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An australian audit of vaccination status in children and adolescents with inflammatory bowel disease

Children and adolescents with inflammatory bowel disease (IBD) are at increased risk of vaccine preventable diseases (VPD). This includes invasive pneumococcal disease and influenza. The primary aim of this study was to describe compliance with current Australian guidelines for vaccination of children and adolescents diagnosed with IBD. A secondary aim was to review the serological screening for VPD.

A random sample of patients (0-18 years at diagnosis), were selected from the Victoria Australia state based Pediatric Inflammatory Bowel Disease Register. A multi-faceted retrospective review of immunization status was undertaken, with hospital records audited, a telephone interview survey conducted with consenting parents and the vaccination history was checked against the primary care physician and Australian Childhood Immunization Register (ACIR) records. The routine primary childhood vaccinations and administration of the recommended additional influenza and pneumococcal vaccines was clarified.

This 2007 audit reviewed the immunization status of 101individuals on the Victorian Pediatric IBD database. Median age at diagnosis was 12.1 years, 50% were on active immunosuppressive therapy. 90% (38/42) [95% confidence intervals (CI) 77%; 97%] with complete immunization information were up-to-date with routine primary immunizations. Only 5% (5/101) [95% CI 2%; 11%] received a recommended pneumococcal vaccine booster and 10% (10/101) [95% CI 5%; 17%] had evidence of having ever received a seasonal influenza vaccine. Those living in rural Victoria (p = 0.005) and younger at the age of diagnosis (p = 0.002) were more likely to have ever received an influenza vaccine Serological testing, reviewing historical protection from VPD, identified 18% (17/94) with evidence of at least one serology sample.

This study highlights poor compliance in IBD patients for additional recommended vaccines. A multi-faceted approach is required to maximize protection from VPD in this vulnerable special risk population.

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The prevention of vaccine preventable diseases (VPD) in all individuals with inflammatory bowel disease (IBD) is increasingly recognised as important[13]. IBD rates have increased globally over the past 30 years, including in the Australian state of Victoria, where the incidence of Crohn's disease rose from 0.128 to 2.0 per 100 000 per year over a 31-year period (1971-2001)[4]. Immunosuppressive therapies such as corticosteroids and azathioprine have long been used in IBD management. Infliximab is one of the biologic anti-TNF-alpha antibody therapies, and an effective treatment for luminal and fistulising Crohn's disease, as well as those with a high inflammatory disease burden[5].

The risk of opportunistic infections in IBD is increased, particularly when on immunosuppressive therapies, including biologics such as infliximab[6, 7]. This risk is increased when combining two or more immunosuppressive medications[8, 9]. There are case reports of VPD in this vulnerable group, including severe varicella and pneumococcal pneumonia[10, 11]. Viral hepatitis is also a significant risk as shown in a number of studies[1214]. Cheveuax et al. found evidence of seroprotection from hepatitis B through immunization in 48.9% of IBD patients (N = 315 patients), with age at diagnosis (> 31 years), duration of disease over 7 years and a diagnosis of Crohn's disease, all associated with the lack of effective vaccination. It is important to consider screening for opportunistic infections as part of the clinical approach to IBD patients and maximise prevention of these infections where possible through immunization[15]. This has been detailed in a number of guidelines, including the European evidence based consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[16, 17].

In the state of Victoria, Australia, pediatric gastroenterologists care for the majority of children and adolescents with IBD. This is predominantly at the states two tertiary gastroenterology centres, The Royal Children's Hospital (RCH) and Monash Medical Centre (MMC), Melbourne. This management in a small number of patients is shared care with a general paediatrician, particularly for those patients living in rural areas. As there is centralised service delivery, Victoria has established a state-based IBD register for children and adolescents with IBD[4].

Vaccinations in early childhood are captured in a hand held person medical record and also on the Australian Childhood Immunization Register (ACIR) for those < 7 years of age. At the time of the study ACIR data was not accessible once a child was greater than 7 years. The Australian routine funded vaccines on the National Immunization Program are detailed in Table 1. Annual influenza immunization is also funded for special risk groups such as children and adolescents with IBD ≥ 6 months of age and vaccination offered from late February through to October. Many immunization guidelines, including those in Australia, are also now recommending additional pneumococcal vaccine for special risk patients, including those with IBD on immunosuppressive medication[18]. The treating gastroenterologist is often the main source of immunization advice in childhood and adolescent IBD, particularly if immunosuppressive therapy is commenced. At RCH there is an Immunization Drop-in Centre open business hours, staffed by nurse immunization specialists, where queries can be directed and vaccines administered on the day of clinic appointments. In Victoria, immunization nurse specialist or primary care physicians administer the majority of vaccines.

Table 1 Australian National Immunization Program (NIP) Schedule

The primary aim of this study was to describe the compliance with current Australian guidelines for vaccination of children and adolescents diagnosed with IBD current at the time of the study[19]. A secondary aim was to review the serological screening for VPD in patients with IBD.

A multi-faceted retrospective review of immunization status was undertaken, with the inclusion criteria being age 0-18 years at diagnosis and on the IBD register. There were a total of nine gastroenterologists working across the two tertiary units [RCH and MMC] at the time of the study. Hospital records of all participants were audited, with any vaccinations administered recorded in the outpatient notes and/or a medication chart if administered at the RCH Immunization Drop-in-centre. A telephone interview survey was conducted with consenting parents using the parent-held child immunization record. The vaccination history was checked against the primary care physician and ACIR records. The routine primary childhood vaccinations and administration of the recommended additional influenza and pneumococcal vaccines was clarified. Therapies were categorized into four groups: ASA derivatives (sulphasalazine, osalazine, mesalazine and balsalazide); oral corticosteroids (prednisolone); immunosuppressive agents (azathioprine, methotrexate) and biologics (infliximab). The RCH patient's hospital laboratory results were reviewed to identify if any baseline serological testing was performed to review the requirement for additional protection against VPD such as varicella and hepatitis B. Multiple sources were reviewed for any vaccine safety concerns or reports of adverse events following immunization, including: hospital records, general practitioner records and parent reports through interviews.

A random sample of 101 participants was taken from the IBD register. The random sample was generated using the statistical software STATA Version 10.0 (StataCorp, TX), which was also used for data analysis. This overall sample size calculation was based on the outcome of routine immunization up-to-date status by hospital medical record audit and allowed a determination of proportions within +/- 10% with 95% confidence. Proportions of up-to-date status and additional vaccines administered were compared using a Pearson chi-square test with point estimate odds ratios (OR) and 95% confidence intervals determined and a p value < 0.05 considered statistically significant. The study was approved by the Royal Children's Hospital Human Research Ethics Committee.

The study was conducted between July-November 2007. The participant demographics are detailed in Table 2. There only difference at baseline between those selected for the study, compared with the rest of the IBD database (N = 534), was proportionally more cases of indeterminate colitis in the database group [11% versus 3%; P = 0.01]. Participants had a median age at diagnosis of 12.1 years [range 1.9 to 17.8 years], 50% were female, and 75% had Crohn's disease. At the time of the immunization audit 43 participants were on 5-ASA derivatives: sulphasalazine (17), mesalazine (21), besalazine (4) and osalazine (1). Reviewing ongoing immunosuppressive therapies, there were 39 participants, some on multiple medications including: oral corticosteroids (7), azathioprine (36) and infliximab (7).

Table 2 Baseline Characteristics

One hundred and one hospital patient records were reviewed as detailed in the study flow diagram. (Figure 1) A telephone immunization survey was completed in 42% and primary care practitioner records obtained in 66% (33/50) of consenting participants. In those whom a complete telephone immunization survey was obtained, 90% (38/42) [95% confidence intervals (CI) 77%; 97%] were up-to date with routine primary childhood immunizations.

Figure 1
figure 1

Flow diagram of study participants.

For additional recommended vaccines, only 5% (5/101) [95% CI 2%; 11%] had received a recommended pneumococcal 'booster' and all were on active therapy including azathioprine (4) and infliximab (1). 10% (10/101) [95% CI 5%; 17%] had evidence of having ever received an influenza vaccination, 7% (7/101) [95% CI 3%; 14%] in the year of the survey. Those living in rural Victoria (Odds ratio 6.51 95% CI 1.33; 41.25, p = 0.005) and younger at the age of diagnosis (Pearson square for trend χ2 = 16.8; p = 0.002) were more likely to have received an influenza vaccine. The reasons for not having received an influenza vaccine (n = 33) included: not being aware of it (24%); concerned about side effects (24%) not necessary (15%); doctor did not offer it (6%), allergy (6%) and unspecified or other reason (25%).

Serological testing, reviewing historical protection from VPD, identified 18% (17/94) with evidence of at least one serology sample. Having serology testing was not associated with age (χ2 for trend = 1.7; p = 0.42). For the patients who had varicella serology, 18% (2/11) were negative. Pre-diagnosis varicella vaccine had been received by 8 participants, with an additional 30 confirming pre-diagnosis clinical infection. There were four participants (none currently on infliximab) who had hepatitis B serology documented, all were negative for hepatitis B surface antigen (HepBsAg) and seropositive for protective anti-HepB surface antibody. On Hepatitis B vaccination history, immunization was confirmed in 93% (39/42), including 86% (6/7) of those currently on infliximab. The quadrivalent human papillomavirus (4vHPV) vaccine had been administered to 14 young women. There were no documented safety concerns or flares of inflammatory bowel disease notified following immunization.

This study highlights good compliance with routine childhood immunizations, with the 90% up-to date status equivalent to the coverage rates of 93.6% at seen in Victoria, Australia overall[20]. The uptake of recommended additional vaccines such as annual influenza vaccination and pneumococcal vaccine boosters was low.

There are very few published studies on compliance guidelines on immunizations in IBD patients[2, 21]. In a United States immunization status survey of 169 adolescents and adults diagnosed with IBD, 86% were using immunosuppressive therapies[22]. On immunization status recall only 28% had received influenza and 13% pneumococcal vaccination. This compares with this study of influenza vaccination confirmed in 10% and additional pneumococcal vaccination 5%. When reviewing other special risk groups' compliance with recommended additional vaccines, a Victorian study of childhood cancer survivors found 47% had 'ever' had an influenza vaccine[23]. This compared to 27% of children < 7 years diagnosed with cystic fibrosis and managed at RCH who had received an annual influenza vaccine and 37% the additional recommended pneumococcal vaccine[24]. In addition in a study of preterm infants at two Victorian tertiary neonatal units, only 20% of infants with chronic lung disease had received an influenza vaccine[25].

The increased risk of morbidity and mortality from influenza infection in those with a chronic illness such as IBD is well described and confirmed in the 2009-10 HINI influenza epidemic[26, 27]. The response to influenza vaccine in patients receiving immunomodulatory therapy is variable, but usually satisfactory, as seen in an influenza vaccine sero-response study in 146 IBD patients, including those who were immunosuppressed[28]. The safety of influenza vaccine in IBD patients has been reviewed in a cohort of adult IBD patients following H1N1 influenza vaccination [29]. Four weeks after immunization, absence of a disease flare was observed in 96.7% patients with Crohn's disease and 95.6% with ulcerative colitis. The importance of physician recommendation for uptake of influenza vaccine, has been highlighted in a parental survey of children with a chronic illness[30].

Invasive pneumococcal disease (IPD) is also increased in those with an underlying chronic illness and on immunosuppressive therapies[31]. It is hence important to protect IBD patients from these VPD. Australia introduced a 7-valent conjugate pneumococcal vaccine (PCV7) on 1 Jan 2005, and the special risk guidelines current in 2007 recommended additional 7vPCV if under 10 years and 23 valent polysaccharide vaccine 23vPPV if > 10 years of age[18]. The response in adults to 23vPPV is variable and IBD patients on biologic therapies such as infliximab achieve lower protective titres[32]. Newer pneumococcal conjugate vaccines with increasing numbers of serotypes are becoming available internationally [33] and the 13 valent conjugate vaccine (PCV13) introduced into a number of countries including the United States and soon in Australia[34, 35]. Importantly the PCV13 vaccine is also being studied in adults > 50 years, and early results from phase III immunogenicity studies found an equivalent or better response than the 23vPPV[36]. Studies are also in progress to determine the efficacy of this vaccine in preventing community acquired pneumonia in adults > 65 years[37]. Studies of these newer PCV are required in special risk populations to assess both their safety and immunogenicity and help determine the best schedule to optimise IPD protection.

The risk of reactivation of diseases such as hepatitis B by these anti-TNFα antibody therapies, has led to recommendations of additional screening before commencing therapy[38, 39]. In this study 20% of participants had been treated with infliximab and other biologic therapies may be used in the future. Reassuringly, 93% (39/42) of those with comprehensive immunization records had documented evidence of hepatitis B vaccination. However, serological testing confirming a negative Hepatitis B infection status was conducted in only four patients. It is possible this is an underestimate as serology samples may have been sent to external laboratories. Another limitation of this serological evaluation was that it was conducted at only one of the two sites (RCH), but included 94% of subjects.

Live vaccines need to be considered with caution in patients who are immunosuppressed, due to the risk of vaccine associated morbidity such as seen with measles vaccine[40]. Varicella vaccine was introduced into Australian on 1 November 2005 for all children aged 18 months with a catch-up program at age 12-13 years. In this study 9% (4/42) were found to not have a history of clinical infection or immunization against varicella placing them at risk, similar to the 10% found in the United States IBD survey[22]. The morbidity of varicella in immunosuppressed patients has led to studies challenging the general recommendation that this live attenuated vaccines not be administered[41]. A case series of six young people with IBD safely receiving varicella vaccination whilst on infliximab therapy[42]. Despite small numbers of patients, it highlights the need to discuss the risk versus benefits of live attenuated vaccines on a case-by-case basis. Another approach is to review the immunization status at diagnosis and give all recommended vaccines (e.g. varicella, hepatitis B) before commencing immunosuppressive therapy. This, however, is becoming a smaller window of opportunity with the earlier use of immunomodulatory therapy in IBD.

A limitation of the study is the retrospective nature of the review in a state based IBD population. It was a representative sample of the database, but some IBD patients may have been excluded if seeing a private and/or adult gastroenterologist and not placed on the register. One major difficulty in conducting the study was uptake of 'opt in' consent, reflected by only 53% of adolescent and young adult patients being contactable. These are common issues in retrospective health related research and a number of surveys have explored the importance of exploring meaningful non-consent and considering 'opt out' consent[43, 44]. The study was powered for 100 audit participants, but the results were affected by the low recruitment for the telephone immunization status review. The reported proportions of participants receiving the additional pneumococcal and influenza vaccines may not reflect the actual immunization status due to this incomplete record obtainment. The study was also underpowered to detect differences in the characteristics of those who received influenza vaccine. Another limitation is that the ACIR only routinely captures and maintains immunization records in children < 7 years of age, so added little information in this predominantly adolescent population. Whilst ACIR can include additional vaccines (e.g. influenza and pneumococcal), this information is not always captured.

Suboptimal compliance with annual influenza vaccination and other additional vaccines recommended could be due to lack of awareness or acceptance of immunization guidelines for IBD patients. Engagement with subspecialists like pediatric gastroenterologists managing these complex patients is important. As the primary source of healthcare related advice for IBD patients, surveying their opinions regarding immunization would be helpful and lack of this information is a study limitation. An Australian survey of adult gastroenterologists found hepatitis B, influenza and pneumococcal vaccines were recommended infrequently and the window before significant immunosuppressive therapies commenced not always being utilised[45].

Strategies to optimise protection from VPD include education of both patients and their parents/carers, as highlighted by the reasons given for influenza vaccination not being received. Education itself needs to be supported by system changes and this may include immunization reminders, which in the form of cards, telephone and electronic, have all been shown to be effective[46, 47]. Protecting IBD patients through "cocooning", by ensuring parents and siblings are protected against VPD (e.g. influenza, pertussis and varicella), is also recommended.

This study highlights a high level of vaccination coverage with routine scheduled vaccinations, but poor compliance with current guidelines for influenza and pneumococcal vaccination in adolescents and children with IBD. Improving serological assessment prior to commencing immunosuppressive therapies can help minimise the risk of reactivation of VPD such as hepatitis B. An approach using both direct and indirect protective immunization strategies is required to maximise protection from vaccine preventable diseases in this vulnerable population.

Conflict of interests disclosure

NWC has investigator-led study support for a study of Guillain-Barre Syndrome Surveillance post H1N1 influenza vaccination [CSL] and been on a Pfizer [Wyeth] advisory board for pneumococcal vaccines and presented at conferences, for which his MCRI research fund has received honoraria. AGC has chaired an advisory board for GSK - rotavirus vaccine and chaired an advisory board for MSD - Infliximab in Crohn's disease. JPB sits on a data safety monitoring board for influenza vaccines [CSL] and MCRI has received conference travel reimbursement from GSK for Rotavirus vaccine conference presentation. MRO and DJSC have no conflicts of interests to declare.

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Thanks to all the participants and their parents/guardians who took part in the study. Thanks also to all the staff that helped with recruitment, including Ms Di Simpson (Gastroenterology Department RCH), staff in outpatient clinics and Immunization research assistants who helped undertake the audit (Catherine Lloyd-Johnson, Susi Rogers, Nicole Davidson)

Dr Nigel Crawford acknowledges support from a National Health and Medical Research Council (NHMRC) of Australia PhD Postgraduate Public Health Scholarship (437031)

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Correspondence to Nigel W Crawford.

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NWC and JPB conceived the study concept. All authors contributed to the study design and audit questionnaire. TCS provided the Victorian IBD database information. NWC undertook the statistical analysis and wrote the initial draft. All authors contributed to the draft review and have read and approved the final manuscript.

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Crawford, N.W., Catto-Smith, A.G., Oliver, M.R. et al. An australian audit of vaccination status in children and adolescents with inflammatory bowel disease. BMC Gastroenterol 11, 87 (2011).

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SNPs and Hox gene mapping in Ciona intestinalis

The tunicate Ciona intestinalis (Enterogona, Ascidiacea), a major model system for evolutionary and developmental genetics of chordates, harbours two cryptic species. To assess the degree of intra- and inter-specific genetic variability, we report the identification and analysis of C. intestinalis SNP (Single Nucleotide Polymorphism) markers. A SNP subset was used to determine the genetic distance between Hox-5 and -10 genes.

DNA fragments were amplified from 12 regions of C. intestinalis sp. A. In total, 128 SNPs and 32 one bp indels have been identified within 8 Kb DNA. SNPs in coding regions cause 4 synonymous and 12 non-synonymous substitutions. The highest SNP frequency was detected in the Hox5 and Hox10 intragenic regions. In C. intestinalis, these two genes have lost their archetypal topology within the cluster, such that Hox10 is located between Hox4 and Hox5. A subset of the above primers was used to perform successful amplification in C. intestinalis sp. B. In this cryptic species, 62 SNPs were identified within 3614 bp: 41 in non-coding and 21 in coding regions. The genetic distance of the Hox-5 and -10 loci, computed combining a classical backcross approach with the application of SNP markers, was found to be 8.4 cM (Haldane's function). Based on the physical distance, 1 cM corresponds to 39.5 Kb. Linkage disequilibrium between the aforementioned loci was calculated in the backcross generation.

SNPs here described allow analysis and comparisons within and between C. intestinalis cryptic species. We provide the first reliable computation of genetic distance in this important model chordate. This latter result represents an important platform for future studies on Hox genes showing deviations from the archetypal topology.

A recent phylogeny placed Tunicata as the sister group of vertebrates [1]. This new position rejects traditional views of a Tunicata – Cephalochordata – Vertebrata succession [26] and it casts new light on comparative studies [7]. The taxonomic status of C. intestinalis L., the tunicate species most widely used for research purposes, was recently resolved with the discovery of two cryptic taxa, named C. intestinalis spp. A and B [811]. This finding prompts careful re-evaluation of research data, as it is reasonable that literature concerning C. intestinalis sensu Linnaeus refers to both cryptic species. Affinities with vertebrates are visible in the body plan organization of ascidian larvae and, despite major morphological rearrangements during metamorphosis, they are also retained in sessile adults [12, 13]. Sequencing of C. intestinalis genome revealed an estimated number of protein-coding genes (15.852 over ca. 160 Mb genome length) similar to invertebrates and only about half of vertebrates [14]. Gene density is estimated to be 1 locus per 7.5 Kb (compared with 9 Kb in fruit fly and 100 Kb in human). C. intestinalis genes contain, on average, 6.8 exons. From the genomic point of view, the presence of several hundred genes having higher sequence similarity with Drosophila melanogaster and Caenorhabditis elegans than with vertebrates [14], as well as the small gene number, are indicative of species ancestry. Moreover, the genome is rich in AT (65%). Two derived features of the C. intestinalis genome are the presence of gene duplication events not detected in vertebrates, and the derived loss of ancestral genes that are conserved in chordates (e.g. paralogy of the Hox groups 7, 8 and 9) [14]. This latter phenomenon has been estimated to be around 35% and 45% more frequent in C. intestinalis than, respectively, in pufferfish and humans. Recent data on the congeneric species C. savignyi [15] revealed an impressive level of genomic variation, such that this species exhibits the "... highest rates of [...] polymorphisms ever comprehensively quantified in a multicellular organism". More specifically, C. savignyi shows a very high level of haplome-specific DNA (16.6%); this degree of variability between single haploid genomes originates from an enormous amount of various size indels throughout the genome [15].

Single nucleotide polymorphisms (SNPs) are one of the most important categories of genetic markers in the field of population genetics and human diseases. SNPs are base pair substitutions in the DNA of individuals [16], and are by far the most common type of molecular polymorphism in living organisms. Given this definition, single base pair insertions/deletions (indels) are not formally considered as SNPs. On the other hand, single base pair substitutions in cDNA are often included in this category of DNA variation, although they may result from errors in mRNA editing. About 1 SNP per Kb and 1 SNP per 125 bp occur in Homo sapiens [17] and Aedes aegypti [18], respectively. This very high variability represents a unique source of molecular markers. Biallelic SNPs are randomly distributed across the genome and have a low mutation rate (10-8 – 10-9) [19]: this property makes it easier to calculate mutational rates in SNPs than, for example, in microsatellites. Although a restriction to four character states makes SNPs less informative than microsatellites for linkage and population genetics, synonymous coding as well as non-coding SNPs are still useful markers for these applications since they are not under natural selection. Non-synonymous SNPs in coding DNA regions are mostly used to enhance understanding of the molecular genetic basis of phenotypic variation, with a particular relevance for human-disease research. Technical progress in SNP detection [20] has turned this polymorphism into the most reliable marker for genomic approaches. In spite of their importance, few data concerning occurrence of SNPs in natural populations of lower chordates (cephalochordates and tunicates) are available [21]. Such analysis would greatly contribute to a deeper knowledge of genetic variability in evolutionary key model organisms, in particular when research is based on natural populations. In C. intestinalis, allelic polymorphism is equal to 1.2% on average, and it may reach peaks of 10–15% within short (100 bp) regions [14], although these data are still matter of debate [15]. Large-scale analysis in C. savignyi revealed an average SNP heterozygosity of 4.5%, with a Ts/Tv ratio of 2.45 and a quasi- equal distribution of the various types of transversions [15].

Genes belonging to the Hox family of transcription factors are control leaders in the definition of the antero-posterior axis of all bilaterians analyzed so far. Usually, Hox genes are structured in chromosomal clusters displaying an ordered succession of paralogy group members. A general dogma states the colinearity rule(s): more 3' located genes possess more anterior and earlier onsets of expression (spatial and temporal colinearity, respectively). Recent studies in tunicates [22, 23] and in the sea urchin Strongylocentrotus purpuratus [24] revealed that coordinated spatial expression of Hox genes persists even in presence of a rearranged distribution of paralogy groups within the cluster. Therefore, understanding the evolutionary and functional scenarios of unclustered or unconventionally clustered Hox genes is a crucial task.

Herein, we studied SNP occurrence in C. intestinalis sp. A and sp. B. We developed exon-primed-intron-centered (EPIC) [25] primers in order to allow inter-specific genetic comparisons in 2 coding and 10 non-coding regions. For two adjacent Hox genes (Hox-10 and -5) displaying an inverted position, genetic distance and linkage disequilibrium (LD) were calculated using SNP markers in a backcross panel.

Results and discussion

DNA amplification and detection of SNPs

A total of 16 EPIC and 2 non-EPIC primer pairs were PCR-tested on 30 genomic DNA samples from different C. intestinalis sp. A populations. Twelve primers were used to amplify reliable products (Fig. 1), ranging from 357 to 1340 bp length (Table 1). Consequently, we analyzed a total of 7966 bp: 5953 from intronic nuclear regions, 1498 from two nuclear exons (Hox13 and Gsx) and 515 from the mt-DNA COI (cytochrome oxydase subunit I) gene. We detected 128 transition/transversion SNPs (110 in non-coding regions) and 32 single base indels (29 in non-coding regions).

Figure 1
figure 1

Structure and location of loci. Exon-intron structure and genomic location of genic loci used for analyses. Empty boxes: amplified regions. Chromosomes are numbered according to [28]. Distances between genes are indicative. All data are inferred from C. intestinalis sp. A.

Table 1 Nucleotide polymorphisms in C. intestinalis sp. A loci.

No SNPs were observed in the mitochondrial COI gene, 3 in the Hox13 and 15 in the Gsx coding regions. Our data indicate that the observed transition/transversion (Ts/Tv) ratio in the coding regions analyzed in this study is equal to 5.0 (Table 1). The Ts/Tv (1.37) observed within C. intestinalis sp. A coding and non-coding regions is considerably lower than the ratio found in C. savignyi (2.45) [15]. This difference is likely due to the regions here analyzed, namely non-coding intragenic and coding genic, whereas previous estimations were based on all genome sequences [15, 16, 26, 27].

SNP frequency distribution in the non-coding DNA amplified regions displays a high degree of variability. Hox-1, -2, -10, -5 and EvxA genes are sequentially located along the chromosome #1 [23, 28]. Notably, the highest SNP frequency occurs within those Hox genes (Hox-5 and -10) that have lost the archetypal genomic organization.

Types and frequencies of SNPs

Overall, SNPs represent 48% of sequence polymorphisms in the 8 Kb region analyzed in this study, with a frequency of 1.61 SNPs every 100 bp (Table 1). The remaining 52% of polymorphisms consists of 1 bp or longer indels and multiple nucleotide polymorphisms. The high level of non-SNPs fits well with previous estimates in other multicellular organisms [15].

The occurrence of SNPs partially reflects the different C. intestinalis populations. For instance, all nine individuals from the Fusaro Lagoon display a T ↔ C transition at position 114 of the Hox5 locus, while only two individuals present an A ↔ G transition at position 111. In particular, most of non-synonymous changes are carried by individuals from geographically disjunct populations (e.g. California, Japan and Italy). Strain-specificity of non-synonymous SNPs is generally assumed to reflect adaptive responses to distinct environmental conditions [2931].

Concerning non-coding DNA regions, the overall number (∑Ts = 59; ∑Tv = 51 – Table 1) and frequency (fTs = 0.01; fTv = 0.008 – Table 1 and Fig. 2) of transition SNPs is slightly predominant. Furthermore, analysis of nuclear exonic regions indicates that the aforementioned transition prevalence is even more evident in the coding DNA (83% of total SNPs). This is not surprising since it is generally accepted that the frequently occurring 5-methylcytosine de-amination reactions cause transition overrepresentation in genomes [32]. Significant differences in the Ts/Tv ratio between non-coding and coding DNA regions have been previously observed [27], indicating that transitions occur more frequently in coding regions. Polymorphisms along the 1.5 Kb nuclear coding DNA causes 4 synonymous and 12 non-synonymous changes (Table 2). The synonymous/non-synonymous (S/nS) ratio (0.333) here reported is in contrast with the average S/nS ratio found in C. savignyi (5.16) [15]. It is assumed that overrepresentation of non-synonymous nucleotide changes is strictly associated with adaptive evolution [33, 34]. In agreement, this mechanism may have played a crucial role in functional divergence of Hox genes after cluster duplications along the vertebrate lineage [35]. Being tunicates the closest relatives of vertebrates, our data lend support to the hypothesis that adaptive evolution on Hox and ParaHox genes was already active in basal chordates.

Figure 2
figure 2

SNPs in Ciona intestinalis sp. A. Percentage of transversion and transition SNPs in C. intestinalis sp. A. Values outside parenthesis indicate the total percentage of each substitution type calculated over all DNA polymorphisms. Values within parenthesis indicate the percentage of each substitution type calculated, respectively, over non-coding and coding regions.

Table 2 C. intestinalis spp. A & B synonymous and non-synonymous substitutions.

Among different genes herein analyzed, non-coding DNA SNPs are highly variable in types and frequency. Transitions are predominant in 4/10 loci, while transversions prevail in 5/10. In one case (Hox2), Ts/Tv ratio is equal to one. The two transition substitution types are comparably represented (57.6% A ↔ G, 42.3% T ↔ C). Concerning transversions, A ↔ T substitutions are prevalent (70.6%), while A ↔ C, G ↔ C and G ↔ T are underrepresented (13.7%, 7.8%, 15.7%, respectively). However, 46.9% of all A ↔ T transversions are from a single intron (Hox10) and all four types of transversions are never observed at a single locus.

Twenty-nine 1 base-pair indels were found in the analyzed non-coding loci, and 3 in the Hox13 coding region. All indels in coding DNA were found in only one individual from an Adriatic sea population. Whether this data reflects partial or total loss of function of Hox13 remains elusive. Unlike coding sequences, all analyzed non-coding regions present a clear quantitative relationship between SNPs and 1 bp indels (Fig. 3), in support of some kind of structural and/or functional correlation between mechanisms leading to the appearance of different types of polymorphism [14].

Figure 3
figure 3

Correlation between SNPs and 1 bp Indels distribution. Quantitative relationships between SNPs and 1 bp indels in 13 genic loci of C. intestinalis sp. A. Except for Gsx and Hox13 coding sequences, the two types of polymorphism appear to be tightly correlated.

SNPs in Ciona intestinalis sp. B

All pairs of EPIC primers used to amplify C. intestinalis sp. A genomic DNA were successfully tested in C. intestinalis sp. B (Table 3). Among them, 5 associated with non-coding (Hox-1, -2, -5, -10 and Xlox - 2503 bp) and 2 with coding regions (Hox13 and COI - 1111 bp) have been used to investigate SNP presence, using 5 to 10 specimens for each locus. In total, 62 SNPs were detected (41 in non-coding, 21 in coding regions). On average, 1.89 SNPs were detected every 100 bp in coding regions, compared with 0.27 SNPs every 100 bp in the same C. intestinalis sp. A loci. In the cryptic species B, Hox13 SNP frequency is higher than the one detected in non-coding regions (f = 0.018 vs 0.016). SNPs cause 1 synonymous and 6 non-synonymous amino acidic changes at the Hox13 locus, and 2 synonymous and 5 non-synonymous at the COI locus (Table 2). Previous analysis indicated the presence of a single mitochondrial haplotype shared by all Mediterranean populations of C. intestinalis sp. A. Conversely, C. intestinalis sp. B displays a variable COI in north European seas, suggestive of fixed populations [8]. Present results support the idea that the actual status of C. intestinalis sp. A in the Mediterranean Sea is due to a colonization by a mitochondrial variant, as well as the existence of more structured C. intestinalis sp. B populations [8].

Table 3 Nucleotide polymorphisms in C. intestinalis sp. B loci.

Among SNPs in non-coding regions, the Ts/Tv ratio is equal to 1.56, and so it is similar to the ratio detected in sp. A (1.18). A ↔ G are more frequent than C ↔ T. A ↔ T are the most common transversion. The ratio of SNP mutations over total polymorphisms (∑SNPs/∑PS) is similar in the two cryptic species [∑SNPs/∑PS = 0.48 (sp. A) and ∑SNPs/∑PS = 0.39 (sp. B)].

In conclusion, all EPIC and non-EPIC primers that were designed taking advantage of the genome sequence of C. intestinalis sp. A, perfectly amplify homologous loci in C. intestinalis sp. B. Opposite to coding regions, frequency and Ts/Tv ratio in non-coding regions are very similar. Altogether, data are suggestive of genome behavior in the two cryptic species with shared and divergent traits.

Genomic location

The organization of the Hox cluster in C. intestinalis [23] is characterized by an atypical arrangement. Two main differences can be observed: a) the cluster is broken (Hox-12 and -13 are located on a different chromosome) and b) paralogy groups do not respect the canonical 3' → 5' succession (Hox-4 and -5 are separated by Hox10) (Fig. 1). In this context, variability at these loci acquires a peculiar relevance. Our analysis shows (Table 1) that, among all analyzed loci (and among the same subset of specimens), Hox5 and Hox10 display the highest nucleotide diversity (π Hox5 = 0.0241; π Hox10 = 0.038) in non-coding regions. Similarly, the total number (∑ Hox5 = 25; ∑ Hox10 = 35) and frequency (f Hox5 = 0.052; f Hox10 = 0.066) of SNPs are the highest ones. In these loci, the number of observed transition substitutions is the highest among all genes analyzed, while the number of A ↔ T transversions is notably overrepresented in the Hox10 non-coding region (15 vs 3.4 on average). Tamura and Nei (TN93) and Lake's Paralinear (L'sP) genetic distance calculated within the same sub-sample of specimens, concordantly assign higher values (TN93 Hox5 = 0.004; TN93 Hox10 = 0.004; L'sP Hox5 = 0.004; L'sP Hox10 = 0.003) to Hox5 and Hox10 than to any other loci screened for SNPs (Table 1).

The Hox2 and Hox4 genes have retained the archetypal genomic topology within the genome of C. intestinalis sp. A. These loci display a low SNP frequency (∑ Hox-2,-4 = 3; f Hox-2,-4 = 0.0034). More generally, the total number of polymorphisms (including indels and multiple base polymorphisms – ∑PS = 5) and nucleotide diversity (∑Hox-2,Hox-4= 0.0022) is notably low.

Haplotypes structure, linkage disequilibrium and genetic mapping

With the aim to genetically characterize populations of C. intestinalis sp. A inhabiting the Fusaro lagoon (FuI) and the harbour of Castellamare di Stabia (CdS) (Tyrrhenian Sea, Italy), we have identified 11 distinct haplotypes for the Hox5 locus, and 7 for the Hox10 one (Fig. 4). Specimens carrying the most frequent haplotypes (named F5-2, C5-1, F10-1, C10-3) have been selected as parental individuals in a backcross for SNP-based LD and genetic mapping of this region. In four (3.4%) of 120 backcross individuals, analysis failed to detect any SNPs in both loci. In addition, detection of Hox5 and Hox10 SNPs failed in, respectively, 6 (5.0%) and 4 (3.4%) individuals. The final matrix consisted of 116 samples, using a 50% threshold for missing genotypes. Observed vs expected heterozygosity was calculated with Haploview using phased haplotype files. We evaluated LD in the backcross generation using the Lewontin's D' [36] and correlation factor r2 [37].

Figure 4
figure 4

Hox5 and Hox10 haplotypes. Maximum Parsimony trees of Hox5 (A) and Hox10 (B) haplotypes in Fusaro (F) and Castellamare di Stabia (C) populations. Individuals carrying the most common haplotypes (F5-1/C5-1 and F10-1/C10-3) were used as parents to generate the backcross progeny.

Linkage disequilibrium [D'] between genic loci is equal to 0.92, and r2 is 0.74 (Fig. 5). The coefficient r2 increases in the backcross generation from the value detected in F0 (0.67). A genetic map was generated anchoring SNP loci to the same chromosome with MapMaker/exp v.3. The obtained cM value (according to Haldane's function) is 8.4, with a 28.59 threshold between the two loci. Linkage was correlated with physical distances by using a genome browser (C. intestinalis v2.0, Joint Genome Institute) [14]. Being Hox10 and Hox5 separated by 331799 Kb, including some unresolved nucleotide stretches (N), 1 cM corresponds to 39.5 Kb (Fig. 6).

Figure 5
figure 5

Linkage disequilbrium. Linkage disequilibrium |D'| (A) and r2 (B) calculated between Hox5 and Hox10 loci using three SNP markers per locus. Values are only referred to orange blocks. (C) Haplotypes and their population frequency. Letter blocks correspond to the six SNP types. Thin lines connect haplotypes with a frequency > 0.1%; thick lines connect haplotypes with a frequency > 10.0%. The recombination D' value between the two blocks is shown.

Figure 6
figure 6

Genetic and physical map. Genetic and physical distances between Hox10 and Hox5 loci computed using 3 SNP markers per gene in the backcross generation. Numbers following SNPs indicate the substitution position within the sequence. Physical distance was inferred from the JGI C. intestinalis v.2.0 genome sequence.

The recombination ratio for the Hox10/Hox5 region in C. intestinalis sp. A is consistent with previous results, despite some ambiguity concerning the taxonomic status of parents [38]. In the highly polymorphic Fu/Hc locus of the colonial tunicate, Botryllus schlosseri, 1 cM corresponds to approximately 90 Kb [39]. Hence, the high recombination rate of the Hox10/Hox5 region is suggestive of a peculiar variability, and it provides an interesting point of discussion about disrupted topology of Hox clusters.

SNP reliability

Different methods were used in order to assess the validity of the SNPs analyzed in the present paper. First, only SNPs confirmed by two independent PCR, three clones for each PCR, were kept for further analysis. This first step was applied in both C. intestinalis sp. A and sp. B. Second, we analyzed segregation ratios in the FuI/CdS F1 crosses. All SNPs displayed the expected 1:1 ratio of Mendelian inheritance. Therefore, all data herein reported are reliably not due to sequencing artifacts.

We identified 128 SNPs through sequencing of 8 Kb genomic DNA of C. intestinalis sp. A. All primers used to amplify genomic DNA were successfully tested in C. intestinalis sp. B, allowing inter-specific comparison. As expected, SNP frequency is lower in coding than in non-coding regions [40, 41]. Also, SNP frequency is not constant in intronic DNA. Variability likely depends on the genomic location of analyzed sequence. In particular, we identified a highly polymorphic region in correspondence of Hox5 and Hox10, two genes that have inverted their paralogy group position within the typical topology of Hox clusters. The dominance of non-synonymous vs synonymous SNPs in Hox coding regions suggests that adaptive evolution is acting on these genes. In order to establish the genetic map of this region, we performed a SNP-based approach to measure cM distance between two Hox genes in a backcross generation. We calculated linkage disequilibrium and correlated genetic and physical distances.

In this report, we analyzed SNP occurrence in C. intestinalis sp. A at intra- and inter-population levels, providing an important source of genetic markers for linkage, population and comparative studies. Our data indeed represent a further step toward the establishment of a unique integrative system for comparative genomics in chordates, consisting of two cryptic (C. intestinalis sp. A & B) and one congeneric (C. savignyi) species.

We also calculated the genetic distance within a genomic region of particular interest. This data will contribute to in-depth investigations concerning the mechanisms the maintain Hox colinearity in absence of a coordinated genomic organization.

Collection of Ciona intestinalis sp. A and B specimens

Individuals of Ciona intestinalis sp. A were collected in the following localities: Fusaro Lagoon (Italy), Castellamare di Stabia (Italy), Villaggio Coppola (Italy), Taranto (Italy), Venice (Italy), Lake Timsah (Egypt) and Alicante (Spain). Californian and Japanese sequences were obtained from the JGI Ciona intestinalis v2.0 genome [42] and the Ghost Database [43].

Ciona intestinalis sp. B specimens were obtained from the following localities: Plymouth Sound and Edinburgh (United Kingdom), Brest (France), Breskens Harbour (The Netherlands) and Fiskebäckskil (Sweden).

DNA amplification and sequencing

Genomic DNA was extracted as previously described [8]. Amplification of DNA fragments was performed as in [8], except for the following loci: Hox-1, -2, -10, EvxA, Xlox, Wd-40 and ci0100134706 (see Table 4 for details). PCR products were extracted and purified using the QIAquick Gel Extraction Kit (Qiagen), and then cloned into TOPO TA Cloning Vector (Invitrogen) following manufacturer's instruction. Two independent PCR and three clones for each gene were sequenced using the Applied Biosystems 3730 DNA Analyzer Apparatus at the Molecular Biology Service (SBM) of the Stazione Zoologica "A. Dohrn" in Naples. Sequences were automatically aligned using ClustalW [44] and hand-checked with Bioedit v. [45].

Table 4 Markers, primers and thermal cycle conditions.

SNP discovery and analysis

SNPs were identified as sequence differences in the alignment. Only SNPs detected in all different trials were considered valid. All analyses were performed using DAMBE v.4.5.33 [46] and DnaSP v.4.0 [47]. Maximum Parsimony trees were inferred using MEGA v.3.1 [48] with 1000 bootstrap replications. To facilitate detection of SNPs for backcross genotyping, we have chosen 3 SNPs for each of the two Hox genes. Marker selection was done considering a) distance between SNPs (primer design), b) character polymorphism (detection of base changes), and c) F1 inheritance (transmission probability). According to these criteria, SNPs in position 114, 158, 328 were selected for Hox5, and those in position 108, 161, 279 for Hox10 (Table 5).

Table 5 SNP primers and FuI vs CdS polymorphic sites.

Fusaro/Castellamare di Stabia hybrids were cultured according to a published protocol [49], with modifications.

Linkage disequilibrium and Genetic mapping

SNP oligos used to perform the backcross analysis are described in Table 5. Sample reactions were prepared in 10 μl containing 5 μl of SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems, Foster City, CA., USA), 3 μl of PCR products, 1 μl of specific primers, 1 μl of deionized water. Thermal cycling was performed on a MJ DNA Engine PTC 200 at SBM, following standard procedure using an annealing temperature of 42°C. Post-extension treatment was performed using the Applied Biosystems 3730 DNA Analyzer. Data were analyzed by eye with GeneMapper v.3.7 (Applied Biosystems).

The metric D is a quantitative measure of allelic association. Given the two sites 1 and 2, x12 is the frequency of the corresponding haplotype and p1, q2 are the marginal allele frequencies. Hence, D = x12 – (p1)(q2) [50]. D' is obtained by normalising D over the theoretical maximum D, given the specific allele frequencies, such that D' = D/Dmax [36] Finally, the correlation factor r2 is equal to D2/(p1)(q2).

Normalized linkage disequilibrium measure [D'] and the correlation coefficient (r2) were calculated and visualized using HaploView v.3.32 [51] with a phased genotype matrix. Genetic distance (cM) between Hox-5 and -10 loci was calculated using MapMaker/EXP v.3.0 [52] with a threshold (LOD) greater than 3 and ordered with a LOD score of 1.44 [38]. The genetic distance was calculated using Haldane's mapping function [53].

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Authors wish to thank: J. Bishop (MBL, Plymouth) for providing C. intestinalis sp. B specimens, the Marine Resources for Research Service at SZN for culturing assistance; Francesco Esposito for providing live food for ascidians; C. Bowler and M. Idris for comments and suggestions. This research was supported by an OPEN University PhD programme and a grant from the European Union (EU) (Marine Genomics Europe, WP36 "The genetic identity of species").

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Correspondence to Luigi Caputi.

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LC (corresponding author) performed acquisition and analysis of data and drafted the manuscript; MB and EB developed the SNP detection procedure; NA gave a fundamental help in revisiting and improving the quality of the manuscript; PS is the PI who conceived and coordinated the project, and made important contributions to the text. All authors read and approved the final manuscript.

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Caputi, L., Borra, M., Andreakis, N. et al. SNPs and Hox gene mapping in Ciona intestinalis. BMC Genomics 9, 39 (2008).

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  • Linkage Disequilibrium
  • Cryptic Species
  • Backcross Generation
  • Base Pair Substitution
  • Paralogy Group

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