the highest density of medical facilities. In contrast, the highest rate (0.72) was found in Mayenne, where the medical density is the lowest. These data suggest that individuals with greater medical access receive fewer AP prescriptions (see Table 2).

We found conflicting evidence in the literature regarding the impact of residence on AP prescriptions. A study from Taiwan found that AP prescriptions for patients aged 18 years or under were more frequent in suburban areas than in urban and rural areas [11], although this relationship was not found in a Canadian study [12]. However, Verdoux and colleagues found an increase in the level of AP prescriptions in areas with a low density of health facilities for children under 10 years old and in areas with low socioeconomic resources (global CMU-C affiliation rate in the area) for adolescents aged 16-20 years [14]. These conflicting results can be explained, again, by their use of data from large groups rather than from individually oriented data.

However, we could not determine a correlation between the density of medical facilities and access to health resources because of imprecisions in our data. Access to health care facilities depends on the exact part of the county where the patient resides (e.g., a family living in southern Mayenne may have better access to the University Hospital of Angers in Maine et Loire than some people in the extreme south of the Angers area). Another limitation of our study is that our data could not determine the purpose of the prescription or the  patients' diagnoses. For obvious privacy reasons, anonymous data prohibit access to these data in French insurance databases. Accordingly, we could not link AP prescriptions to specific diagnoses. The higher level of AP prescriptions among children and adolescents from low-income families can be explained by two hypotheses:

1. Patients from low SES families are known to exhibit more psychiatric disorders, especially those associated with behavioural disturbances [25,26]. This correlation is stronger for anxiety and mood disorders (which may be associated with behavioural disturbances in youth) and ADHD than for diseases such as autism, schizophrenia and Tourette's syndrome [25,26], which are core indications for AP prescription. Moreover, a higher incidence of behavioural problems has been found among preschool children from low SES families [27] and adolescents living in poverty [28]. Unfortunately, we were not able to verify this hypothesis, as we could not determine diagnoses in our sample. 2. The second hypothesis is related to the specific situations regarding child and adolescent psychiatry in France. A large percentage of child and adolescent psychiatrists in France do not commonly prescribe psychotropic medications, particularly APs. This practice is associated with the strong psychoanalytic culture in France along with a lack of current scientific knowledge about medications. Most of our colleagues consider themselves psychotherapists. Therefore, a psychiatrist may be more likely to prescribe medications for behavioural reasons than for developmental disorders (e.g., autism spectrum disorders, schizophrenia [29], Tourette's). Additionally, there are few hospitalizations that occur for child and adolescent psychiatric disorders due to the limited number of beds in France for emergencies or short periods (13.5/100,000 inhabitants), especially in Pays de la Loire (7/100 000 in Loire Atlantique, 9 in Maine et Loire and 12 in Sarthe, with other counties having only long-term hospitalizations, which are more similar to boarding school) [30]. There is also a lack of non-medical psychiatric healthcare professionals such as clinical psychologists, as in contrast to psychiatrists, psychologists' interviews are not reimbursed by social security [15].

These hypotheses may be synergistic. APs are frequently used to treat behavioural issues and may provide a quick solution to complex situations that would benefit from individual and family evaluation and support. When patients from low SES families exhibit more behavioural disorders, they encounter psychiatrists with few therapeutic options other than medications. Patients from higher SES families may be more likely to seek psychologists in private practice or re-educators (whose visits are not free).

Further studies that evaluate the specific level of care (medical and social) and the exact purpose for AP prescriptions are necessary to confirm this hypothesis.

FGAPs vs. SGAPs

The rates of FGAP compared to those of SGAPs were particularly high among younger patients: 86% vs. 32%, respectively, for ages 0-4 years and 69% vs. 44%, respectively, for ages 5-9 years. Remarkably, the rates remained high in children 10 to 13 years old (39.2% FGAP vs. 34.4% SGAP) and adolescents from 15 to 17 years old (36.1% FGAP vs. 34.1% SGAP). Indeed, recent AMMs (Market Release Authorizations) for SGAPs have made them available to 13-to 15-year-old patients instead of only 18-year-old patients (for aripiprazole, olanzapine and risperidone). Moreover, in 2007, an AMM was obtained for the use of risperidone in 5-yearolds for behavioural disorders involving intellectual deficiency. Therefore, SGAPs could have been prescribed more extensively. Our results indicate higher rates of FGAP than those reported in the most recent comparable studies in France [14,31] for all ages, with rates of FGAPs vs. SGAPs for infants, children and adolescents of 86.3% vs. 53.6%, 68.9% vs. 41.5% and 83.9% vs. 58.8%, respectively [14]. However, these previous studies involved a representative national sample that did not reflect regional disparities (see explanation for Pays de la Loire above).

This high level of FGAP prescription may be harmful because the side effect profile of APs differs drastically, especially in the paediatric population [6,32]. In other countries, longitudinal studies have shown an increase in AP prescriptions in the last two decades as well as a switch from FGAPs to SGAPs [7][8][9].

The high use of cyamemazine, which was the most commonly prescribed AP in our study, is likely related to French habits. In France, cyamemazine is mainly used as a sedative and anxiolytic drug, despite its poor tolerance due to metabolic syndrome as well as its limited AP effects [33]. Cyamemazine was also originally developed by a French laboratory and was marketed heavily in the 1990s. However, cyamemazine has a low affinity for dopaminergic receptors and therefore poor AP activity. The sedative effects of cyamemazine are mediated through its 5HT2A activity [34]. The main scientific literature regarding cyamemazine has investigated its efficacy in treating benzodiazepine withdrawal, particularly for alcoholic patients (for a review, see [35]). The very high level of cyamemazine use is likely related to a lack of knowledge of the efficacy and side effects as well as to prescribers' habits. The lack of knowledge about new SGAPs and their AMMs for younger patients may also be a reason for the low level of SGAP prescription, particularly during adolescence. Our results are comparable with those of previous studies showing a high rate of cyamemazine use in France [14].

Conclusion

Our epidemiological study in an exhaustive population demonstrated that the consumption of APs is concerning, occurring in 33 children and adolescents out of 10,000. This overall rate is admissible, but the qualitative analysis suggests a few concerning findings that should be addressed.

There was a misuse of APs, with an excessive number of FGAP prescriptions, especially for cyamemazine. This practice appears to be related to a lack of knowledge regarding the efficacy and side effects of this drug. The FGAP/SGAP ratio in northwest France was higher than the national mean [14] and was not compliant with the latest recommendations for drug use in this population. This discrepancy should be addressed through an information campaign directed towards medical practitioners and in the teaching of medical students. As our work was conducted with the French National Health Insurance organization, a full partnership to undertake this type of campaign is already in place.

Despite the lack of information regarding the reasons for prescription, our results strongly suggest an overrepresentation of patients from low SES families in our sample of youth receiving APs. These patients were mainly living in areas with a low physician density and received drugs to treat their conditions more often than other individuals. Therefore, it is important to communicate these findings to medical practitioners, and political action to address these discrepancies may be necessary. Ethics approval and consent to participate All data were obtained from the French National Health Insurance Information System (SNIIRAM). The access to data and the data analysis were granted by decree for a period of only 3 years plus the current year and authorization was obtained from the French Data Protection Authority (Commission nationale de l'informatique et des libertés, CNIL). Arrêté du 19 juillet 2013 relatif à la mise en oeuvre du Système national d'information interrégimes de l'assurance maladie NOR: AFSS1318985A Version consolidée au 07 avril 2016 https://www.legifrance. gouv.fr/affichTexte.do?cidTexte=JORFTEXT000027830713. Approval for data use was provided by the owner of the data: CPAM (France Healthcare Insurance).

Author details

Abbreviations ADHD: Attention deficit hyperactivity disorder; AP: Antipsychotic; ATC: Anatomical therapeutic chemical; BP: Bipolar disorder; CNIL: Commission Natioanale Informatique et Liberté = national commission for liberty and informatics; DCIR: Datamart de Consommation Inter Regimes; EMA: European medicine agency; ESCAPAD: Enquete sur la Santé et les Consommations lors de l'appel de la Preparation à la Defense Nationale = survey on health and consummation during army conscription mandatory day; FDA: Food and drug administration; FGAP: First generation antipsychotic; INN: International non-proprietary name; SGAP: Second generation antipsychotic

Table 1
1Raw 2013 prevalence rates of antipsychotic delivery by age and sex in Pays de la Loire and its 5 countiesGender
Age
(years)

Loire Atlantique
Maine et Loire
Mayenne
Sarthe
Vendée
Pays de la Loire (All)

n
% Pop
n
% Pop
n
% Pop
n
% Pop
n
% Pop 85
n
% Pop

MALE
0-4
4
0.01
12
0.05
9
0.09
4
0.02
-
-
29
0.02

5-9
89
0.21
105
0.39
104
0.98
35
0.19
29
0.14
362
0.30

10-14
221
0.52
233
0.89
183
1.75
114
0.63
123
0.62
874
0.74

15-17
216
0.88
170
1.14
114
1.93
99
0.93
108
0.98
707
1.05

Total
530
0.35
520
0.55
410
1.10
252
0.38
260
0.36
1972
0.47

FEMALE
0-4
5
0.01
4
0.02
2
0.02
3
0.02
1
0.01
15
0.01

5-9
13
0.03
25
0.10
15
0.15
15
0.08
11
0.06
79
0.07

10-14
61
0.15
97
0.38
52
0.52
33
0.19
23
0.12
266
0.24

15-17
113
0.48
99
0.68
44
0.80
61
0.58
57
0.53
374
0.57

Total
192
0.13
225
0.25
113
0.32
112
0.18
92
0.13
734
0.18

ALL
0-4
9
0.01
16
0.03
11
0.05
7
0.02
1
<0.01
44
0.02

5-9
102
0.12
130
0.25
119
0.57
50
0.14
40
0.10
441
0.19

10-14
282
0.34
330
0.64
235
1.15
147
0.41
146
0.38
1140
0.50

15-17
329
0.68
269
0.91
158
1.38
160
0.75
165
0.75
1081
0.82

Total
722
0.24
745
0.40
523
0.72
364
0.28
352
0.25
2706
0.33

Table 2 Medical
2facility density (/100 000 inhabitants) and raw antipsychotic prescription rate in all Pays de la Loire department
(n = 3 658 000) from July 1st 2013 to June 30th 2014

Mayenne
Vendée
Sarthe
Maine et Loire
Loire Atlantique

Medical practionner density
179.8
201.3
212
283.7
308.3

Rate of AP prescriptions
0.72
0.25
0.28
0.40
0.24

Table 3
3Patients with CMU-C in our sample and in general populationPatients with CMU-C
affiliation (n)

Patients with NO CMU-C
affiliation (n)

Total Proportion of individuals with CMU-C
in general population (%)

95% CI/Chi2
p

Total Pays de la Loire
Region

972
1734
2706 3.7
0.34-037/7875.1 <0.001

Loire Atlantique
250
472
722
4.6
0.31-0.38/1476.4 <0.001

Maine et Loire
255
490
745
4.9
0.31-038/1368.8 <0.001

Mayenne
190
333
523
4.2
0.32-0.40/1333.8 <0.001

Sarthe
132
232
364
6.2
0.31-041/560.5
<0.001

Vendée
122
230
352
3.1
0.29-0.39/1156.6 <0.001

Comparison test with chi2. All p are 0.20 10 −16

Table 4
4Type of antipsychotic and thymoregulator prescribed by age in all Pays de la Loire areas(n = 3 658 000) from July 1, 2013

Table 5
5Type of prescribers (alone or associated with others) in all Pays de la Loire regions (n = 3 658 000) for antipsychotic prescriptions from July 1, 2013 toJune 30, 2014    Specialty
Only prescriber for a particular
patient

Other prescribers are in charge of the
patient

Total n = 2706

n
%
n
%
n
%

Hospital any type (mostly paediatricians and psychiatrists) 1252
46.3
729
26.9
1981
73.2

General Practice
309
11.4
581
21.5
890
32.9

Psychiatrist
250
9.2
257
9.5
507
18.7

Paediatrician
15
0.6
26
1.0
41
1.5

Other
9
0.3
52
1.9
61
2.3

No Info
42
1.6
164
6.1
206
7.6

Table is to read by line, and not per columns (for example 507 patients (18.7% of our 2 706 patient's sample had prescription by a psychiatrist, among them 250
had only one prescriber, and 257 had more then one)

AcknowledgementsNone -all work was performed by the authors.FundingNo funding was used and was not necessary for this survey.Availability of data and materialsWe do not wish to share the data as they are exclusive property of the French National Health Insurance Information System (SNIIRAM) and the statistics were generated directly in the datamart base (not connected to an outside network and only readable in FHIS main offices).Authors' informationAuthors' contributions MD, OB, CV, PH, AS, AC, JP and EM were involved in the study conception and design and the analysis and interpretation of data. OB, MD, PH drafted the article; CV, AC, AS, JP and EM critically revised it for important intellectual content; tables were constructed by MD, OB and CV. Data were extracted by MD, EM, AC and JP (CPAM team). All authors provided final approval of the version to be published.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable. Data are anonymized irreversibly by the FOIN system (Fonction d'Occultation des Identifiants Nominatifs) of the French National Health Insurance Information System (SNIIRAM).• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
Practice parameter for the assessment and treatment of children and adolescents with schizophrenia. J Mcclellan, S Stock, J Am Acad Child Adolesc Psychiatry. 52American Academy of Child and Adolescent Psychiatry (AACAP) Committee on Quality Issues (CQI)McClellan J, Stock S, American Academy of Child and Adolescent Psychiatry (AACAP) Committee on Quality Issues (CQI). Practice parameter for the assessment and treatment of children and adolescents with schizophrenia. J Am Acad Child Adolesc Psychiatry. 2013;52:976-90.

Pharmacologic treatment of bipolar disorder in children and adolescents. B I Goldstein, R Sassi, R S Diler, Child Adolesc Psychiatr Clin N Am. 21Goldstein BI, Sassi R, Diler RS. Pharmacologic treatment of bipolar disorder in children and adolescents. Child Adolesc Psychiatr Clin N Am. 2012;21:911-39.

Off-Label prescribing of antipsychotics in adults, children and elderly individuals: A systematic review of recent prescription trends. L Carton, O Cottencin, M Lapeyre-Mestre, P A Geoffroy, J Favre, N Simon, Curr Pharm Des. 21Carton L, Cottencin O, Lapeyre-Mestre M, Geoffroy PA, Favre J, Simon N, et al. Off-Label prescribing of antipsychotics in adults, children and elderly individuals: A systematic review of recent prescription trends. Curr Pharm Des. 2015;21:3280-97.

Treatment of young people with antipsychotic medications in the United States. M Olfson, M King, M Schoenbaum, JAMA Psychiatry. 72Olfson M, King M, Schoenbaum M. Treatment of young people with antipsychotic medications in the United States. JAMA Psychiatry. 2015;72:867-74.

Second-generation antipsychotic use in children and adolescents: a 6-month prospective cohort study in drug-naive patients. C Arango, M Giráldez, J Merchán-Naranjo, I Baeza, J Castro-Fornieles, J A Alda, 1190.e1J Am Acad Child Adolesc Psychiatry. 53Arango C, Giráldez M, Merchán-Naranjo J, Baeza I, Castro-Fornieles J, Alda JA, et al. Second-generation antipsychotic use in children and adolescents: a 6-month prospective cohort study in drug-naive patients. J Am Acad Child Adolesc Psychiatry. 2014;53:1179-90,1190.e1.

Adverse effects of second-generation antipsychotics in children and adolescents: a Bayesian meta-analysis. D Cohen, O Bonnot, N Bodeau, A Consoli, C Laurent, J Clin Psychopharmacol. 32Cohen D, Bonnot O, Bodeau N, Consoli A, Laurent C. Adverse effects of second-generation antipsychotics in children and adolescents: a Bayesian meta-analysis. J Clin Psychopharmacol. 2012;32:309-16.

Increasing off-label use of antipsychotic medications in the United States. G C Alexander, S A Gallagher, A Mascola, R M Moloney, R S Stafford, Pharmacoepidemiol Drug Saf. 20Alexander GC, Gallagher SA, Mascola A, Moloney RM, Stafford RS. Increasing off-label use of antipsychotic medications in the United States, 1995-2008. Pharmacoepidemiol Drug Saf. 2011;20:177-84.

Epidemiologic and clinical perspectives on antipsychotic treatment of children and adolescents. M Olfson, Can J Psychiatry Rev Canadienne Psychiatrie. 57Olfson M. Epidemiologic and clinical perspectives on antipsychotic treatment of children and adolescents. Can J Psychiatry Rev Canadienne Psychiatrie. 2012;57:715-6.

Antipsychotic prescribing trends: a review of pharmaco-epidemiological studies. H Verdoux, M Tournier, B Bégaud, Acta Psychiatr Scand. 121Verdoux H, Tournier M, Bégaud B. Antipsychotic prescribing trends: a review of pharmaco-epidemiological studies. Acta Psychiatr Scand. 2010;121:4-10.

National trends in the office-based treatment of children, adolescents, and adults with antipsychotics. M Olfson, C Blanco, S M Liu, S Wang, C U Correll, Arch Gen Psychiatry. 69Olfson M, Blanco C, Liu SM, Wang S, Correll CU. National trends in the office-based treatment of children, adolescents, and adults with antipsychotics. Arch Gen Psychiatry. 2012;69:1247-56.

Trends, correlates, and disease patterns of antipsychotic use among children and adolescents in Taiwan. Y C Hsu, I C Chien, H K Tan, C H Lin, S W Cheng, Y J Chou, Soc Psych Psych Epid. 48Hsu YC, Chien IC, Tan HK, Lin CH, Cheng SW, Chou YJ, et al. Trends, correlates, and disease patterns of antipsychotic use among children and adolescents in Taiwan. Soc Psych Psych Epid. 2013;48:1889-96.

Ten years of antipsychotic prescribing to children: a Canadian population-based study. S Alessi-Severini, R G Biscontri, D M Collins, J Sareen, M W Enns, Can J Psychiat. 57Alessi-Severini S, Biscontri RG, Collins DM, Sareen J, Enns MW. Ten years of antipsychotic prescribing to children: a Canadian population-based study. Can J Psychiat. 2012;57:52-8.

Prescribing of antipsychotics in UK primary care: a cohort study. L Marston, I Nazareth, I Petersen, K Walters, D P Osborn, BMJ Open. 46135Marston L, Nazareth I, Petersen I, Walters K, Osborn DP. Prescribing of antipsychotics in UK primary care: a cohort study. BMJ Open. 2014;4, e006135.

Antipsychotic prescribing in youths: a French community-based study from. H Verdoux, E Pambrun, S Cortaredona, M Tournier, P Verger, Eur Child Adoles Psy. 24Verdoux H, Pambrun E, Cortaredona S, Tournier M, Verger P. Antipsychotic prescribing in youths: a French community-based study from 2006 to 2013. Eur Child Adoles Psy. 2015;24:1181-91.

Répartition des médecins et chirurgiens. INSEE. INSEE, Répartition des médecins et chirurgiens. 2016. https://www.insee.fr/ fr/statistiques/2012677. Accessed 13 Feb 2017.

ATC Classification. ATC Classification 2015. http://www.whocc.no/atc_ddd_index/. Accessed 13 Feb 2017.

. Les Idres, Cmu-C Bénéficiaires De La, IDRES, LEs bénéficiaires de la CMU-C. 2015. http://www.irdes.fr/ EspaceEnseignement/ChiffresGraphiques/Cadrage/CMU/CMUC.htm. Accessed 13 Feb 2017.

. Démographie Cnom, Médicale, CNOM, Démographie médicale. 2015. http://www.conseilnational.medecin. fr/sites/default/files/pays_de_la_loire_2013_0.pdf. Accessed 13 Feb 2017.

Psychotropic medication in the French child and adolescent population: prevalence estimation from health insurance data and national self-report survey data. E Acquaviva, S Legleye, G R Auleley, J Deligne, D Carel, B B Falissard, BMC Psychiatry. 972Acquaviva E, Legleye S, Auleley GR, Deligne J, Carel D, Falissard BB. Psychotropic medication in the French child and adolescent population: prevalence estimation from health insurance data and national self-report survey data. BMC Psychiatry. 2009;9:72.

Observatoire Français des Drogues et Toxicomanies. Estimation 2014 des consommations de produits psychoactifs à 17 ans. Observatoire Français des Drogues et Toxicomanies. Estimation 2014 des consommations de produits psychoactifs à 17 ans. 2016. http://www.ofdt.fr/ BDD/publications/docs/eisxfbv4b.pdf. Accessed 13 Feb 2017.

Psychotropic medication use in French children and adolescents. V Kovess, S Choppin, F Gao, M Pivette, M Husky, E Leray, J Child Adolesc Psychopharmacol. 25Kovess V, Choppin S, Gao F, Pivette M, Husky M, Leray E. Psychotropic medication use in French children and adolescents. J Child Adolesc Psychopharmacol. 2015;25:168-75.

Antipsychotic prescription in children and adolescents: an analysis of data from a German statutory health insurance company from. C J Bachmann, T Lempp, G Glaeske, F Hoffmann, Deutsches Arzteblatt International. 111Bachmann CJ, Lempp T, Glaeske G, Hoffmann F. Antipsychotic prescription in children and adolescents: an analysis of data from a German statutory health insurance company from 2005 to 2012. Deutsches Arzteblatt International. 2014;111:25-34.

Antidepressant and antipsychotic use in an Italian pediatric population. A Clavenna, M Andretta, P Pilati, M Dusi, M Gangemi, M B Gattoni, BMC Pediatr. 1140Clavenna A, Andretta M, Pilati P, Dusi M, Gangemi M, Gattoni MB, et al. Antidepressant and antipsychotic use in an Italian pediatric population. BMC Pediatr. 2011;11:40.

A three-country comparison of psychotropic medication prevalence in youth. J M Zito, D J Safer, L T De Jong-Van Den Berg, K Janhsen, J M Fegert, J F Gardner, Child Adolesc Psychiatry Ment Health. 226Zito JM, Safer DJ, de Jong-van den Berg LT, Janhsen K, Fegert JM, Gardner JF, et al. A three-country comparison of psychotropic medication prevalence in youth. Child Adolesc Psychiatry Ment Health. 2008;2:26.

The Christchurch Health and Development Study. The Christchurch Health and Development Study: Review of findings on child and adolescent mental health. D M Fergusson, L J Horwood, Aust NZ J Psychiatry. 35Fergusson DM, Horwood LJ, The Christchurch Health and Development Study. The Christchurch Health and Development Study: Review of findings on child and adolescent mental health. Aust NZ J Psychiatry. 2001;35:287-96.

Epidemiology of mental disorders in children and adolescents. K R Merikangas, E F Nakamura, R C Kessler, Dial Clin Neurosci. 11Merikangas KR, Nakamura EF, Kessler RC. Epidemiology of mental disorders in children and adolescents. Dial Clin Neurosci. 2009;11:7-20.

The performance of low-income, African American children on the Preschool Language Scale-3. C H Qi, J Speech Lang Hear Res. 463Qi CH, et al. The performance of low-income, African American children on the Preschool Language Scale-3. J Speech Lang Hear Res. 2003;46(3):576-90.

Poverty and adolescent mental health. C Dashiff, W Dimicco, B Myers, K Sheppard, J Child Adolesc Psychiatr Nurs. 22Dashiff C, DiMicco W, Myers B, Sheppard K. Poverty and adolescent mental health. J Child Adolesc Psychiatr Nurs. 2009;22:23-32.

Neurodevelopmental model of schizophrenia: update 2012. J L Rapoport, J N Giedd, N Gogtay, Mol Psychiatry. 17Rapoport JL, Giedd JN, Gogtay N. Neurodevelopmental model of schizophrenia: update 2012. Mol Psychiatry. 2012;17:1228-38.

Eco-Données Santé. Score Sante. Score Sante. Eco-Données Santé. 2016. http://www.scoresante.org/ score2008/sindicateurs.html. Accessed 13 Feb 2017.

A review of pharmacoepidemiologic studies of antipsychotic use in children and adolescents. S B Patten, W Waheed, L Bresee, Can J Psychiat. 57Patten SB, Waheed W, Bresee L. A review of pharmacoepidemiologic studies of antipsychotic use in children and adolescents. Can J Psychiat. 2012;57:717-21.

Second-generation antipsychotic use in children and adolescents: a 6-month prospective cohort study in drug-naïve patients. C Arango, M Giráldez, J Merchán-Naranjo, I Baeza, J Castro-Fornieles, J A Alda, 10.1016/j.jaac.2014.08.009J Am Acad Child Adolesc Psychiatry. 53Arango C, Giráldez M, Merchán-Naranjo J, Baeza I, Castro-Fornieles J, Alda JA, et al. Second-generation antipsychotic use in children and adolescents: a 6- month prospective cohort study in drug-naïve patients. J Am Acad Child Adolesc Psychiatry. 2014;53:1179-90,1190.e1. doi:10.1016/j.jaac.2014.08.009.

Pharmacological management of anxiety in patients suffering from schizophrenia. F J Baylé, O Blanc, I De Chazeron, J Lesturgeon, C Lançon, H Caci, Encéphale. 371SupplBaylé FJ, Blanc O, De Chazeron I, Lesturgeon J, Lançon C, Caci H, et al. Pharmacological management of anxiety in patients suffering from schizophrenia. Encéphale. 2011;37 Suppl 1:S83-9.

Preclinical and clinical pharmacology of cyamemazine: anxiolytic effects and prevention of alcohol and benzodiazepine withdrawal syndrome. M Bourin, E Dailly, M Hascöet, CNS Drug Rev. 10Bourin M, Dailly E, Hascöet M. Preclinical and clinical pharmacology of cyamemazine: anxiolytic effects and prevention of alcohol and benzodiazepine withdrawal syndrome. CNS Drug Rev. 2004;10:219-29.

Potential role of cortical 5-HT (2A) receptors in the anxiolytic action of cyamemazine in benzodiazepine withdrawal. A Benyamina, M Naassila, M Bourin, Psychiatry Res. 198Benyamina A, Naassila M, Bourin M. Potential role of cortical 5-HT (2A) receptors in the anxiolytic action of cyamemazine in benzodiazepine withdrawal. Psychiatry Res. 2012;198:307-12.