Conservation and functional influence of alternative splicing in wood formation of Populus and Eucalyptus

Peng Xu
Yimeng Kong
Dongliang Song
Cheng Huang
Xuan Li
Laigeng Li
Conservation and functional influence of alternative splicing in wood formation of Populus and Eucalyptus
R E S E A R C H A R T I C L E Open Access
Background: Wood formation in tree species is regulated by multiple factors at various layers. Alternative splicing (AS) occurs within a large number of genes in wood formation. However, the functional implications and conservation of the AS occurrence are not well understood. Results: In this study, we profiled AS events in wood-forming tissues of Populus and Eucalyptus, and analyzed their functional implications as well as inter-species conservation. 28.3% and 20.7% of highly expressed transcripts in the developing xylem of Populus and Eucalyptus respectively were affected by AS events. Around 42% of the AS events resulted in changes to the original reading frame. 25.0% (in Populus) and 26.8% (in Eucalyptus) of the AS events may cause protein domain modification. In the process of wood formation, about 28% of AS-occurring genes were putative orthologs and 71 conserved AS events were identified in the two species.Conclusion: Through analysis of AS events in developing xylem of two tree species, this study reveals an array of new information regarding AS occurrence and function in tree development.

Background

Alternative splicing (AS), which generates multiple transcript variants from one gene, is a key scheme in multicellular eukaryotes to enhance the functional diversity of the proteome [1]. In flowering plant species, up to 61% of multiexonic genes in Arabidopsis and 48% of genes in rice are affected by AS [2,3]. AS has been shown to regulate plant development in photosynthesis, disease resistance, floral transition, circadian rhythm, starch metabolism, auxin biosynthesis, and temperature response [4][5][6]. AS can cause alterations to protein function through modulating protein structure. For example, frame shifts caused by AS often result in truncated proteins that can form nonfunctional heterodimers and act as dominant-negative regulators [7,8]. AS occurrence in functional domain structures can affect protein-protein interaction, transcriptional activation or DNA binding [9][10][11]. In addition to modulate protein functions, AS can influence transcript stability through nonsense-mediated decay (NMD) or miRNA linked regulation [12,13].

Wood, a unique structural and storage tissue in tree species, is derived from the meristematic activities of the vascular cambium during secondary growth [14,15]. Wood formation involves a coordinated progression of cell differentiation, expansion, secondary cell wall formation, and programmed cell death [16]. AS has been found to play important roles in the process of wood formation. NAC transcription factor PtrWND1B / PtrSND1-A2, which controls secondary wall biosynthesis, was shown to undergo AS in Populus [17,18]. The splice variant of PtrWND1B/ PtrSND1-A2 IR contains a retained intron and encodes a protein lacking DNA binding and transactivation activity but retaining dimerization ability. PtrSND1-A2 IR functions as a dominant negative of PtrSND1 members through heterodimerization [17]. This dominant negative regulation was demonstrated as a specific mechanism controlling fiber cell wall thickening during wood formation in Populus [18]. AS may have evolved as a regulatory mechanism of the isochorismate synthase gene, PtiICS, which encodes an enzyme for phylloquinone biosynthesis, in Populus [19].

Analyzing how AS is conserved across species can help to identify AS events important in general for plant development. Conserved AS events in several gene families such as ribulosebisphosphate carboxylase/oxygenase activase, SR protein, MYB transcription factor and transthyretin-like protein have been characterized in plant species [20][21][22][23]. Genome-wide analysis have identified 56 conserved AS events between Arabidopsis and rice, and 49 conserved AS events between rice and maize [24]. Between two or more legume species, 22 conserved AS events were detected [25]. More conserved AS events (527 cases) were reported between closely related species Brassica and Arabidopsis [26]. Meanwhile, it is not known how AS events are conserved and involved in modulating protein function in wood formation of tree species.

Recently, transcriptome of Populus xylem was analyzed to identify AS events in wood formation tissue. Up to 36% of expressed genes were detected to undergo AS [27], indicating a wide influence of AS in the process of wood formation. On the other hand, great variations of AS events, which are likely caused by genotypic polymorphism, were observed among different Populus populations [27]. Although AS has shown to influence diverse groups of genes in Populus, how the AS events are involved in protein function modulation is little known. In this study, we compared the occurrence of AS in Eucalyptus and Populus and analyzed the functional implications and conservation of AS during wood formation in tree species.

Results

AS profiles in wood formation tissues

To detect AS events during wood formation, we first constructed transcriptomes of developing xylem in Populus (P. × euramericana) and Eucalyptus (E. grandis). Two biological replicates were sampled from the two species for transcriptome analysis using the Illumina platform based RNA-seq technology [28]. The sequencing analysis generated approximate 30~45 million paired-end reads (100-nucleotide length) from each sample of the two species. 77.6%~80.6% of these reads could be mapped onto the reference genome via the TopHat program [29]. More than 60% of the mapped reads located in the exon regions of the two genomes (Additional file 1). In Populus, 26.2% of the reads were mapped to an exon-intron/intronexon junction region, among which 21.3% were not annotated in the reference genome. This part of reads could be from the products caused by AS variants. In Eucalyptus, 36.8% of the junction reads had no annotations. Some reads were found to map to intergenic regions. 6.8% of the total reads in Eucalyptus were mapped to the intergenic regions of the genome while 0.9% of reads in Populus showed such mapping. This may reflect the genome annotation difference in two species.

Expressed transcripts were assembled by the Cufflinks program with the cutoff of 0.1 FPKM in both biological replicates [30]. Considering that the weakly expressed transcripts could result in false AS identification [2], highly expressed transcripts with an expression level higher than 5% of the most abundant transcript were subsequently used as the basis for follow up analysis. In Populus, 31,984 highly expressed transcripts were assembled. Among them, 13.8% (4,397) of transcripts had no annotation in the reference genome (Additional file 1). In Eucalyptus, the number of highly expressed transcripts was 21,372 and 19.5% of them were not annotated in the genome. The assembled transcripts were transcribed from 23,735 (57.4% of the total predicted genes) genes in the Populus genome and 17,202 (47.3% of the total predicted genes) genes in the Eucalyptus genome (Additional files 1 and 2), suggesting that a large portion of the genes in the two species are expressed in developing xylem tissue.

On the basis of the assembled transcripts, AS events among the transcriptome was analyzed. The ASTALA-VISTA tool [31] was used to establish the repertoire of AS events. In total, 6,031 AS events in Populus and 2,987 events in Eucalyptus were detected in developing xylem tissue (Additional file 3). These AS events affected 28.3% and 20.7% of the highly expressed transcripts in the two species ( Figure 1A), respectively, which were transcribed from 17.2% (4,079 in Populus) and 11.9% (2,039 in Eucalyptus) of the xylem expressed genes. The majority of AS transcripts (93% in Populus and 97% in Eucalyptus) displayed expression levels higher than 1.0 FPKM ( Figure 1B). Over 85% of the AS events occurred in the transcript isoforms with expression ratios of 0. 11 .0 compared to the major transcript of a same gene (Additional file 4). For verification of the AS events, 16 representative AS events were selected for RT-PCR analysis and 14 of them (about 87.5%) were confirmed (Additional file 5), indicating the reliability of the AS detection in our RNA-seq analysis.

The constructed AS events were categorized into five major AS types [32]: intron retention (IR), alternative acceptor site (AltA), alternative donor site (AltD), exon skipping (ES) and alternative position (AltP) for further analysis. IR, which encompassed 45.0% and 33.9% of the AS events in Populus and Eucalyptus respectively ( Figure 1C), represented a much higher portion of AS events than ES. AltA was over-represented and IR under-represented while the proportions of the other three AS types were similar in Eucalyptus compared to Populus ( Figure 1C).

AS features in wood formation

Based on the identified AS events in Populus and Eucalyptus, AS features in woody tissues were analyzed, including AT content of the introns, and nucleotide change caused by AS. In Populus, the average AT content of the total introns of xylem expressed genes is 66.4%, higher than 62.4% in Eucalyptus (p-value < 0.0001, t-test). Similarly, the average AT content across all alternative introns (AS introns) in Populus was 65.6%, higher than that in Eucalyptus (61.1%) (Figure 2A). This difference between species was also consistent in the introns among various AS types. Nevertheless, the nucleotide composition at splicing junction site was conserved between Populus and Eucalyptus (Additional file 6).

Selection of different splice sites may result in a string of nucleotides changed between transcript variants. The changed nucleotides could interrupt the original reading frame if they are not multiples of 3 base pairs. About 67% of the AS events were found to be located in the open reading frame (ORF) region of the two species. Analysis of ORF located AS events revealed that 79% of the changed nucleotides were between 1 and 200 bps ( Figure 2B). A change of 3 nucleotides occurred most frequently in both Populus and Eucalyptus. Similar phenomenon was reported in Arabidopsis, rice and Brassica [26,33]. The 3 nucleotide change was mainly generated from AltA, AltD and AltP, while IR or ES largely caused changes of 50-150 bp (Additional file 7). We observed 37% of the changed lengths fitting in multiples of 3 nucleotides in the two species, which could maintain the original reading frame. In other words, up to 63% of ORF located AS events (42% of the total AS events) in the two species could cause frameshift. A similar frequency of ORF change was observed in the AS isoforms with different expression ratios (Additional file 4).

Protein domain modification caused by AS

One of the major effects of AS is to create protein isoforms that increase proteome diversity [5,34]. Previous studies suggest that protein functions could be affected if their functional domains are interrupted by AS [9][10][11]17,18]. We conducted in silico transcript translation and then analyzed whether AS occurrence caused protein domain alteration. In the two species, 25.0% (1507 events in Populus) and 26.8% (801 events in Eucalyptus) of the AS events occurred within domain sequences which would cause domain modification (Additional file 8). A similar frequency of such domain modification was observed in the AS isoforms with different expression ratios (Additional file 4). In detected  Figure 3A). To exclude the potential influence of domain number in the background genome, Fisher's exact test was used to obtain the enriched domains in the AS affected genes (Additional file 9). 51 enriched domains showed a close association with AS modification in the two species (p-value < 0.05), including domains RRM_1, PP2C and Ndr which were identified among the most frequently modified domains. This suggests that these protein domains may be particularly modified by AS events in wood formation process.

To understand how AS impacts wood formation, the AS modified domains from Populus and Eucalyptus were mapped to individual genes associated with wood formation according to their annotation (Additional file 10). These genes could be categorized into five fundamental processes for wood formation including: cell wall formation, hormone signaling, transcriptional regulation, cellular transportation and programmed cell death ( Figure 3B). For example, cellulose synthase (CESA) and caffeoyl-CoA 3-Omethyltransferase (CCOAMT) are involved in secondary cell wall thickening during wood formation [35][36][37]. The occurrence of AS was observed in the catalytic domains of these enzymes. The functional domains of many other wood formation-related genes was also detected to be affected by AS.

Conservation of AS-occurring genes in wood formation

To investigate conservation of AS-occurring genes, putative orthologous genes in Populus and Eucalyptus were searched by the BLAST program (E-value < 1e-50, identity > 0.4 and coverage > 0.6). A total of 1159 Populus and 887 Eucalyptus putative orthologous genes were identified with AS-occurrence, which represents 28% and 44% of the AS-occurring genes in the two species, respectively ( Figure 4A). Gene expression patterns established via microarray analysis [38,39] revealed that 56.3% of these orthologous genes were highly expressed in xylem ( Figure 4B), indicating their association with wood formation. These orthologs in the two species constituted 716 independent gene groups with different biological functions (Additional file 11). Gene ontology (GO) annotations suggested several important processes for plant development such as transportation, cell cycle and cell growth were significantly enriched (FDR < 0.001) ( Figure 4C). Genes involved in the process responding to abiotic stimulus were also enriched, highlighting the potential impact of environmental cues on AS-occurring genes.

Conserved AS events in wood formation

Among orthologous genes with AS occurrence, conserved introns were searched to identify conserved AS events across the two species. A total of 71 conserved AS events (1.2% of total AS events in Populus and 2.4% in Eucalyptus) were identified. These conserved AS events occurred between 65 Populus and 55 Eucalyptus genes (Additional file 12). Among them, 37 events belonged to the AltA type, ranking it as the most frequent type of AS. Approximately, 60% of the conserved AS events caused frameshift and 48% of the conserved AS events were detected to cause modification of functional domain structures (Additional file 12).

Two examples of the conserved AS events in the two species were further analyzed in greater detail. AS was observed in an orthologous pair of AP2 genes in Populus and Eucalyptus ( Figure 5A). The AP2/EREBP family encodes transcription factors that play a role in a variety of plant regulatory processes [40]. The AP2 domain of this gene family consists of 60 amino acids that are responsible for DNA binding. In Populus, one isoform of the PtAP2 transcript has two AP2 domains while the second isoform only has one AP2 domain due to a skipped exon. The conserved AS event was also observed in Eucalyptus. Another example of conserved AS event occurred in the functional domain of Aux/IAA gene family, which encode short-lived nuclear proteins that negatively regulate auxin signaling transduction [41]. Aux/IAA proteins are likely involved in the early response to auxin signaling, which controls the development of wood-forming tissues [42]. Based on our analysis, a conserved intron is retained in the IAA8 genes of Populus and Eucalyptus. Importantly, the conserved IR event resulted in a truncated protein as it introduced a premature termination codon in the transcript ( Figure 5B). RT-PCR verified the two cases of conserved AS events in the two species ( Figure 5C).

To study how AS can regulate the function of the Aux/ IAA family, sequences of IAA8 orthologs from Arabidopsis, rice, Populus and Eucalyptus were aligned. Multiple sequence alignment showed the truncated proteins, PtIAA8 IR and EgIAA8 IR , specifically lack the conserved subdomain IV of Aux/IAA proteins, but contain intact subdomains I~III ( Figure 6A). Subdomain IV contains a sequence which is critical for nucleus localization [43]. To examine whether the location of the protein is affected by AS, IAA8 isoforms from Populus were fused with GFP for localization analysis. When expressed in tobacco leaves, the protein encoded by full transcript PtIAA8 was shown to locate in the nucleus, while the truncated protein PtIAA8 IR displayed ubiquitous location in cells ( Figure 6B). This indicates AS can modulate protein functions of Aux/IAAs through regulating their intercellular location.

Discussion

Landscape of AS in wood formation of tree species

This study established a comprehensive profile of AS events during wood formation. The results show that in both Populus and Eucalyptus more than 20% of the highly expressed transcripts were affected by AS, indicating extensive influence of AS on wood formation. In Populus, 6,031 AS events were detected while 2,987 events were detected in Eucalyptus. Based on current information, however, it is unknown whether this difference is implicated by the characteristics of the species. Five AS types were detected but their composition ratio was different in the two species. AltA was more frequently in Eucalyptus while IR was observed with a relatively greater frequency in Populus. Interestingly, intron sequences in Eucalyptus have a significantly lower AT content compared to those in Populus. Variations of AT content may be responsible for the different AS profiles in the two species, as UArichness of plant introns is important for splicing efficiency [44]. AS identification in the developing xylem of Populus trichocarpa has recently reported [27]. About 71 1% of the total annotated genes were found to undergo AS in 20 Populus individuals from different populations, while around 46% of the AS events were not conserved within individuals, which may be caused by genotypic variations [27]. In the present study, the occurrence of AS was detected in about 10% of the total annotated genes and about 40% of the AS-occurring genes were the same in the reported individuals (PT17 and PT18) [27], indicating a consistency of AS occurrence in different Populus species. The results may also reflect some of the detected AS events being specific to the gene type of hybrid poplars (P. × euramericana cv.'Nanlin895').

Functional implications of AS in wood formation

AS occurs extensively in various plant species, however, comprehensive analysis of their functional impact has been little studied. AS affect protein functions in a variety of ways by altering amino acid composition, subcellular location, secondary structure stability, binding property, and posttranslational modification [45]. The diversified aspects of AS influence can be assessed through in silico analysis. In this study, we primarily focused on analysis of AS- occurring genes which bring about modification of domain structures. Results show that about 42% of the AS events caused ORF disruptions or change, suggesting that AS extensively affects protein sequence. A large proportion (25.0% in Populus and 26.8% in Eucalyptus) of AS events resulted in protein domain modifications, which may be involved in wood formation at various levels. For example, the protein domains modified included Cellulose_synt, Glyco_transf and Glyco_hydro, which function in cell wall biosynthesis. Protein domains like proteasome and peptidase are involved in programmed cell death. AS modification to the AUX/IAA and PP2C domains indicates that AS may play a role in auxin and ABA signaling in wood formation tissue. We also observed a similar pattern of ORF change or domain modification in the AS events with different isoform ratios.

Apart from generating functionally tailored proteins, AS could also affect mRNA transport, localization and stability prior to protein translation. For example, some intron-containing transcript variants could remain within the nucleus resulting in decreased mobility [46]. This indicates a subset of AS transcripts which cannot be translated into proteins as in silico predictions would suggest. In addition, aberrant transcripts caused by AS are often degraded by RNA surveillance pathways such as nonsensemediated decay (NMD) [12]. In theory, NMD sensitive transcripts would generally display low expression levels. A group of such AS events might be excluded from this study as weakly expressed transcripts were filtered out in analysis. Thus, a normalized cDNA library should be constructed in order to estimate NMD targets during wood formation [2]. Intriguingly, typical NMD targets in Arabidopsis include mRNA with a 3'UTR sequence longer than 350 bp [47][48][49]. Our analysis suggests that the average length of 3'UTR in both Populus and Eucalyptus is over 400 bp. And 3'UTR sequences are usually longer than 700 bp when they contained AS events. This may suggest a difference between Arabidopsis and tree species in the precise length of 3'UTR that could trigger NMD pathway. Taken together, our results indicate that the AS events may function in regulating wood formation through modifying protein domain structures.

Conservation of AS in Populus and Eucalyptus

By comparing AS profiles in Populus and Eucalyptus, this study provides a sketch of AS events conserved in wood formation tissue. In our results, AS occurred within 1159 Populus and 887 Eucalyptus putative orthologous genes. GO analysis revealed that the orthologous genes were featured with functions related to the regulation of cell growth and differentiation. Over half of the orthologous genes were preferentially expressed in the developing xylem of Populus, indicating their involvement in wood formation.

Comparison of AS events identified 71 AS events that were conserved in Populus and Eucalyptus. Among them, 47.9% of the conserved AS events caused domain modifications. For instance, a conserved IR event was demonstrated to affect subdomain IV of Aux/IAA isoforms, which is critical to regulate protein subcellular location. The intron-retained Aux/IAA isoforms contain a premature termination codon and have a long 3'UTR sequence (>700 bp), suggesting they may be regulated by the NMD pathway that is coupled with AS occurrence. Consistent with this, these transcripts displayed reduced expression level according to RT-PCR. Interestingly, no equivalent AS regulation has been reported for Aux/IAA family genes in other plant species such as Arabidopsis and rice. Whether this AS event regulation of Aux/IAA family genes is specific to wood formation is yet to be further characterized.

Conclusions

AS plays a critical role in the development of multicellular organisms, though much remains to be learned about its function in tree-specific processes. AS is extensively involved in wood formation as revealed by profiles of AS-occurring genes, AS events and AS modification of protein structures in the developing xylem of Populus and Eucalyptus. The results of this study highlight a number of new avenues to explore how AS is employed as a mechanism to modulate gene functions in woodforming tissues.

Methods

RNA-seq experiment

The Populus (P. × euramericana cv.'Nanlin895') and Eucalyptus (E. grandis) were grown in experimental fields in Shanghai (31°N) and Guangxi Province (21°N). Developing xylem from 3-year old trees was sampled at midday (June, 2012) and razor blades were used to collect xylem tissues after peeling off the bark. Xylem tissues from three individual trees of each species were mixed together for sample preparation. Two biological replicates of xylem samples in each species were used for RNA extraction following the CTAB method. RNA concentration and quality were determined by the Agilent Bioanalyzer 2100. The cDNA library was constructed by polyA enriched RNA. 200-300 bp fragments were selected for non-directional 100 nucleotide paired-end sequencing, which was performed on an Illumina HiSeq 2000 according to Illumina protocols (San Diego, CA, USA).

Detection of AS events

The reads from two biological replicates in each species were mapped and assembled independently. Reference genomes of Populus (v3.0) [50] and Eucalyptus (v1.1) [51] were obtained from Phytozome database. RNA-seq reads were mapped onto reference genomes via TopHat (v2.0.10) [29]. The maximum intron length was set to 6000 bp and other parameters were left as defaults. Unique mapped reads were extracted by samtools (v0.1.18) with a MAPQ cutoff of 20 [52]. Cufflinks (v2.1.1) was used for transcript assembly with supplied genome annotations [30]. Expression abundance of assembled transcripts was obtained from the Cufflinks output, which was measured in the unit of FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Expression of 99% of the transcripts were consistent between two replicates in each species (FDR < 0.05), indicating a good reproducibility. Downstream analysis are based on the genes that could be detected by RNA-seq (>0.1 FPKM) in both biological replicates. To ensure the reliability of AS detection, weakly expressed transcripts with expression levels less than 5% of the most abundant transcript format from a gene, were filtered out before AS detection. Transcripts that remained in both replicates were subjected to ASTALAVISTA web server to discover AS events [31]. AS transcripts were extracted from each AS event and the percentage of AS transcripts was calculated through dividing the number of AS transcripts by that of the total transcripts. Within a gene, isoform ratio of the AS event was set as the expression level of the minor transcript divided by that of the most abundant transcript. Five major types of AS previously described in Wang et al. 2006 [32], including intron retention (IR), alternative acceptor site (AltA), alternative donor site (AltD), exon skipping (ES) and alternative position (AltP), were extracted for further analysis.

Analysis of AS features

Intron sequences with different AS types were extracted according to the coordinates reported by ASTALAVISTA. Transcript isoforms were translated based on the longest ORF. AS events were classified as 5'UTR, ORF and 3'UTR location according to their positions in different mRNA features. Perl scripts were written for analysis including AT content and changed nucleotides. The length of changed nucleotides was computed by comparing the alternative introns from two isoforms in a single AS event. For IR and ES, it was the length of the retained intron and skipped exon. For AltA, AltD and AltP, the length corresponded to the altered nucleotides that were present in the exon of one isoform, but included in the intron of the other isoform. The Pfam 27.0 database was searched to identify the domains affected by AS, with an E-value cutoff of 1e-10. The genes with AS modified domains were examined for their function annotations (on the basis of information in Phytozome) and classified into different biological processes. To determine significantly enriched protein domains associated with the AS occurrence, Fisher's exact test was conducted with a p-value cutoff of less than 0.05.

Conservation analysis of AS-occurring genes

Orthology of the AS-occurring genes in Populus and Eucalyptus was investigated by the BLAST program (v2.2.21) with parameters of E-value < 1e-50, identity > 0.4 and coverage > 0.6. Expression patterns of the orthologous genes in Populus were established by microarray data from the NCBI Gene Expression Omnibus (GEO) database under accession numbers GSE30507 and GSE13990. Robust Multiarray Averaging (RMA) method was used to normalize microarray data across different tissues in Bioconductor according to previously described methods [53]. Enriched genes were defined as those with the highest expression level in xylem, compared to other tissues. The other genes were included as expressed genes. Heatmap.2 in R (v2.15.1) program was used to generate heatmaps of enriched expressed genes. Populus genes were converted to their orthologous genes in Arabidopsis and then used for GO annotation by agriGO [54]. Plant GOslim database and Fisher's exact test were implemented to identify enriched GO processes (FDR < 0.001).

Identification of conserved AS events

Similar to previous reports [24,32], conserved AS events in the two species were defined mainly by identification of the conserved introns in orthologous AS genes. To get conserved introns, the introns flanked by homologous exonic sequences were searched. In every AS event from Populus and Eucalyptus, 100 bp exonic sequences flanking the alternative introns were extracted. The tblastx program was used to search homologous exonic sequences across the two species with an E-value cutoff of 1e-5. If both of the flanking exonic sequences of a Populus intron were homologous to that of a Eucalyptus intron, the alternative introns were regarded as conserved introns. If the conserved introns were present in orthologous genes with the same AS type, the pair of AS event was considered a conserved AS event.

Subcellular localization and RT-PCR analysis

Coding sequences of PtIAA8 and PtIAA8 IR were cloned into pCAMBIA2300 backbone fused in frame with GFP under the control of the CaMV 35S promoter to determine their subcellular location. Tobacco leaves were transformed with constructs mediated with Agrobacterium strain GV3101 according to previous descriptions [55]. GFP fused proteins were examined with a confocal microscope (LSM 510 META; Zeiss) using 488 nm for excitation and 505 to 555 nm for emitted light capture. For RT-PCR, the xylem of Populus (P. × euramericana) and Eucalyptus (E. grandis) was used for RNA extraction. 500 ng RNA was reverse transcribed into cDNA by PrimeScript RT Master Mix (TaKaRa, Dalian, China) according to the manufacturer's protocols. Primers that span alternative introns were designed to perform PCR reactions in each species (Additional file 13).

Figure 1
1AS profiles in developing xylem of Populus and Eucalyptus. (A) Percentages of expressed transcripts undergoing AS in two species. (B) Expression levels of AS transcript isoforms. The expression abundance was measured with unit of FPKM (Fragments Per Kilobase of transcript per Million mapped reads). (C) Distribution of different AS types. IR, intron retention; AltA, alternative acceptor site; AltD, alternative donor site; ES, exon skipping; AltP, alternative position. AS events, 789 and 500 kinds of domains might be modified in Populus and Eucalyptus, respectively. Moreover, 16.6% (1003 events in Populus) and 18.2% (545 events in Eucalyptus) of AS events could result in domain gain or loss. Among the 15 most frequently modified domains, 8 domains were present in both Populus and Eucalyptus, namely Pkinase, Pkinase_Tyr, RRM_1, PP2C, zf-DHHC, Ras, Ndr and DnaJ (

Figure 2
2AS profile features in developing xylem of Populus and Eucalyptus. (A) Comparison of AT content in constitutive introns (non-AS introns) and different types of alternative introns (AS introns). IR, intron retention; AltA, alternative acceptor site; AltD, alternative donor site; ES, exon skipping; AltP, alternative position. (B) Histogram indicating frequency of ORF located AS events at different changed lengths of nucleotides.

Figure 3
3Influence of AS on protein domains during wood formation. (A) The 15 most frequently modified domains in Populus and Eucalyptus. Protein domains were identified from the Pfam database with the E-value cutoff of 1e-10. (B) Association of AS modified domains, AS affected genes and their potentially involved biological processes.

Figure 4
4Othologous genes affected by AS during wood formation. (A) Orthology of AS-occurring genes in the xylem of Populus and Eucalyptus. (B) Tissue expression patterns of orthologous AS-occurring genes in Populus. Enriched genes showed the highest expression level in xylem tissue. (C) Enriched GO processes of orthologous AS-occurring genes in Populus (FDR < 0.001).

Figure 5
5Characterization of two conserved AS events in developing xylem of Populus and Eucalyptus. Gene structures are shown for the conserved AS events in AP2 family (A) and AUX/IAA family (B). Boxes represent exons and lines represent introns. Hatched regions indicate coding sequences. Red and blue colors indicate predicted domains. Primers designed for RT-PCR are shown by black arrows. (C) RT-PCR verification of the two conserved AS events. The upper bands indicate the full transcripts in AP2 lanes and the intron-retained transcripts in AUX/IAA lanes.

Figure 6
6Regulation of subcellular locations of IAA8 proteins by a conserved AS event. (A) Protein sequence alignment of IAA8 orthologs in Arabidopsis, rice, Populus and Eucalyptus. Nuclear localization sequence (NLS) and four subdomains are shown on the top. Green box indicates the domain lost by the conserved IR event in Populus and Eucalyptus. (B) Subcellular localization of PtIAA8 isoforms. GFP fused PtIAA8 and PtIAA8 IR were transiently expressed in tobacco leaves and examined by confocal microscope. Bar: 20 μm.
http://www.biomedcentral.com/1471-2164/15/780
Availability of supporting dataAll sequencing data were deposited in the Short Read Archive at NCBI under accession number SRA108028.Additional filesAdditional file 1: Summary of reads mapping and transcript assembly.Competing interestsThe authors declare that they have no competing interests.Authors' contributions PX designed the study, conducted the experiments and prepared the manuscript. YK performed the statistical analysis and participated in the design of the study. LL directed the research project and composed the manuscript. XL directed data analysis and edited the manuscript. DS prepared the tissue samples. CH participated in the confocal microscopy analysis. All the authors have read and approved the final manuscript.Submit your next manuscript to BioMed Central and take full advantage of:
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Efficacy of cognitive rehabilitation on psychosocial functioning in Borderline Personality Disorder: a randomized controlled trial

Juan C Pascual
Nerea Palomares
Ángela Ibáñez
Maria J Portella
Rocío Arza
Raquel Reyes
Albert Feliu-Soler
Marina Díaz-Marsá
Jerónimo Saiz-Ruiz
Joaquim Soler
Jose L Carrasco
Efficacy of cognitive rehabilitation on psychosocial functioning in Borderline Personality Disorder: a randomized controlled trial
10.1186/s12888-015-0640-5R E S E A R C H A R T I C L E Open Access
Background: Follow-up studies revealed that subjects with borderline personality disorder (BPD) present high rates of clinical remission, although psychosocial functioning often remains impaired. The aim of this study is to evaluate the efficacy of a cognitive rehabilitation intervention versus a psychoeducational program on psychosocial functioning in subjects with BPD.Methods: A multicenter, randomized, and positive-controlled clinical trial was conducted. Seventy outpatients with BPD were randomized to cognitive rehabilitation or psychoeducational group interventions. Participants were evaluated after completion of the intervention period (16 weeks) and after the follow-up period (6 months). Psychosocial functioning, clinical and neuropsychological outcomes were evaluated. Results: No main effects of group or group x time were observed on functionality but a significant effect of time was found. Post-hoc analyses showed that only cognitive rehabilitation increased psychosocial functioning significantly at endpoint. Psychoeducation showed a significant enhancement of depressive symptoms. Conclusions: Cognitive rehabilitation and psychoeducational interventions appeared to show good efficacy in improving disabilities in daily life in subjects with BPD. These interventions are easily implemented in mental health settings and have the advantage of improving general functioning and clinical symptoms.

Background

Borderline Personality Disorder (BPD) is a common and severe disorder that has long been considered a chronic and untreatable disorder for many clinicians [1,2]. Nevertheless, recent long follow-up studies offer a more optimistic scenario indicating high rates of clinical remission [2][3][4]. However, remission does not appear to be equivalent to full recovery as symptoms improvement is not necessarily associated with amelioration of psychosocial dysfunctioning. These same studies point out that the challenge for the next generation of therapies is to redirect the focus onto improving functional outcomes as well as clinical symptoms [2][3][4]. Most psychotherapies such as Dialectical Behaviour Therapy (DBT) or Mentalization Based Therapy have proven their efficacy to treat emotional dysregulation, impulsivity and interpersonal difficulties, but not cognitive deficits or psychosocial functioning [1].

It is often accepted that psychosocial dysfunction is partly caused by cognitive deficits that remained impaired after clinical remission in other mental disorders such as schizophrenia and bipolar disorder [5,6], but there are few studies addressing this issue in BPD. Some recent studies have established that neuropsychological dysfunction of BPD might be affecting domains such as attention, cognitive flexibility, memory, planning, processing speed and visuo-spatial skills [7][8][9][10]. Accordingly, recent neuroimaging studies in BPD have reported structural and functional abnormalities in many brain areas supporting the assumption of a dysfunctional frontolimbic network in subjects with BPD [1,[11][12][13][14][15]. Such abnormalities are compatible with the cognitive impairment observed in BPD patients [7][8][9][10]. Nevertheless, some authors suggest that these neurocognitive dysfunctions in BPD may owe more to the impact of transient mood states or emotional distress than to underlying primary cognitive deficits per se [16,17].

Cognitive rehabilitation strategies have usually been applied in schizophrenia [18], and, more recently, their efficacy has been demonstrated in affective disorders [19,20]. To our knowledge, there are no consistent data on the application of neuropsychological remediation in patients with BPD, apart from preliminary communications of positive findings in case series [21,22]. The aim of the present randomized and controlled study is to evaluate the efficacy of a cognitive rehabilitation group therapy as compared to a psychoeducational group intervention in subjects with BPD on psychosocial functioning.

Method

Participants A total of 70 outpatients with BPD were included from September 2011 to July 2013, 30 from the Hospital Clinico San Carlos, 20 from the Hospital Santa Creu i Sant Pau, and 20 from the Hospital Ramón y Cajal. Inclusion criteria were the following: 1) Outpatients aged 18 to 45 years; 2) Diagnoses of BPD according to DSM-IV-TR [23] criteria and evaluated by two semi-structured diagnostic interviews -Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II) [24] and the Revised Diagnostic Interview for Borderlines (DIB-R) [25]-to guarantee a correct diagnosis; 3) Clinical severity measured with Clinical Global Impression for BPD (CGI-BPD) [26] higher than 4; and 4) Functional impairment measured with a Global Assessment Functioning (GAF) [23] lower than 65.

Exclusion criteria were the following: 1) Severe physical conditions, such as organic brain syndrome or neurological disease that could affect neuropsychological performance; 2) Intelligence Quotient IQ < 85; 3) Major Depression Disorder (MDD) or substance misuse within the last 6 months evaluated with DSM-IV criteria and SCID-I specific sections; 4) DSM-IV criteria for Schizophrenia, severe psychotic disorder or bipolar disorder evaluated by SCID-I specific sections; 5) Previous participation in any psychoeducation or cognitive rehabilitation intervention.

Study design and procedure

Study design was a multicenter, randomized, rater-blind clinical trial (see Additional file 1: Figure S1). There were two-parallel arms (1:1) to evaluate functional, clinical and cognitive efficacy of a specific cognitive rehabilitation group intervention (CR) compared with a psychoeducational group intervention (PE) in subjects with BPD. To ensure the reliability among centers regarding the evaluation and the treatment fidelity, two meetings were organized before the start of the study to train therapists.

Clinical and neuropsychological evaluations before interventions were administered on different days since they lasted in general more than one hour each and the effect of fatigue or boredom might have affected the results. Experienced psychiatrists and psychologists performed clinical interviews over 3 months to ensure the follow-up of all participants prospectively. The following sociodemographic and clinical variables were collected: age at recruitment, gender, education level, occupational status, and pharmacological treatment. All participants were randomized to receive CR or PE in a 1:1 ratio stratified by centre, age, and education level. Generation of random allocation sequence was done with the Research Randomizer (www.randomizer. org). The present study was powered to test hypotheses about potential between-treatment differences on the primary outcome. With a sample size of 70 and an expected attrition from assessments of 30 %, the study had power of 65 % and a level of significance of 5 % to detect a moderate effect (d = .6).

Participants were evaluated at baseline, after the intervention (16-week period), and after the follow-up period (six months after the intervention). During the whole study period, subjects did not receive any other individual or group psychotherapy. All patients continued pharmacological treatment if it had been initiated prior to inclusion. Type and doses of medication could not be modified at any time during the whole study period. Both interventions were applied in a group format and were conducted by two psychologists with experience in managing patients with BPD. Subjects were instructed not to disclose any information about the intervention to maintain blind conditions. Adverse events such as severe self-harm, suicide attempt, hospitalization and death during the trial were collected.

Ethics

The study was approved by the Ethics Committee of the Hospital Clínico San Carlos and carried out in accordance with the ethical principles of Declaration of Helsinki. All subjects received extensive information about the study and provided written informed consent before they were enrolled in the study. This study was registered at clinicaltrials.gov (NCT02033044).

Interventions

Cognitive Rehabilitation (CR)

CR consisted of group sessions (5 individuals per group) of 120 min, twice a week during a total period of 16 weeks (32 sessions). The exercises addressed neurocognitive issues related to sustained attention, processing speed, memory and executive functioning. The whole program aimed at getting new strategies to improve functional adaptation, thus tasks were carried out in the clinical setting and at home. Some homework tasks were based on their daily life difficulties and problems. The main objective of this program was the generalization of rehabilitated cognitive functions to daily life activities. Most of the techniques were based on a previous program for bipolar disorder [20].

Psychoeducation (PE)

The psychoeducational intervention consisted of 16 weekly group sessions of 5 individuals of 120 min each (16 sessions). This therapy aimed at improving awareness of illness, interpersonal abilities, family balance, therapeutical adherence, emotional management in frustrating situations, problem solving, and lifestyle regularity. During this intervention, no homework tasks were required. This intervention was based on the first step of the Systems Training for Emotional Predictability and Problem Solving (STEPPS) program: "Awareness of Borderline Personality Disorder" [27]. Any other STEPPS's contents were included in this psychoeducation intervention. In order to provide a more rigorous comparison condition and for controlling nonspecific effects of the CR (e.g., attending in a regular basis to a group therapy), PE was elected instead of other common comparisons such as treatment as usual (TAU) or waiting list.

Instruments

Diagnostic interviews and severity assessment -Structured Clinical Interview for DSM-IV Axis II Personality Disorders (SCID-II) [24]. A semi-structured interview to assess personality disorders according to DSM-IV criteria. The Spanish version has good discrimination between Axis II personality disorders, as well as good reliability between raters as indicated by an overall Kappa of 0.85. -Revised Diagnostic Interview for Borderlines (DIB-R) [25]. The DIB-R is a semi-structured interview that brings the diagnosis of BPD within the last two years. Scores range from 0 (no BPD severity) to 10 (high BPD severity). The Spanish version has shown good psychometric properties regarding internal consistency (Cronbach's alpha: 0.89), sensitivity (0.81) and specificity (0.94). The interviewers were experienced psychologists and presented a high inter-rater reliability (within-class correlation: 0.94).

The inter-test-reliability of DIB-R and SCID-II was moderate (Kappa: 0.59).

-Clinical Global Impression Scale for BPD (CGI-BPD) [26]. This clinician-rated scale assesses global severity for BPD and symptomatic dimensions using a 7-point Likert scale, which ranges from 1 (absence of illness) to 7 (high severity of illness). Primary outcome: psychosocial functioning -Functioning Assessment Scale Test (FAST) [28]. This was the primary outcome measure. It is a 24-item clinician-rated scale that measures the level of functioning of patients in daily life situations in the past two weeks. It assesses six functional domains: autonomy, occupational functioning, cognitive functioning, financial issues, interpersonal relationships, and leisure time. Scores range from 0 (none) to 3 (higher functional impairment). This instrument has shown good psychometric properties and sensitivity to change.

Secondary outcomes: clinical and neuropsychological effects

Clinical assessment -Borderline Symptom List -23 (BSL-23) [29]. A 23item self-rating instrument used to assess the typical symptomatology and severity of BPD. It is rated by using a 5-point scale from 0 ("not at all") to 4 ("very strong"). This instrument has shown good psychometric properties. -Hamilton Anxiety Rating Scale (HARS) [30]. It is a clinician-rated scale to evaluate current anxious symptoms. -Montgomery-Asberg Depression Rating Scale (MADRS) [31]. This is a self-rated questionnaire for the evaluation of depression severity. -Barrat Impulsivity Scale (BIS) [32]. This is a 30-item self-rated scale to assess behavioral impulsivity.

Neuropsychological assessment

Subjects were evaluated with a complete neuropsychological battery based on the literature from previous research that explored cognitive functions in subjects with BPD [7][8][9][10]. The estimated IQ was evaluated with the WAIS-III vocabulary subtest. The battery consisted of several tests to evaluate three different domains: attention, memory, and executive function. In order to sum up redundant neuropsychological tests assessing these domains, three indexes were further calculated with composite score.  [39] to assess the capacity of abstraction, cognitive flexibility, elaboration of concepts and planning.

Statistical analysis

All analyses were performed with the SPSS 19.0 software package for Windows and all hypotheses were tested with a two-sided significance level of 0.05. First, descriptive analyses were performed using chi-square test for categorical variables and t-test for continuous variables. Given the high number of dropouts in post-intervention and follow-up, the planned repeated measures ANOVA was substituted by the Hierarchical Linear Modeling (HLM) to investigate treatment, time and the interaction with the MIXED procedure of SPSS [40]. Post hoc analyses were performed with HLM to evaluate groupspecific changes related to intervention. Subjects were included only if they had baseline measure and at least one post-baseline measure and all analyses were conducted on an intention-to-treat basis. The primary outcome was the change in total score of FAST but this same procedure was applied to test changes on clinical and neuropsychological variables.

Results

Sociodemographics and clinical characteristics

Patient flow is presented in Fig. 1 (Fig. 1). There were no significant differences at baseline between individuals that finalized the intervention and those that dropped out in terms of demographic and clinical characteristics. As shown in Table 1, there were no significant differences between the two interventions at baseline in terms of demographic characteristics, clinical severity or pharmacological treatment. The majority of individuals were women (74 %) and more than 40 % of the sample had a long-term sick-leave. The sample had a moderate to severe clinical profile and a poor psychosocial functioning. Most participants were taking pharmacological treatment and had participated in previous psychotherapies (mainly Dialectical Behaviour Therapy) but did not have previous experience in cognitive rehabilitation or psychoeducation programme. Table 2 presents a summary of mean scores corresponding to pre-and post-intervention as well as 6-month follow-up measurements. HLM was carried out including number of sessions as a covariable and main effects can be found in Table 2. The primary outcome of the trial (FAST) did not show main effects of group, group x time or number of sessions. There was a significant main effect of time for both treatments (baseline, post-treatment, and 6-month follow-up assessments) [F(2, 40.04) = 6.34, p = .004]. Post hoc analyses showed that CR was the intervention which showed greater improvement in the FAST (p = .018) (Fig. 2).

Primary outcome Psychosocial functional improvement

Secondary outcomes Clinical improvement

No significant changes related to CR or PE were observed in BPD psychopathology measured by BSL-23 (p > .6). Regarding impulsivity measured by BIS, no significant differences were observed either (p = .

Discussion

This is the first randomized clinical trial that evaluates the efficacy and long-term effects of a cognitive rehabilitation group intervention compared with a psychoeducational group intervention in subjects with borderline personality disorder. The results show that cognitive rehabilitation exerted the greatest change on the primary outcome six months after the intervention finished but not at its end, demonstrating long-term effects of cognitive remediation. By contrast, psychoeducational intervention also showed a significant enhancement of depressive symptoms and attention functioning.

It has been suggested that cognitive remediation programs for psychiatric disorders should aim at improving general functioning beyond cognition [41]. As in a previous trial with patients suffering bipolar disorder [20], inclusion criteria of the present study required functional disability of patients in their daily activities but not necessarily an impaired cognitive profile. Therefore, this could explain the beneficial effects of cognitive rehabilitation on psychosocial functioning without observing neuropsychological changes, neither at the end of the intervention nor in the Fig. 1 The CONSORT diagram shows the randomization of patients to cognitive rehabilitation or psychoeducation and the progress through the study follow-up assessment. Strikingly, our results showed that some cognitive deficits, as measured with neuropsychological tests, remained unchanged after cognitive rehabilitation although patients exhibited fewer difficulties and better functioning after intervention, regardless of whether it was cognitive rehabilitation or psychoeducation. This was unexpected, as the hypothesis was that enhancement of cognitive functioning should have accounted for improvements in functional outcomes, at least in part. Two explanations are possible: the deficits of these patients were too mild to be improved with the programs or there may have been a type II error, i.e., the sample was not big enough to detect differences in neuropsychological data. Alternatively, and considering recent evidences [16,17], it is also possible that the neuropsychological performance of subjects with BPD would be affected by emotional distress. Further studies that evaluate intervention effects on the cognitive function in BPD should control for this variable. In any case, cognitive rehabilitation emphasized the use of compensatory skills to cope with daily life difficulties. Psychoeducational intervention would have exerted similar effects as patients acquired deeper insight into their illness and new strategies to cope with stress.

Amongst psychiatric disorders, neurocognitive rehabilitation has been efficiently tested for schizophrenia with the Integrated Psychological Therapy or IPT [42] and the Cognitive Remediation Therapy or CRT [18]. More recently, it has been tested for affective and bipolar disorders with significant efficacy [19,20]. To date, there are no consistent data on neuropsychological remediation for subjects with BPD and only preliminary and uncontrolled communications of positive experiences have been published [21,22]. No significant differences between CR and PE were found, but both interventions improved general functioning, which provides evidence of the need for therapeutic strategies focused on psychosocial difficulties of psychiatric patients. The lack of an inactive arm such as  Our results suggest that the general functional improvement observed in BPD is independent of clinical and neuropsychological changes. Long-term follow-up studies also show that clinical and general functioning improvements are not always related because high rates of clinical remission are not always associated with better psychosocial functioning [3,4]. These results are in concordance with a recent study in bipolar disorder where a delayed improvement on general functioning was also found without clinical or neuropsychological changes [20], and are strengthened even more by the fact that conditions of the trial were controlled until the end of the follow-up. What is clear is that psychosocial functioning needs to be specifically addressed to obtain a global recovery of BPD patients.

Psychoeducation impacted on clinical depressive and anxiety symptoms as well as on attention domain compared with CR. Our PE was partially based on "Awareness of Borderline Personality Disorder", the first step of STEPPS programme that had previously been demonstrated to improve clinical symptoms in BPD [27]. A previous study comparing psychoeducational training with waiting list also showed efficacy in symptoms such as impulsivity or unstable relationships but no improvement in psychosocial functioning [43]. It is worth mentioning that PE efficacy for clinical and neuropsychological enhancement was observed with half of the sessions of CR intervention.

The following limitations have to be taken into consideration. The final sample size was limited due to the high number of dropouts. Therefore, the present findings need replication in larger samples. Such small numbers did not allow us to perform analyses by domains of the FAST, as done in a previous study [20]. Given that this was a multicenter trial, the representativeness of the sample with regard to subjects with BPD from Spain can be acknowledged. Individual motivation to change and groupal procedure are key factors for some types of psychotherapeutic interventions. These factors were not evaluated in this study, and could have affected the findings, particularly for CR, which normally requires additional efforts and a more personalized intervention. In any case, CR was compared with an active intervention although with lower duration and intensity, and not a weaker treatment condition such as treatment as usual or waiting list. The presence of a third arm with an inactive treatment would probably have been useful to confirm a significant efficacy of both interventions. The influence of pharmacological effect was not evaluated, but most patients were treated without differences between treatment arms. Finally, we did not control other non-specific factors that could affect psychological functioning as social relationships, family support, regular social activity stimulated by trainings, etc.

Conclusions

In summary, preliminary results from the present study seem to suggest that both cognitive rehabilitation and psychoeducational interventions could be efficacious in subjects with BPD to treat functional disabilities in daily life. These interventions can be easily implemented in mental health settings and provide advantages to improve general functioning, which frequently remains affected after clinical remission in long-term follow-up [2][3][4]. The present results highlight the need for further research in order to better determine the specific impact of these interventions on psychosocial functioning in BPD. Specifically, investigating the combined effect of cognitive rehabilitation with other BPD-specific psychotherapeutic models (i.e., DBT or Mentalization-Based Therapy) would be of special interest. Doing so may provide useful information to better determine if adding cognitive rehabilitation would amplify the efficacy of well-established BPD treatments.

-
Global Assessment Functioning (GAF) [23], from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition Text Revision (DSM-IV-TR). This scale allows the evaluation of the global functioning of the patient and ranges from 1 to 100.

1). Significant time effects were observed in the HLM analyses for HARS [F(2, 46.29) = 9.29, p < .001] and MADRS [F(2,45.63) = 8.92, p < .001]. An effect of group [F(1, 64.14) = 7.37, p = .009] and a tendency for group x time [F(2, 45.63) = 2.96, p = .062] was also found in MADRS scores. Post hoc analyses indicated significant improvements on anxiety and depression in PE group (p = .006 and p < .001, respectively) and on anxiety in the CR group (p = .023).Neuropsychological improvementNo effect of the interventions was found for the Memory and Executive Indexes (p > .05). Regarding the Attention Index, a group x time interaction was found [F(2, 39.66) = 4.33, p = .02], although post hoc tests indicated no significant differences in Attention Index (p > .05).

Fig. 2
2General functional improvement measured by FAST scores in CG and PE groups in pre-, post-and 6 months follow-up measurements. Note Fig. 2: Rounded and squared dots represent means and bars, standard errors. CR = Cognitive Rehabilitation; PE = Psychoeducational intervention. A time effect was observed in the HLM analysis [F(2, 41.14) = 7.54, p = .002]. Post hoc analyses showed that FAST only improved significantly in the CR condition (p = .018)

Table 1
1Summary of demographics and clinical variables at baselineCR (n = 36)
PE (n = 34)
P

Gender (n/% females)
28/77.8
24/70.6
n.s.

Age
32.4 (6.04)
32.8 (8.8)
n.s.

Years of education
11.78 (4.12)
11.0 (3.33)
n.s.

Sick-leave/Unemployed (n/%)
16/44.4
13/38.2
n.s.

DIB-R
7.65 (1.35)
7.33 (1.31)
n.s.

GAF
54.74 (7.45)
56.03 (8.94)
n.s.

CGI-BPD
4.94 (0.79)
4.74 (0.93)
n.s.

Pharmacological treatment (n/%)
27 (75)
23 (67.6)
n.s.

Antidepressant
24 (66.7)
22 (64.7)
n.s.

Benzodiazepine
16 (44.4)
13 (38.2)
n.s.

Mood Stabilizer
20 (55.6)
13 (38.2)
n.s.

Antipsychotic
11 (30.6)
11 (32.4)
n.s.

Note: Values represent mean scores (SD between brackets) or otherwise
specified. No significant differences between groups were observed in χ 2 test
for categorical variables neither in t-test for quantitative ones (n.s)
Sick-leave/Unemployed: long-term sick-leave (>3 months) or for being unemployed,
DIB-R Diagnostic Interview for Borderlines-Revised, GAF Global Assessment
Funcioning, CGI-BPD Clinical Global Impression Scale for BPD

Table 2
2Summary of pre-post-intervention and 6-months follow-up measures and significant interactions on HLM analysis Post-hoc were obtained by HLM per each group. FAST Functioning Assessment Scale Test, BSL-23 Borderline Symptom List -23, HARS Hamilton Anxiety Rating Scale, MADRS Montgomery-Asberg Depression Rating Scale, BIS Barrat Impulsivity Scale treatment as usual (TAU) might have prevented the observation of more robust differences.CR
PE

Baseline
(n = 36)

Post-treatment
(n = 22)

Follow-up
(n = 20)

Post-hoc
tests

Baseline
(n = 34)

Post-treatment
(n = 24)

Follow-up
(n = 22)

Post-hoc
tests

HLM

Group Time
Group
x time

Psychosocial Functioning

FAST
37.86 (1.81) 36.86 (2.55)
29.55 (2.92) p = .018 42.94 (1.89) 38.01 (2.51)
35.61 (3.03) n.s.
n.s.
p = .004 n.s.

Clinical variables

BSL-23
42.32 (3.90) 42.56 (5.00)
34.91 (5.25) n.s.
40.42 (4.14) 38.89 (4.86)
39.59 (5.53) n.s.
n.s.
n.s.
n.s.

HARS
24.89 (1.94) 19.76 (2.62)
16.53 (2.61) p = .023 21.60 (2.02) 18.61 (2.65)
11.52 (2.87) p = .006 n.s.
p < .001 n.s.

MADRS
22.54 (1.60) 19.16 (1.83)
20.02 (1.98) n.s.
18.67 (1.65) 16.58 (1.85)
9.91 (2.13) p < .001 p = .009 p < .001 n.s.

BIS
64.53 (2.70) 65.93 (3.34)
57.14 (4.04) n.s.
70.59 (2.84) 67.92 (3.33)
67.06 (4.35) n.s.
n.s.
n.s.
n.s.

Neuropsychological variables

Memory
Index

-.10 (.11)
-.13 (.09)
-.15 (.11)
n.s.
.20 (.11)
.01 (.09)
.11 (.11)
n.s.
n.s.
n.s.
n.s.

Attention
Index

.01 (.13)
-.07 (.15)
-.18 (.15)
n.s.
.10 (.14)
.29 (.15)
.35 (.15)
n.s.
n.s.
n.s.
p
= .020

Executive
Index

-.04 (.20)
.08 (.24)
-.08 (.24)
n.s.
-.02 (.21)
.38 (.23)
.13 (.25)
n.s.
n.s.
n.s.
n.s.

t-test * < .05, ** < .01, *** < .001
Note: HLM analyses correspond to pre-, post-and follow-up measures.
AcknowledgementsThis study was supported by Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and by grants from Instituto de Salud Carlos III (PI11/00725, FIS PI09/90751, PI09/90773 and PI14/00214). One of the authors (Maria J. Portella) has a Miguel Servet research contract from the Spanish Agency "Fondo de Investigación Sanitaria" of the Spanish Ministry of Health (CP10/00393).Additional fileAdditional file 1:Figure S1. CONSORT checklist. (PDF 684 kb)Competing interestThe authors declare that they have no competing interests.Authors' contributionsAll authors contributed to the design of the study and reviewed drafts of the report. JCP, NP and JLC were responsible for the conception and overall supervision of the trial. NP, RA, AI, RR and AF managed the project, data gathering and data entry. JS, JS, MJP and MD gave technical support throughout the trial. AF, MJP and JCP did the data analyses. NP performed the literature research. JCP, MJP, NP and JLC wrote the first draft of the report, and were responsible for subsequent collation of inputs and redrafting. All authors read and approved the final manuscript.
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