Gene silencing of the tick protective antigens , Bm86 , Bm91 and subolesin , in the one-host tick Boophilus microplus by RNA interference The use of RNA interference -LRB- RNAi -RRB- to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick , Boophilus microplus , has not been reported . We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens : Bm86 , Bm91 and subolesin . Gene-specific double-stranded -LRB- dsRNA -RRB- was injected into two tick stages , freshly molted unfed and engorged females , and specific gene silencing was confirmed by real time PCR . Gene silencing occurred in injected unfed females after they were allowed to feed . Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs , but not in adults that developed from these larvae . dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition , demonstrating that unprocessed dsRNA was incorporated in the eggs . Eggs produced by engorged females injected with subolesin dsRNA were abnormal , suggesting that subolesin may play a role in embryonic development . The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis . 1 Introduction The cattle tick Boophilus microplus is an important pest of cattle in subtropical and tropical regions of the world -LRB- Estrada-Pena et al. , 2006 -RRB- . Although all Boophilus species including B. microplus have been reclassified to the genus Rhipicephalus -LRB- Murrell and Barker , 2003 -RRB- , we maintain use of the previous genus assignment for the purpose of biological clarity . Besides causing direct production losses and leather damage , B. microplus transmits several cattle pathogens , including Babesia bovis , Babesia bigemina and Anaplasma marginale . Control of B. microplus depends primarily on the use of acaricides or genetically resistant animals . Both approaches have limitations , including development of acaricide resistance , environmental contamination , pesticide residues in food products , the expense of developing new pesticides and the difficulty of producing tick-resistant cattle while maintaining desirable production characteristics -LRB- Willadsen , 2004 -RRB- . Other tick control approaches which show promise are the use of biological control agents -LRB- reviewed by Samish et al. , 2004 -RRB- and anti-tick vaccines -LRB- reviewed by de la Fuente and Kocan , 2003 ; Willadsen , 2004 -RRB- . Two commercial vaccines have been developed for control of tick infestations on cattle , TickGARD Plus ® in Australia and Gavac ® in Cuba . Both are based on the same recombinant antigen named Bm86 , a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells -LRB- Gough and Kemp , 1993 -RRB- . This ` concealed ' antigen is not naturally exposed to the host 's immune system . Lysis of midgut digest cells occurs in ticks that feed on vaccinated cattle , resulting in leakage of blood meal into the tick hemocoel . The overall effect of the vaccine is on engorging female ticks and includes a decrease in the number and weight of replete ticks and oviposition . While Bm86-based vaccines were effective against several other tick species , including Boophilus annulatus -LRB- Fragoso et al. , 1998 ; Pipano et al. , 2003 -RRB- , Boophilus decoloratus , Hyalomma anatolicum anatolicum and Hyalomma dromedarii , they were not effective against Amblyomma variegatum , Amblyomma cajennense and Rhipicephalus appendiculatus -LRB- de Vos et al. , 2001 ; Rodriquez and Jongejan , unpublished data -RRB- . Another ` concealed ' antigen , Bm91 , was shown to increase the efficacy of the Bm86 vaccine for B. microplus when co-administered -LRB- Willadsen et al. , 1996 -RRB- . Bm91 is a low-abundance glycoprotein located in the salivary glands and midgut of B. microplus -LRB- Riding et al. , 1994 -RRB- . The protein , a homologue of carboxydipeptidase , shares many biochemical and enzymatic properties with mammalian angiotensin converting enzyme , but its natural substrate has not been identified -LRB- Jarmey et al. , 1995 -RRB- . More recently , a protein first labeled 4D8 and now called subolesin was identified through Ixodes scapularis cDNA expression library immunisation as a potential tick-protective antigen . Immunisation trials using recombinant subolesin caused reductions of larval , nymphal and adult I. scapularis infestations -LRB- Almazan et al. , 2003 , 2005a , b -RRB- . The protein was later found to be conserved among ixodid tick species . Characterisation of its function by RNA interference -LRB- RNAi -RRB- in I. scapularis , Amblyomma americanum , Rhipicephalus sanguineus , Dermacentor variabilis and Dermacentor marginatus suggested involvement of this protein in the modulation of blood ingestion and reproduction . Therefore , the generic name `` subolesin '' was introduced for the 4D8 proteins and `` subA '' for the subolesin-encoding gene -LRB- de la Fuente et al. , 2006a -RRB- . Gene silencing by RNAi of subA and Rs86 , the homologue of Bm86 , in R. sanguineus , revealed a synergistic effect in which the expression of both genes was silenced and resulted in decreased tick attachment , feeding and oviposition -LRB- de la Fuente et al. , 2006c -RRB- . RNAi is a conserved post-transcriptional gene-silencing mechanism present in ticks and a wide range of eukaryotes in which double-stranded RNA -LRB- dsRNA -RRB- triggers a sequence-specific degradation of cognate mRNAs . It has been an effective tool to study the function of tick proteins at the tick -- pathogen interface in a number of three-host tick species such as in I. scapularis which transmits Anaplasma phagocytophilum and Borrelia burgdorferi -LRB- Pal et al. , 2004 ; Ramamoorthi et al. , 2005 ; Sukumaran et al. , 2006 -RRB- . RNAi was used to study the function of several tick salivary gland proteins involved in feeding of A. americanum -LRB- Aljamali et al. , 2003 ; Karim et al. , 2004 -RRB- , Haemaphysalis longicornis -LRB- Miyoshi et al. , 2004 -RRB- and I. scapularis -LRB- Narasimhan et al. , 2004 -RRB- . The inducer of RNAi , dsRNA , is injected into nymphal or adult ticks which are then allowed to feed normally . Capillary feeding of dsRNA -LRB- Soares et al. , 2005 -RRB- or incubation of isolated tick tissues with dsRNA -LRB- Aljamali et al. , 2003 ; Karim et al. , 2005 -RRB- are other methods used successfully to silence genes in ticks . These studies suggest that RNAi is systemic and effects gene silencing throughout the tick . In one-host Boophilus ticks , with all life stages feeding and molting on the same host , alternative strategies are required to conduct gene silencing by RNAi as compared with three-host tick species which spend their non-parasitic life stages off-host . Herein , we examined two methods of dsRNA delivery and its effect on the one-host tick B. microplus : -LRB- i -RRB- injection of dsRNA into freshly molted females and -LRB- ii -RRB- injection of dsRNA into engorged females . The latter method caused gene-specific silencing in the oviposited eggs and larvae that hatched from these eggs . We believe this is the first report of the silencing of the expression of Bm86 , Bm91 and subolesin in Boophilus ticks as quantified by real-time RT-PCR using two routes of dsRNA delivery . 2 Materials and methods 2.1 Experimental animals Three Holstein -- Friesian calves , 5 months of age -LRB- # 7793 , # 7794 and # 7799 -RRB- , were used . All animals had no previous exposure to ticks . All tick feedings were approved by the Animal Experiments Committee -LRB- DEC -RRB- of the Faculty of Veterinary Medicine , Utrecht University -LRB- DEC No. 0111.0807 -RRB- . 2.2 Ticks and tick feeding Boophilus microplus ticks originating from Mozambique were provided by ClinVet International -LRB- Pty -RRB- , Bloemfontein , South Africa . The ticks were subsequently maintained on cattle at our tick rearing facility . Larvae were kept off-host at 20 °C with 95 % relative humidity . Patches used for tick feeding with inner dimensions of 60 × 85 mm and sewn to an open cotton bag were glued to the shaved back of calf # 7793 and # 7794 using Pattex ® contact glue -LRB- Henkel Nederland , Nieuwegein , The Netherlands -RRB- . A batch of larvae eclosed from 1500 mg of pooled eggs oviposited by 25 females -LRB- approximately 24,000 larvae -RRB- , was divided on day 0 between two patches on calf # 7794 . Since males appear earlier from the nymphal stage than females , approximately 500 unfed males were collected on days 13 and 14 and 600 unfed females on days 14 and 15 and incubated at 27 °C with 95 % relative humidity . Freshly molted females were subjected to injection of dsRNA on day 15 as described below . For gene silencing in engorged females and their progeny , 25 engorged females with an average weight of 261 mg -LRB- 248 -- 272 mg -RRB- fed on calf # 7794 were collected on day 21 . Larvae which hatched from eggs laid by mock-injected , Bm86 - and Bm91-dsRNA injected engorged females were fed in three patches on calf # 7799 . 2.3 RNA extraction and synthesis of tick cDNA for dsRNA preparations The viscera of five partially fed B. microplus females were dissected in ice-cold PBS and immediately stored in 1 ml Tri reagent -LRB- Sigma -- Aldrich , Zwijndrecht , The Netherlands -RRB- at − 80 °C . Total RNA was isolated and subsequently purified using the Nucleospin RNA II kit -LRB- Macherey-Nagel , Düren , Germany -RRB- in accordance with the reagent and kit manufacturer 's directions . Total RNA concentration was determined spectrophotometrically and the material was stored at − 80 °C before use . Complementary DNA was made with the Revertaid first strand cDNA synthesis kit -LRB- Fermentas , St. Leon-Rot , Germany -RRB- in accordance with the manufacturer 's protocol using random hexamer primers . Control reactions were performed using the same procedures but without RT as a control for DNA contamination in the RNA preparations . 2.4 Cloning and sequencing of the B. microplus subolesin gene Cloning and sequencing of the subA gene from the Mozambiquan B. microplus strain was performed as described elsewhere -LRB- de la Fuente et al. , 2006a -RRB- . The sequence has been submitted to GenBank and can be retrieved under Accession No . DQ923495 . 2.5 dsRNA synthesis Oligonucleotide primers containing T7 promotor sequences at the 5 ′ - end for in vitro transcription and synthesis of dsRNA were used to PCR-amplify cDNA encoding B. microplus Bm86 -LRB- 421 bp -RRB- , Bm91 -LRB- 417 bp -RRB- and subolesin -LRB- 381 bp -RRB- . All oligonucleotide primers used in this study were synthesised by Isogen Life Science , IJsselstein , The Netherlands and their sequences are shown in Table 1 . PCR products were purified using the GfX PCR purification kit -LRB- Amersham -RRB- and used as templates to produce dsRNA using the T7 Ribomax Express RNAi system -LRB- Promega , Leiden , The Netherlands -RRB- . dsRNA aliquots were stored at − 80 °C until used . 2.6 Injection of ticks with dsRNA Freshly molted females were placed on double-sided sticky tape with the ventral sides upwards and injected into the anal aperture with 0.5 μl Bm86 , Bm91 or subolesin dsRNA alone or a combination of Bm86 and subolesin dsRNA -LRB- 6 -- 9 × 1011 molecules/μl -RRB- using a 10 μl syringe with a 33 G needle -LRB- Hamilton , Bonaduz , Switzerland -RRB- mounted on a MM3301-M3 micromanipulator -LRB- World Precision Instruments -LRB- WPI -RRB- , Berlin , Germany -RRB- and connected to an UMPII syringe pump -LRB- WPI -RRB- . The tip of a 27 G needle was used to slightly pierce the anal aperture before the 33 G needle was inserted . The dsRNA was dissolved in injection buffer -LRB- 10 mM Tris -- HCl , pH 7 and 1 mM EDTA -RRB- . A control group was injected with injection buffer alone . The ticks were placed in an incubator at 27 °C with 95 % relative humidity for 3 -- 10 h following injection , before they were examined for mortality and placed in five separate patches , one for each group , on calf # 7793 . One hundred male ticks were placed in each patch simultaneously with the injected females . The ticks were checked twice daily and collected when they dropped from the host . Ticks still attached 14 days after the dsRNA-injection -LRB- day 29 after application of the larvae -RRB- were removed manually . All ticks were weighed separately within 1 h after collection and stored individually in 1.5 ml Eppendorf tubes with pierced lids at 27 °C and 95 % relative humidity for oviposition . For the second experiment , engorged B. microplus females were injected with 5 μl of Bm86 , Bm91 or subolesin dsRNA -LRB- 1 -- 2 × 1012 molecules/μl -RRB- or injection buffer alone in the right spiracular plate within 6 h after dropping off the host , using the same methods as described above , or left uninjected . They were stored individually in 2 ml Eppendorf tubes with pierced lids in an incubator at 27 °C and 95 % relative humidity . Eggs were removed daily and each daily egg batch was stored separately under the same conditions . 2.7 Analysis to confirm gene silencing by quantitative RT-PCR Viscera was dissected from five females of each dsRNA-injected or mock-injected group after 6 days of feeding . Total RNA was isolated from these samples using Tri reagent and subsequently purified using the Nucleospin RNA II kit in accordance with the reagent and kit manufacturer 's directions . Total RNA was isolated from 100 mg eggs -LRB- 14 days after injection -RRB- , 50 mg larvae -LRB- at 6 days and 5 weeks after hatching -RRB- laid by/eclosed from the dsRNA - and mock-injected engorged females , 50 mg larvae at 10 weeks after hatching laid by the dsRNA - and mock-injected unfed females and from the dissected viscera of five females and five males which developed from 7-week-old larvae fed on animal # 7799 using the same methods . cDNA from 1 μg of RNA -LRB- adults , eggs and 6-day-old larvae -RRB- and 0.3 μg of RNA -LRB- 5-week-old larvae -RRB- was prepared using the Revertaid first strand cDNA synthesis kit -LRB- Fermentas -RRB- using random hexamer primers in accordance with the manufacturer 's protocol . All samples were analyzed for transcription of target genes by quantitative real-time RT-PCR using primers Bm86h-F6 and Bm86h-R4 , amplifying a 117 bp section of the Bm86 gene ; Bm91-F2 and Bm91-R3 , amplifying a 129 bp section of the Bm91 gene and Bm-subA-F2 and Bm-subA-R2 , amplifying a 166 bp section of the subolesin gene . Tick β-actin was included as a control and used for normalisation . A 126 bp fragment was amplified using primers Actin-F2 and Actin-R . All primer combinations amplified a different part of the targeted genes than the sections which were used for dsRNA synthesis , circumventing re-amplification of any unprocessed dsRNA . Twenty-five microlitres of real-time PCRs were performed using the Quantitect SYBR green PCR kit in accordance with the manufacturer 's protocol -LRB- Qiagen , Venlo , The Netherlands -RRB- on an iCycler real-time detection system -LRB- Bio-Rad Laboratories , Veenendaal , The Netherlands -RRB- . Real-time PCR data were analyzed by iCycler IQ software version 1.0 . 2.8 Analysis to check for the presence of dsRNA in eggs cDNA from 1 μg of total RNA of eggs 14 days post-oviposition was screened by quantitative real-time RT-PCR for the presence of non-processed Bm86 , Bm91 and subolesin dsRNA to see whether the injected dsRNA was incorporated into the eggs and could be re-amplified . Oligonucleotide primers located within the region used for dsRNA synthesis of the Bm86 , Bm91 and subolesin genes were used for this purpose . The following primer combinations were used : Bm86-F7 and Bm86h-R3 , amplifying a 121 bp section of the Bm86 gene , primers Bm91-F3 and Bm91-R1 , amplifying a 128 bp region of the Bm91 gene , primers BmsubA-F2 and BmsubA-R1 , amplifying a 121 bp section of the subolesin gene . Real-time RT-PCR conditions were identical to those used to confirm gene silencing . 2.9 Statistical analysis Statistical analysis of data from two quantitative RT-PCR experiments , the weights of ticks after feeding and oviposited egg masses , was performed using Microsoft Excel and consisted of an unpaired t-test with unequal variances . Tick mortality was compared between the dsRNA - and mock-injected ticks by χ2-test . P values of 0.05 or less were considered statistically significant . 3 Results 3.1 Cloning and sequencing of the subolesin gene from B. microplus The subolesin gene -LRB- subA -RRB- from the Mozambiquan B. microplus strain was cloned and sequenced . This gene was found to be 99 -- 100 % identical to subA from B. microplus strains from Mexico and Brazil -LRB- de la Fuente et al. , 2006a ; our unpublished data -RRB- . 3.2 RNAi in freshly molted B. microplus females Five groups consisting of 120 freshly molted B. microplus females were each injected with 0.5 μl of injection buffer in the following groups : -LRB- i -RRB- injection buffer alone , -LRB- ii -RRB- Bm86 , -LRB- iii -RRB- Bm91 , -LRB- iv -RRB- subolesin and -LRB- v -RRB- both Bm86 and subolesin-dsRNA . An average of 81 -LRB- 76 -- 82 ; 32.8 % overall mortality -RRB- females were alive in each group 3 -- 10 h following injection . These ticks were subsequently fed together on a calf with an excess of B. microplus males until the females became replete or for a maximum of 14 days . Tick weight after engorgement or manual removal , mortality rate , egg mass and hatching rate is presented in Table 2 . A significant decrease in tick weight and oviposited egg mass , together with a higher mortality rate , was observed in the subolesin dsRNA injected groups compared with the control group -LRB- P < 0.01 -RRB- . Hatching rates were uniformly constant in the control , Bm86 - and Bm91-dsRNA-injected groups -LRB- > 90 % -RRB- , while in the ticks injected with subolesin dsRNA the hatching rate was lower -LRB- < 20 % -RRB- . Gene silencing was confirmed by quantitative real-time RT-PCR -LRB- Fig. 1 -RRB- . The normalised transcript level of Bm86 was reduced with 86 % in the Bm86-dsRNA-injected ticks and with 79 % in the combined Bm86/subolesin-dsRNA-injected ticks , compared with the normalised transcript level of the mock-injected group -LRB- Fig. 1A -RRB- . For Bm91 , the normalised transcript level was reduced with 90 % in the Bm91 RNAi-silenced ticks compared with the control ticks . A significant decrease in the Bm91 transcript level of 58 % was observed in the subolesin dsRNA-injected ticks -LRB- P < 0.01 -RRB- as well , but this reduction was not observed in the combined Bm86/subolesin-dsRNA-injected ticks -LRB- Fig. 1B -RRB- . Normalised transcript levels of subolesin in the subolesin dsRNA - and combined Bm86/subolesin dsRNA-injected ticks compared with the control group were reduced with 90 % and 80 % , respectively -LRB- Fig. 1C -RRB- . No differences were observed in the normalised Bm86 and Bm91 transcript levels of 10-week-old larvae which hatched from the Bm86 - and Bm91-dsRNA-injected ticks compared with the mock-injected ticks -LRB- results not shown -RRB- . 3.3 RNAi in engorged B. microplus females Five groups of five engorged females each with an average weight of 261 mg -LRB- 248 -- 272 mg -RRB- were injected with 5 μl of Bm86 dsRNA , Bm91 dsRNA , subolesin dsRNA , injection buffer alone or left uninjected within 6 h after dropping from the host . No reflux of the injected solution or hemolymph was observed from the puncture when the needle was gently withdrawn . Oviposition began in all groups within 3 days , except for one tick from the control-injected group and another tick from the Bm91 dsRNA-injected group which did not lay any eggs . The course of oviposition was not significantly influenced by the injection of dsRNA -LRB- Table 3 -RRB- , and dried or shriveled eggs were not observed , indicating that all eggs were successfully coated with a secretion from Gené 's organ . Interestingly , nearly all -LRB- > 99.4 % -RRB- eggs oviposited by engorged females injected with subolesin dsRNA showed an aberrant phenotype compared with those from the other groups . A typical example is shown in Fig. 2 . Development of embryos in these eggs was not observed while many undifferentiated cells with some yolk cells were seen in Giemsa-stained egg crush smears . Most eggs did not hatch and eventually dried up and shriveled after 6 -- 7 weeks of incubation at 27 °C / 95 % relative humidity . The few eggs from this group which did develop and hatched normally -LRB- < 0.6 % -RRB- were all laid during the first day of oviposition . A 64 % decrease of subolesin transcript levels was found in eggs from the subolesin dsRNA-injected engorged females -LRB- Fig. 3C -RRB- . Interestingly , a 30-fold increase in the Bm86 transcript level and a 56 % decrease in Bm91 transcript level were also found in these aberrant eggs -LRB- Figs. 3A and B -RRB- . An 84 % decrease in the number of Bm86 copies was observed in eggs laid by the engorged females injected with Bm86 dsRNA -LRB- Fig. 3A -RRB- and the transcript level of Bm91 was reduced by 97 % in eggs from the Bm91 dsRNA-injected engorged females compared with the mock-injected control group -LRB- Fig. 3B -RRB- , confirming gene-specific silencing in the eggs of dsRNA-injected engorged females . Injected dsRNA could be re-amplified from the eggs of dsRNA-injected engorged females when primers located within the dsRNA sections were used , instead of primers located downstream of the dsRNA region which were used to demonstrate gene silencing -LRB- Table 1 -RRB- . Results of quantitative real-time RT-PCR performed with these primers showed levels of Bm86 , Bm91 and subolesin which were 10.5 , 4.3 and 4.5 times higher , respectively , in the Bm86 , Bm91 and subolesin injected groups than levels found in the mock-injected group -LRB- Fig. 4 -RRB- . Gene silencing was observed in larvae 6 days after hatching ; a decrease of 86 % in Bm86 transcript level and of 91 % in Bm91 transcript level in the Bm86 - and Bm91 dsRNA-injected groups , respectively -LRB- Figs. 5A and B , grey bars -RRB- . Subolesin transcript levels were not measured in the few larvae which hatched from the subolesin dsRNA-injected females due to their small number . Quantitative real-time RT-PCR analysis of RNA extracted from larvae 5 weeks after hatching -LRB- 9 weeks after the initial injection of engorged females with dsRNA -RRB- revealed that genes remained silenced in both Bm86 - and Bm91-silenced groups -LRB- Figs. 5A and B , black bars -RRB- . This effect diminished over time , in particular for the Bm86-silenced group , in which transcript levels were now 67 % lower compared with the control group . Bm91 transcript levels were still 90 % lower in the Bm91-silenced group compared with the control group . When 7-week-old larvae from these three groups were fed in separate patches on a calf and total RNA from the viscera of adults which had developed from these larvae was analyzed by quantitative real-time RT-PCR , gene silencing was not observed -LRB- results not shown -RRB- . 4 Discussion The production characteristics of the subolesin-silenced female B. microplus ticks corresponded with previous results from subolesin RNAi studies in other ixodid tick species , and typically resulted in decreased tick and egg mass weights and high mortality -LRB- de la Fuente et al. , 2005 , 2006a , c -RRB- . The synergistic effect of combined silencing of the Bm86 and subolesin gene reported previously in R. sanguineus -LRB- de la Fuente et al. , 2006c -RRB- could not be confirmed for B. microplus -LRB- Table 2 -RRB- . Significant changes in the production characteristics of the Bm86 - and Bm91-silenced females were not observed , suggesting that the deleterious effect on ticks feeding on Bm86 - or Bm91-vaccinated cattle is not caused by a loss of function of the Bm86 or Bm91 protein alone . In fact , the protective effect of Bm86-based vaccines is through gut damage mediated by anti-Bm86 antibodies during tick feeding -LRB- Willadsen et al. , 1989 -RRB- . These results suggest that the protection mechanisms of Bm86 and subolesin-based vaccines are different and may contribute to the increased efficacy of Bm86 and subolesin combined vaccines . Quantitative real-time RT-PCR performed on samples taken 6 days after dsRNA injection and feeding resulted in a decrease of 79 -- 90 % of the targeted gene transcript level compared with the mock-injected group . This result was comparable with previous semi-quantitative measurements of the gene silencing effect by dsRNA-injection in A. americanum . In this RNAi study , a decrease of ∼ 90 % to 50 % in cystatin transcript level was observed from 24 h to 9 days of feeding -LRB- Karim et al. , 2005 -RRB- . In the present study , although gene silencing was specific in the Bm86 and Bm91 dsRNA-injected groups , subolesin dsRNA injection resulted in both significantly decreased subolesin as well as Bm91 expression levels . This effect was not observed in the combined Bm86/subolesin silenced group , which may be explained by a slight difference in midgut : salivary gland ratio in the dissected viscera between the groups . Since Bm91 is present in relatively high concentrations in salivary glands compared with midgut -LRB- Riding et al. , 1994 -RRB- , a shift in the midgut : salivary gland ratio of the dissected viscera in favor of midgut tissue could result in the lower Bm91 expression levels we found in this specific sample . Alternatively , the effect of silencing subolesin expression may affect the expression of other genes . The pleiotropic effect on tick tissues in which subolesin expression has been silenced suggests that this gene may be involved in the regulation of multiple pathways in ticks -LRB- de la Fuente et al. , 2006a -RRB- . Injection of pilocarpine solution into the hemocoel via the spiracular plate of engorged ticks has been described for inducement of salivation in engorged B. microplus ticks -LRB- Bechara et al. , 1988 -RRB- . These openings of the tracheae are sclerotised structures located posterior to the fourth pair of legs . Their rigid structure allows for puncturing and injection of small quantities of fluid by a fine needle without subsequent reflux of the injected solution , hemolymph or tissue . When we injected Bm86 , Bm91 or subolesin dsRNA into the hemocoel of engorged females , the course of oviposition appeared to be unaffected . Only one Bm91 dsRNA-injected female and one mock-injected female tick died before ovipositing . Interestingly , embryogenesis was undisturbed in eggs oviposited by the Bm86 dsRNA - , Bm91 dsRNA - and mock-injected engorged females , but an aberrant development was seen in the majority of egg masses oviposited by the subolesin dsRNA-injected engorged females . This egg phenotype has not been described previously and suggests that subolesin plays a role in embryonic development . When total RNA isolated from these aberrant eggs was analyzed by real-time RT-PCR , significantly higher Bm86 levels and decreased Bm91 and subolesin levels were found . Again , these results suggest that subolesin may be a regulator of transcription in ticks . Injected dsRNA was detected in eggs from dsRNA-injected engorged females by real-time RT-PCR using primers located within the dsRNA section , indicating that unprocessed dsRNA is incorporated in the eggs . The suggested route of incorporation of exogenously produced yolk directly from the hemolymph into oocytes -LRB- Saito et al. , 2005 -RRB- may be followed for the incorporation of dsRNA into oocytes as well . Further experiments are needed to determine whether this dsRNA forms a reservoir of mobile silencing signals inducing gene-specific silencing in eggs , or whether small interfering RNAs are responsible for this effect . Other possible routes for the mobile silencing signal to enter the oocyte are through the pedicel in the ovary or , once the oocyte has ovulated , by contact with cells from the genital tract . Some eggs -LRB- < 0.6 % -RRB- found in the batches laid during the first day of oviposition by subolesin dsRNA-injected engorged females , hatched normally . It is likely that these eggs developed to a stage which was not accessible by the dsRNA prior to injection of the dsRNA . They may have been ovulated eggs which were not in direct contact with the hemolymph or ones in which the shell was hardened during the oocyte passage through the oviduct and thus became impermeable to dsRNA during this process -LRB- Diehl et al. , 1982 -RRB- . After dsRNA was injected into the body cavity of B. microplus , the gene silencing effect spread throughout the organism and its progeny . This systemic RNAi has been associated with the sid-1 protein , a transmembrane protein which enables passive cellular uptake of dsRNA -LRB- Winston et al. , 2002 ; Feinberg and Hunter , 2003 -RRB- . Unfortunately , our attempts to detect a B. microplus sid-1 homologue , as described previously in studies on presence of a sid-1 homologue in grasshopper species Schistocerca americana , were not successful -LRB- data not shown -RRB- -LRB- Dong and Friedrich , 2005 -RRB- . The only protein currently present in the B. microplus expressed sequence tag -LRB- EST -RRB- database -LRB- Guerrero et al. , 2005 -RRB- which is associated with the RNAi machinery is the nuclease Argonaute-2 -LRB- Ago-2 -RRB- , the central catalytic component of the RNA-induced silencing complex -LRB- RISC -RRB- in mammals and Drosophila -LRB- Liu et al. , 2004 ; Miyoshi et al. , 2005 -RRB- . Homologues of other RNAi-associated proteins such as Dicer , which is responsible for the cleavage of exogenous long dsRNA into short interfering RNA -LRB- siRNA -RRB- , remain to be identified in B. microplus and other tick species as well . RNAi described herein provides an important tool to screen for tick-protective antigens in this one-host tick species , B. microplus -LRB- de la Fuente et al. , 2005 -RRB- and allowed for characterisation of the effect and function of tick protective antigens , as well as the role of genes involved in tick -- host -- pathogen interactions and the transmission of tick-borne pathogens -LRB- de la Fuente et al. , 2006b -RRB- . Initiation and completion of the B. microplus genome sequencing project would greatly enhance these kinds of studies -LRB- Guerrero et al. , 2006 -RRB- , as well as the availability of the genomes from cattle and the pathogens transmitted by B. microplus , most notably : A. marginale -LRB- Brayton et al. , 2005 -RRB- , B. bigemina and B. bovis , which are currently being sequenced . Although sequences of the dsRNAs used in this study do not contain any significant overlap with other known B. microplus genes , the possibility of off-target gene silencing effects can not be excluded due to the limited amount of sequence data available . Availability of the complete B. microplus genome sequence data will facilitate screening for potential off-target effects . These can subsequently be minimised by avoiding the use of dsRNAs or siRNAs containing sequences which are present in multiple genes . The effect of silencing tick genes suggested to be involved in embryogenesis such as vitellin degrading cysteine endopeptidase -LRB- Seixas et al. , 2003 -RRB- , in the transovarial transmission of Babesia spp. or genes associated with acaricide resistance , which is measured in larvae by the Larval Packet Test -LRB- Li et al. , 2003 -RRB- , could be studied using our method to silence genes in B. microplus embryos and larvae by injecting engorged females with dsRNA . These experimental approaches are more efficient and less labour intensive than the three other dsRNA delivery approaches into oocytes and embryos using microinjection -LRB- Wargelius et al. , 1999 -RRB- , transgenic RNAi -LRB- Tavernarakis et al. , 2000 -RRB- and electroporation -LRB- Grabarek et al. , 2002 -RRB- .

O O O O O O O O B O B O B O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O B O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O


Differential expression of HOX genes upon activation of leukocyte sub-populations The HOX genes are key determinants of cellular identity both in early development and in the renewal and differentiation of adult blood cells . Although a number of studies have examined the expression of individual HOX genes in defined blood cell lineages , we have undertaken a comprehensive analysis of HOX gene expression in resting and activated lymphocytic and monocytic subpopulations . This has revealed distinct patterns of expression between different cell types and resting and activated states . -LRB- Main category A : Erythrocytes , Leukocytes and Hematopoiesis , subcategory : 8 : Lymphocytes -RRB- . Introduction The HOX genes are a family of homeodomain-containing transcription factors that define specific positional identities , both in vertebrate and invertebrate embryos -LSB- 4 -RSB- . They are also involved in the regulation of haemopoiesis -LRB- reviewed by -LSB- 13 -RSB- -RRB- , and blocking HOX gene function by the use of antisense oligos or gene knock out perturbs a number of different haemopoietic events . Thus for example myeloid , erythroid and lymphoid haemopoiesis are all defective in HoxA9 knock out mice -LSB- 8 -RSB- , and the antisense ablation of HoxB5 , HoxB6 , HoxB7 -LSB- 15 -RSB- , or HoxA5 -LSB- 5 -RSB- blocks erythroid differentiation . Forced expression of HOXA10 in CD34 + progenitor cells purified from cord blood results in a significant reduction in the number of B cells and natural killer cells but a marked increase in monocytes -LSB- 14 -RSB- . There are also examples of specific regulatory functions for HOX genes , including the modulation of globin transcription by HOXB6 -LSB- 12 -RSB- . In addition , HOX genes can also act to regulate the proliferation of haemopoietic stem cells -LRB- HSCs -RRB- . Hence HOXB4 is a strong , positive regulator of HSC self renewal -LRB- reviewed by -LSB- 10 -RSB- -RRB- , and an estrogen-dependant version of HOXB8 can be used to drive the production of neutrophils and macrophages from their respective progenitor cells -LSB- 16 -RSB- . Despite these studies , relatively little is still known about the expression of HOX genes in the mature blood lineages -LRB- reviewed by -LSB- 6 -RSB- -RRB- . In order to address this we purified different cell populations from human peripheral blood on the basis of their expression of the surface markers CD4 , CD8 , CD14 and CD19 that are present on T-lymphocytes -LRB- CD4 and CD8 -RRB- , monocytes , and B-lymphocytes respectively . HOX expression was compared in ` resting ' cells and cells that had been activated by challenging with specific antigenic stimuli -- antibodies against CD2 , CD3 and CD28 for CD4 + and CD8 + cells , Lipopolysaccharide for CD14 + cells and anti-IgG for CD19 + cells . Each population of cells underwent proliferation in response to this treatment , as measured by -LSB- 3H -RSB- thymidine incorporation -LRB- Fig. 1 -RRB- . RNA was extracted from each cell type and the expression of each of the 39 HOX genes was assed by quantitative PCR -LRB- QPCR -RRB- , relative to the amount of Beta-actin transcript present -LRB- Fig. 2 -RRB- . Fig. 1Proliferation of activated lymphocyte populations . Proliferation was measured by -LSB- 3H -RSB- thymidine incorporation 4 h after activation . Values shown are the fold increase in incorporation compared to resting cells . Error bars : SEM from triplicate experimentsFig . 2HOX gene expression in resting -LRB- a -RRB- and activated -LRB- b -RRB- after 6 h , -LRB- c -RRB- after 24 h -RRB- lymphocytic and monocytic populations . Expression was assessed by quantitative PCR and is shown as a relative value to the number of beta-actin transcripts -LRB- ` Relative expression ' -RRB- . HOX genes are abbreviated to show only their family and paralogue position , hence HOXA2 is shown as ` A2 ' . HOX genes for which no expression was detected are not shown in the figure . Each value shown is the mean from three experiments . Significant changes in expression levels -LRB- p < 0.05 -RRB- 6 and 24 h after activation are marked -LRB- * -RRB- . Error bars : SEM The highest level of expression is generally shown by the HOXA and HOXC genes , with HOXD genes generally exhibiting a tenfold lower expression level and HOXB genes a 100 fold lower expression level , a feature that is also true for the other cell populations examined . For all of the HOX genes expressed in CD4 + cells there is a significant reduction in HOX expression upon activation . This is particularly striking for the most posterior genes of the HOXA , HOXC and HOXD clusters . A similar pattern is also observed in CD8 + cells , although there is a general reduction in HOX expression with every HOX gene except HOXC10 being present at lower levels in these cells . There are a number of notable differences between the HOX expression profile of CD14 + cells and the CD4 + / CD8 + populations . HOXC4 , HOXC6 and HOXC8 are not expressed . The most striking difference though is the change of HOX expression upon activation -- in contrast to CD4 + / CD8 + cells , CD14 + cells show a large increase in HOXA2 , HOXB13 , HOXC10 , HOXC13 , HOXD1 and HOXD9 . Other HOX genes such as HOXB4 and HOXA10 show no significant change in expression upon activation , which again contrasts with the response of CD4 + / CD8 + cells . CD19 + cells show a broadly similar pattern of HOX expression to CD14 + cells prior to activation , but have a distinct response to activation . Like CD4 + / CD8 + cells , CD19 + cells exhibit a large reduction in expression of most HOX genes with the exception of HOXD10 , HOXD11 and HOXD12 that show a very strong increase in expression upon activation . To our knowledge this is the first comprehensive analysis of HOX gene expression in these leukocyte populations . Taking the data as a whole , each population shows a unique pattern of HOX expression that may define cellular identity . Generally the expression levels are in agreement with those previously reported for individual HOX genes in other studies -LSB- 6 , 7 , 9 -RSB- . However the changes of HOX gene expression upon cellular activation have not previously been reported . The magnitude of these changes is striking , with the activated T cell subsets exhibiting an almost complete loss of expression of many HOX genes especially the more posterior members of the groups -LRB- HOXA10 , HOXC13 , and HOXD12 -RRB- . Conversely , activated monocytic and B-lymphocyte cells show a large increase in expression of the same genes . Given the key regulatory roles of HOX genes it is likely that these changes in expression facilitate the cellular changes associated with activation . One of these is increased proliferation , a response that is known to be mediated by HOX genes in a number of hematopoietic lineages -LRB- reviewed by -LSB- 1 -RSB- -RRB- . Indeed the antisense mediated ablation of HOXB2 or HOXB4 in T-lymphocytes was previously shown to block the proliferation of activated cells -LRB- Care et al. 1995 -RRB- . Furthermore HOXA10 deficiency causes a severe immunological disturbance in uterine tissues characterised by rapid T cell proliferation and a failure of progesterone-mediated immunosuppression -LSB- 17 -RSB- . The HOX genes are also likely to regulate lineage-specific transcription upon activation , and in this context the contrasting expression levels of these genes presumably reflect the dramatically different response of each cell type . Materials and methods CD4 + , CD8 + , CD14 + and CD19 + cells were isolated using the Dynabead system -LRB- Invitrogen -RRB- and cultured as previously described -LSB- 2 -RSB- . Quantitative PCR was performed as previously described -LSB- 11 -RSB- . -LSB- 3H -RSB- thymidine incorporation was evaluated by a standard procedure . Briefly , 2 μCi of -LSB- 3H -RSB- thymidine -LRB- 20 Ci/mmol -RRB- was added for 4 h to each well . Cells were then recovered , washed , and processed for the determination of TCA precipitable radioactivity . Each evaluation was performed in triplicate . T cell activation was achieved using anti-CD2 , anti-CD3 and anti-CD28 antibodies as supplied in the T cell activation/expansion kit -LRB- Miltenyi Biotec -RRB- .

O O O B O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O


Hereditary

diffuse

gastric

cancer

association

with

lobular

breast

cancer

Hereditary

diffuse

gastric

cancer

-LRB-

HDGC

-RRB-

has

been

shown

to

be

caused

by

germline

mutations

in

the

gene

located

at

which

encodes

the

cell

cell

adhesion

molecule

E-cadherin

Not

only

does

loss

of

expression

of

E-cadherin

account

for

the

morphologic

differences

between

intestinal

and

diffuse

gastric

cancer

-LRB-

DGC

-RRB-

variants

but

it

also

appears

to

lead

to

distinct

cellular

features

which

appear

to

be

common

amongst

related

cancers

that

have

been

seen

in

the

syndrome

As

in

most

hereditary

cancer

syndromes

multiple

organ

sites

may

be

commonly

affected

by

cancer

in

HDGC

lobular

carcinoma

of

the

breast

-LRB-

LBC

-RRB-

and

possibly

other

organ

sites

have

been

shown

to

be

associated

with

the

familial

cancer

syndrome

Given

the

complexity

of

HDGC

not

only

with

regard

to

the

management

of

the

DGC

risk

but

also

with

regard

to

the

risk

for

other

related

cancers

such

as

LBC

a

multi-disciplinary

approach

is

needed

for

the

management

of

individuals

with

known

mutations

Introduction

Despite

an

overall

decrease

in

the

global

incidence

of

gastric

cancer

-LRB-

GC

-RRB-

-LSB-

-RSB-

the

incidence

of

the

subtype

diffuse

gastric

cancer

-LRB-

DGC

-RRB-

has

remained

stable

and

may

even

be

increasing

-LSB-

-RSB-

Within

the

past

ten

years

germline

mutations

in

which

encodes

E-cadherin

have

been

found

-LSB-

-RSB-

in

over

of

hereditary

diffuse

gastric

cancer

-LRB-

HDGC

-RRB-

families

with

at

least

two

cases

of

GC

with

one

diagnosed

as

DGC

before

the

age

of

years

-LSB-

-RSB-

Within

these

HDGC

families

we

and

others

have

noted

an

overrepresentation

of

lobular

breast

cancer

-LRB-

LBC

-RRB-

-LSB-

-RSB-

This

observation

has

led

to

efforts

to

determine

whether

or

not

is

a

breast

cancer

susceptibility

gene

distinct

from

its

gastric

cancer

risk

Recently

our

group

has

reported

a

novel

germline

truncating

mutation

-LRB-

-RRB-

in

an

LBC

family

with

no

known

history

of

GC

-LSB-

-RSB-

Within

this

review

we

report

a

germline

mutation

in

a

second

family

in

which

breast

cancer

is

the

predominant

cancer

diagnosis

The

management

of

HDGC

in

all

patients

with

a

particular

focus

on

the

management

of

the

breast

cancer

risk

associated

with

germline

mutations

will

be

discussed

Methods

The

described

family

was

referred

to

the

ongoing

HDGC

study

at

the

British

Columbia

Cancer

Agency

from

a

cancer

genetics

clinic

in

Seattle

WA

USA

Informed

consent

was

obtained

from

the

proband

by

the

referring

genetic

counselor

following

ascertainment

of

a

detailed

cancer

family

history

and

appropriate

genetic

counseling

prior

to

germline

mutation

testing

Our

laboratory

carried

out

the

molecular

genetic

testing

for

the

mutation

on

a

research

basis

Approval

for

the

HDGC

study

is

by

the

clinical

research

ethics

board

of

the

University

of

British

Columbia

The

proband

-LRB-

-RRB-

was

diagnosed

with

widely

metastatic

lobular

breast

cancer

at

age

years

-LRB-

Fig.

-RRB-

Her

family

of

European

ancestry

had

a

history

of

breast

cancer

diagnoses

occurring

in

an

autosomal

dominant

fashion

on

the

maternal

side

of

the

family

where

her

mother

aunt

and

first

cousin

developed

breast

cancer

in

their

's

Due

to

her

high-risk

pedigree

and

PTEN

genetic

testing

was

undertaken

and

all

were

negative

testing

was

also

pursued

Fig.

-LRB-

a

-RRB-

Pedigree

of

family

reported

showing

a

predominance

of

breast

cancer

-LRB-

b

-RRB-

Sequence

from

family

carrying

A

mutation

Results

All

exons

were

amplified

for

DHPLC

analysis

-LSB-

-RSB-

For

exon

of

the

initial

amplicon

failed

and

was

therefore

analyzed

by

direct

sequencing

and

thus

revealed

a

donor

splice

site

mutation

A

-LRB-

Fig.

-RRB-

Due

to

its

position

at

a

donor

splice

site

this

mutation

is

regarded

as

pathogenic

-LSB-

-RSB-

The

proband

's

sisters

-LRB-

and

-RRB-

participated

in

all

aspects

of

the

proband

's

genetic

consultation

They

were

appropriately

concerned

about

their

risk

of

breast

cancer

but

had

not

thought

much

about

the

possibility

of

getting

gastric

cancer

until

the

mutation

was

found

and

had

predictive

genetic

testing

for

the

mutation

testing

and

both

were

found

to

be

negative

Other

family

members

are

being

informed

about

the

availability

of

predictive

genetic

testing

Discussion

E-cadherin

-LRB-

OMIM

-RRB-

located

on

chromosome

encodes

E-cadherin

an

epithelial

transmembrane

cell

cell

adhesion

molecule

and

member

of

the

cadherin

superfamily

of

glycoproteins

In

a

zipper-like

fashion

its

extracellular

domain

forms

calcium-dependent

homodimers

between

the

E-cadherin

molecules

of

adjacent

epithelial

cells

to

act

as

the

primary

mediator

of

epithelial

cell

adhesion

at

the

adherens

junction

complex

-LSB-

-RSB-

Through

interactions

of

its

cytoplasmic

tail

with

multiple

signalling

and

structural

molecules

such

as

the

catenins

E-cadherin

maintains

cellular

adhesion

and

epithelial

architecture

with

this

link

to

the

cytoskeleton

The

cytoplasmic

tail

of

E-cadherin

directly

associates

with

β-catenin

and

γ-catenin

which

in

turn

binds

to

the

f-actin

microfilaments

of

the

cytoskeleton

directly

or

through

α-catenin

-LSB-

-RSB-

also

associates

with

E-cadherin

's

cytoplasmic

tail

at

a

different

site

the

juxtamembrane

domain

and

acts

to

both

strengthen

the

adhesion

between

cells

and

regulate

cadherin

membrane

trafficking

and

degradation

-LSB-

-RSB-

E-cadherin

is

considered

to

have

an

invasion

suppressor

role

where

decreased

expression

permits

cells

to

dissociate

from

each

other

in

order

to

migrate

and

invade

-LSB-

-RSB-

In

cancers

this

manifests

as

increased

infiltrative

and

metastatic

potential

-LSB-

-RSB-

E-cadherin

is

also

thought

to

act

as

a

tumour

suppressor

potentially

through

its

interaction

with

the

multipurpose

β

catenin

molecule

which

is

an

effector

of

the

WNT

signalling

pathway

-LSB-

-RSB-

Loss

of

E-cadherin

can

result

in

β-catenin

release

from

the

membrane

and

translocation

to

the

nucleus

where

it

complexes

with

Tcf/Lef

transcription

factors

to

initiate

transcription

of

WNT

responsive

genes

-LSB-

-RSB-

Activation

of

these

genes

have

been

implicated

in

tumourigenesis

through

the

WNT

signalling

pathway

as

seen

in

adenomatous

polyposis

coli

-LRB-

APC

-RRB-

-LSB-

-RSB-

In

support

of

the

role

of

E-cadherin

as

a

tumour

suppressor

is

the

observation

of

abnormal

or

absent

E-cadherin

expression

in

precursor

lesions

of

DGC

and

LBC

where

the

phenomenon

is

seen

in

in

situ

signet

ring

cell

carcinomas

found

in

prophylactic

gastrectomy

specimens

from

germline

mutation

carriers

-LSB-

-RSB-

and

the

lobular

carcinoma

in

situ

lesions

seen

adjacent

to

invasive

lobular

breast

cancers

-LSB-

-RSB-

These

examples

suggest

that

loss

of

E-cadherin

is

an

early

or

even

tumour-initiating

event

however

the

actual

molecular

basis

of

such

a

potential

role

of

E-cadherin

in

such

cases

is

unknown

Inactivating

mutations

are

found

in

of

sporadic

DGCs

-LSB-

-RSB-

and

cluster

between

exons

seven

and

nine

-LSB-

-RSB-

in

contrast

with

the

low

percentage

of

mutations

seen

in

sporadic

intestinal

type

GCs

-LSB-

-RSB-

Decreased

expression

of

E-cadherin

in

DGCs

may

account

for

morphologic

differences

between

intestinal

and

DGC

variants

-LSB-

-RSB-

Unlike

somatic

mutations

germline

mutations

associated

with

DGC

are

distributed

throughout

the

gene

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

In

the

cancers

from

individuals

with

mutations

acts

as

a

classic

tumour

suppressor

gene

with

loss

of

expression

of

the

wildtype

allele

-LSB-

-RSB-

In

a

single

study

of

hereditary

DGC

cancers

inactivation

of

the

wild-type

allele

could

be

attributed

to

promoter

hypermethylation

in

-LRB-

-RRB-

of

cases

-LSB-

-RSB-

This

finding

warrants

verification

in

a

larger

cohort

as

abnormal

promoter

methylation

in

early

cancers

could

potentially

form

the

basis

of

a

screening

test

Fig.

and

LBC

associated

germline

mutations

Mutations

shown

above

gene

schematic

occur

in

families

with

DGC

history

and

those

below

occur

in

families

with

an

additional

or

exclusive

LBC

history

In

addition

to

the

known

germline

mutations

compiled

by

Kaurah

et

al.

-LSB-

-RSB-

the

recent

mutation

in

an

LBC

family

-LSB-

-RSB-

and

novel

mutation

from

this

paper

are

shown

and

identified

below

the

symbol

denoting

mutation

type

Denotes

the

halfway

point

of

the

coding

sequence

-LRB-

or

the

start

of

exon

-RRB-

Lobular

breast

cancer

and

diffuse

gastric

cancer

loss

of

E-cadherin

Currently

germline

mutations

in

single

genes

account

for

approximately

of

breast

cancer

-LSB-

-RSB-

High

penetrance

genes

such

as

and

account

for

and

and

PTEN

as

seen

in

Li-Fraumeni

and

Cowden

syndrome

together

only

account

for

of

breast

cancer

diagnoses

-LSB-

-RSB-

Other

medium

and

low

penetrance

genes

such

as

and

ATM

-LSB-

-RSB-

have

been

identified

however

there

still

remains

a

proportion

of

hereditary

breast

cancer

not

yet

determined

LBC

accounts

for

approximately

of

all

breast

cancers

compared

to

the

other

major

histologic

subtype

invasive

ductal

carcinoma

-LRB-

IDC

-RRB-

-LSB-

-RSB-

Several

factors

suggest

that

LBC

has

a

stronger

hereditary

basis

relative

to

IDC

such

as

the

higher

frequency

of

bilateral

disease

-LSB-

-RSB-

and

also

where

excess

familiality

of

LBC

has

been

observed

in

population

studies

-LSB-

-RSB-

LBCs

compose

only

and

of

the

breast

cancer

tumour

types

seen

in

germline

BRCA

and

mutation

carriers

respectively

-LSB-

-RSB-

illustrating

that

the

genetic

risk

factors

for

the

majority

of

cases

are

unaccounted

for

by

these

genes

The

histology

of

LBC

is

characterized

by

infiltrative

cancer

cells

which

are

isolated

highly

dispersive

and

demonstrate

a

growth

pattern

with

scattered

and

single

files

of

tumor

cells

dispersed

in

stromal

tissue

-LSB-

-RSB-

This

pathologic

appearance

is

remarkably

similar

to

DGCs

and

both

LBC

and

DGC

demonstrate

characteristic

mucinous

signet

ring

cells

This

is

not

unexpected

as

E-cadherin

staining

is

absent

in

of

sporadic

invasive

LBC

-LSB-

-RSB-

and

somatic

mutations

have

been

identified

in

of

sporadic

LBCs

-LSB-

-RSB-

Furthermore

in

IDC

somatic

mutations

are

not

found

-LSB-

-RSB-

and

complete

loss

of

E-cadherin

expression

is

an

uncommon

feature

As

loss

of

E-cadherin

expression

is

a

distinctive

trait

of

both

LBCs

and

DGCs

it

likely

contributes

to

the

unique

histopathologic

features

shared

by

the

two

cancers

There

are

some

differences

with

regard

to

the

nature

of

the

mutations

seen

in

LBC

and

DGC

Generally

mutations

associated

with

sporadic

LBC

have

been

found

to

be

nonsense

or

frameshift

mutations

-LSB-

-RSB-

which

encode

truncated

non-functional

proteins

whereas

in

sporadic

DGC

mutations

have

generally

been

found

to

be

splice

site

and

in-frame

mutations

-LSB-

-RSB-

In

sporadic

LBC

mutations

in

are

spread

throughout

the

gene

-LSB-

-RSB-

compared

with

the

mutations

seen

in

sporadic

DGC

which

tend

to

cluster

Germline

mutations

associated

with

DGC

and/or

LBC

occur

throughout

the

gene

-LRB-

Fig.

-RRB-

However

when

the

DGC

and

LBC

associated

mutations

are

tabulated

and

compared

based

on

their

or

positions

relative

to

the

halfway

point

of

the

coding

sequence

-LRB-

or

the

start

of

exon

-RRB-

LBC

associated

mutations

show

a

statistically

significant

trend

towards

clustering

at

the

end

-LRB-

Fisher

's

exact

test

two-tailed

P-value

equals

-RRB-

-LRB-

Fig.

-RRB-

As

this

association

is

of

weak

statistical

significance

it

is

unlikely

to

impact

clinical

testing

strategies

Future

analyses

of

novel

germline

LBC-associated

mutations

should

help

to

confirm

this

observation

Another

difference

between

the

molecular

genetics

of

the

two

types

of

cancers

is

that

in

sporadic

LBC

silencing

of

E-cadherin

expression

is

generally

accomplished

by

a

mutation

in

one

allele

in

combination

with

loss

of

heterozygosity

-LRB-

LOH

-RRB-

or

promoter

hypermethylation

in

the

remaining

allele

-LSB-

-RSB-

This

is

in

contrast

to

sporadic

DGC

where

biallellic

inactivation

is

achieved

by

mutations

in

one

allele

in

concert

with

promoter

hypermethylation

in

the

other

-LSB-

-RSB-

We

have

recently

identified

a

truncating

germline

mutation

in

an

LBC

family

where

analysis

of

the

tumour

was

suggestive

of

partial

LOH

in

the

WT

allele

-LSB-

-RSB-

Our

current

case

demonstrates

a

germline

mutation

-LRB-

A

-RRB-

in

a

predominantly

breast

cancer

family

which

is

predicted

to

disrupt

splicing

The

mutation

is

in

the

same

conserved

position

as

a

previously

reported

mutation

-LRB-

T

-RRB-

which

was

found

in

an

Arabian

HDGC

family

with

no

recorded

history

of

breast

cancer

-LSB-

-RSB-

Moreover

a

previous

study

reported

a

germline

missense

mutation

in

a

proband

with

LBC

but

did

not

detail

family

history

or

functionally

characterize

the

missense

mutation

-LSB-

-RSB-

These

examples

demonstrate

the

need

for

further

studies

of

germline

mutations

in

LBC

families

in

order

to

determine

the

mutation

frequency

and

potential

genotype-phenotype

correlations

Lobular

breast

cancer

and

HDGC

Breast

cancer

has

been

observed

in

HDGC

kindreds

to

the

extent

where

clustering

of

LBCs

within

HDGC

families

has

led

to

the

misclassification

of

families

as

breast

cancer

kindreds

who

test

negative

for

mutations

-LSB-

-RSB-

In

Keller

described

the

first

case

of

histologically

defined

LBC

in

association

with

HDGC

-LSB-

-RSB-

Since

then

several

more

HDGC

families

with

associated

breast

cancer

were

reported

where

it

was

observed

that

these

cases

were

LBCs

when

pathology

was

available

-LSB-

-RSB-

Prior

to

establishment

of

the

association

between

HDGC

and

LBC

several

efforts

to

determine

whether

was

a

breast

cancer

susceptibility

gene

were

attempted

in

view

of

the

well-recognised

phenotype

of

loss

of

E-cadherin

expression

displayed

by

the

breast

cancer

subtype

For

various

reasons

these

studies

failed

to

demonstrate

the

link

Rahman

et

al.

examined

cases

of

lobular

carcinoma

in

situ

however

did

not

pre-screen

the

cases

based

on

family

history

and

included

a

wide

age

range

from

to

years

not

necessarily

in

keeping

with

the

usual

age

of

onset

seen

in

hereditary

cancer

syndromes

-LSB-

-RSB-

Salashor

examined

breast

cancer

tumours

exhibiting

LOH

at

the

locus

however

of

those

only

were

confirmed

to

be

pure

LBC

or

mixed

LBC/IDC

pathology

-LSB-

-RSB-

Lei

examined

familial

LBC

cases

and

found

no

mutations

however

did

not

define

the

extent

of

the

family

history

-LSB-

-RSB-

Penetrance

data

based

on

HDGC

families

estimated

the

cumulative

risk

for

LBC

for

female

mutation

carriers

to

be

-LRB-

CI

-RRB-

by

years

of

age

-LSB-

-RSB-

More

recently

we

have

published

an

estimated

cumulative

risk

for

breast

cancer

for

females

by

the

age

of

years

as

being

-LRB-

CI

-RRB-

from

analysis

of

predominantly

gastric

cancer

pedigrees

from

Newfoundland

with

the

founder

mutation

-LSB-

-RSB-

This

is

with

the

caveat

that

LBC

risk

for

mutation

carriers

has

been

assessed

within

high

risk

HDGC

families

leading

to

a

potential

ascertainment

bias

and

underestimation

of

the

role

of

mutations

in

LBC

development

To

accommodate

for

this

we

have

begun

analysis

of

mutations

within

familial

lobular

breast

cancer

families

or

those

families

ascertained

through

a

relatively

young

index

case

with

confirmed

LBC

and

have

found

germline

mutations

in

these

kindreds

-LSB-

-RSB-

Clinical

implications

of

associated

LBC

risk

At

this

time

it

seems

reasonable

to

conclude

that

at

least

four

groups

of

women

are

at

increased

risk

for

LBC

women

with

LBC

and

a

family

history

of

breast

cancer

women

with

a

known

mutation

women

from

families

with

diffuse

gastric

cancer

in

whom

no

mutation

has

yet

been

identified

and

women

with

a

germline

mutation

Since

there

has

not

yet

been

a

large

population

based

study

of

the

prevalence

of

mutations

among

women

with

lobular

breast

cancer

it

is

premature

to

recommend

genetic

evaluation

to

women

with

a

family

history

of

breast

cancer

unless

at

the

very

least

one

of

the

breast

cancers

can

be

shown

to

have

been

lobular

Additional

research

can

be

expected

to

provide

better

guidance

for

these

families

Although

there

are

not

yet

definitive

data

available

on

surveillance

or

risk

reduction

programs

for

women

with

known

mutations

or

untested

women

from

families

the

high

lifetime

risk

of

LBC

-LRB-

-RRB-

-LSB-

-RSB-

that

these

women

face

mandates

their

careful

management

We

suggest

that

they

follow

the

recommendations

for

other

high-risk

women

with

hereditary

breast

cancer

predisposition

This

subgroup

should

be

advised

to

practice

breast

self-examination

and

to

have

annual

mammograms

and

semiannual

clinical

breast

examination

beginning

at

least

by

age

There

is

certainly

interest

in

regular

bilateral

breast

MRI

as

lobular

breast

cancer

are

known

to

frequently

elude

mammographic

detection

because

they

do

not

form

masses

or

develop

calcifications

These

women

can

also

be

counseled

to

consider

hormonal

chemoprevention

since

most

LBCs

are

estrogen

receptor

positive

-LSB-

-RSB-

and

both

tamoxifen

and

raloxifene

reduce

the

risk

of

estrogen

receptor

positive

-LSB-

-RSB-

breast

cancers

in

randomized

trials

In

addition

the

risk

reduction

was

greatest

with

both

agents

in

women

with

lobular

carcinoma

in

situ

-LSB-

-RSB-

Prophylactic

mastectomy

may

also

be

considered

an

option

by

some

women

particularly

those

who

have

been

previously

diagnosed

with

breast

cancer

in

one

breast

or

those

who

have

had

to

undergo

multiple

biopsies

for

abnormal

clinical

findings

Several

studies

have

reported

a

reduction

in

breast

cancer

incidence

with

prophylactic

mastectomy

among

women

with

a

strong

family

history

or

with

a

germline

or

mutation

-LSB-

-RSB-

The

published

series

include

some

lobular

breast

cancers

but

not

at

numbers

sufficient

to

permit

meaningful

subset

analysis

at

this

time

Management

of

hereditary

diffuse

gastric

cancer

Penetrance

studies

examining

data

from

HDGC

families

have

estimated

the

lifetime

risk

of

developing

gastric

cancer

by

age

and

respectively

to

be

from

in

men

to

in

women

-LSB-

-RSB-

Although

identification

of

germline

mutations

has

enabled

a

significant

proportion

of

HDGC

families

to

utilise

predictive

testing

to

determine

their

individual

risks

of

GC

within

mutation

positive

pedigrees

unfortunately

screening

for

DGC

is

ineffective

and

the

current

recommendation

is

for

consideration

of

prophylactic

gastrectomy

in

mutation

positive

individuals

Positron

emission

tomography

-LSB-

-RSB-

and

chromoendoscopic-directed

biopsies

-LSB-

-RSB-

have

been

proposed

over

basic

endoscopy

as

more

sensitive

means

of

screening

carriers

however

screening

methods

have

been

consistently

undermined

by

the

recurrent

discovery

of

multifocal

DGC

lesions

underlying

normal

mucosa

in

prophylactic

gastrectomy

specimens

of

individuals

with

recent

negative

screening

-LSB-

-RSB-

Regardless

of

the

current

limitations

of

screening

it

is

currently

recommended

that

consideration

for

genetic

testing

and

screening

begin

in

at

risk

individuals

in

the

late

teens

or

early

twenties

-LSB-

-RSB-

and

that

prophylactic

total

gastrectomy

be

considered

in

the

early

twenties

for

mutation

carriers

Female

mutation

carriers

will

need

specialized

counseling

to

the

potential

nutritional

effects

on

pregnancy

following

gastrectomy

-LSB-

-RSB-

Further

studies

are

currently

underway

to

examine

the

quality

of

life

impact

of

prophylactic

gastrectomies

In

the

case

report

herein

although

there

was

a

GC

in

the

maternal

grandfather

the

family

history

was

more

striking

for

the

large

number

of

breast

cancer

cases

This

highlights

the

particular

challenges

we

currently

face

with

regard

to

counseling

these

families

which

appear

to

be

mainly

breast

cancer

as

it

is

unknown

if

the

penetrance

of

DGC

in

this

family

is

as

high

as

it

is

in

other

HDGC

pedigrees

Conclusion

HDGC

is

one

of

a

number

of

hereditary

cancer

syndromes

that

feature

both

an

increased

breast

and

gastric

cancer

risk

-LRB-

Table

-RRB-

In

general

a

lack

of

shared

genetic

risks

for

most

breast

and

GI

cancers

was

suggested

through

a

recent

study

of

genes

in

breast

and

colon

cancer

cell

lines

in

which

the

only

commonly

mutated

gene

between

these

two

cancer

types

is

-LSB-

-RSB-

This

likely

reflects

underlying

differences

in

the

biology

of

these

diseases

however

also

highlights

the

unique

nature

of

germline

mutations

in

the

gene

which

are

strongly

associated

with

specific

histologically

defined

subtypes

of

breast

and

GI

cancer

namely

LBC

and

DGC

which

are

both

part

of

the

HDGC

syndrome

Table

syndromes

with

familial

susceptibility

to

breast

and

gastric

cancersSyndromeMode

of

inheritanceAssociated

gene

-LRB-

s

-RRB-

Sites

of

primary

cancer

-LRB-

s

-RRB-

Evidence

for

association

with

the

syndrome

Hereditary

Breast/Ovarian

is

considered

an

integral

tumor

of

the

syndrome

with

an

average

cumulative

risk

in

carriers

by

age

years

of

-LRB-

confidence

interval

-LRB-

CI

-RRB-

-RRB-

-LSB-

-RSB-

Familial

aggregations

of

both

BC

and

GC

have

been

reported

-LSB-

-RSB-

In

a

study

of

families

relative

risk

for

GC

was

-LSB-

-RSB-

Among

Ashkenazi

Jewish

GC

patients

the

frequency

of

mutation

is

five

times

that

of

the

general

Ashkenazi

Jewish

population

frequency

-LSB-

-RSB-

Among

other

cancers

GC

occurred

in

first

degree

relatives

when

mutations

were

located

in

the

ovarian

cancer

cluster

region

of

exon

of

-LSB-

-RSB-

Hereditary

Breast/Ovarian

ProstateBC

is

considered

an

integral

tumor

of

the

syndrome

with

an

average

cumulative

risk

in

carriers

by

age

years

of

-LRB-

confidence

interval

-LSB-

CI

-RSB-

to

-RRB-

-LSB-

-RSB-

There

is

a

times

increased

risk

for

GC

-LSB-

-RSB-

Peutz-Jeghers

SyndromeADSTK

-LRB-

GI

-RRB-

tractGC

is

considered

an

integral

tumor

of

the

syndrome

with

a

relative

risk

of

-LRB-

confidence

interval

-RRB-

-LSB-

-RSB-

BC

is

considered

an

integral

tumor

of

the

syndrome

with

a

relative

risk

of

-LRB-

CI

-RRB-

-LSB-

-RSB-

Cowden

SyndromeADPTENBreastThyroidEndometriumBC

is

considered

an

integral

tumor

of

the

syndrome

with

an

incidence

of

-LSB-

-RSB-

GC

in

situ

has

been

reported

in

a

patient

with

Cowden

Syndrome

-LSB-

-RSB-

Li-Fraumeni

cortexConnective

tissueKidneyNervous

systemPancreas

White

blood

cellsBC

is

frequently

found

in

families

with

this

cancer

susceptibility

syndrome

-LSB-

-RSB-

Chompret

expanded

the

spectrum

of

cancers

to

include

GC

-LSB-

-RSB-

Germline

mutations

have

been

found

in

GC

families

without

mutations

-LSB-

-RSB-

The

allele

confers

a

fold

risk

of

BC

to

carriers

-LSB-

-RSB-

however

germline

mutations

in

GC

kindreds

have

not

been

identified

-LSB-

-RSB-

Familial

Adenomatous

PolyposisADAPCColon

and

rectumDuodenumThyroidPancreasLiterature

review

by

Shimoyama

et

al.

totalled

reported

cases

of

GC

and

FAP

-LSB-

-RSB-

to

of

primary

BCs

had

promoter

hypermethylation

at

the

APC

locus

-LSB-

-RSB-

of

LBCs

have

been

shown

to

have

LOH

of

APC

-LSB-

-RSB-

Lynch

Syndrome

-LRB-

Hereditary

Nonpolyposis

Colon

Cancer

-LRB-

HNPCC

-RRB-

-RRB-

and

rectumEndometriumStomach

Small

intestineUrotheliumKidneyOvarySkinPancreasBrainWhite

blood

cells

Biliary

tractGastric

cancer

accounted

for

of

cancers

in

families

harboring

or

mutations

-LSB-

-RSB-

mutations

in

large

kindred

segregated

with

BCs

exhibiting

microsatellite

instability

-LRB-

MSI

-RRB-

-LSB-

-RSB-

A

slight

increased

incidence

of

BC

was

seen

in

mutation

carriers

-LSB-

-RSB-

Germline

mutation

carrier

with

BC

exhibited

LOH

for

in

tumors

analyzed

-LSB-

-RSB-

Analysis

of

primary

invasive

BCs

demonstrated

that

of

tumours

were

immunonegative

for

staining

-LSB-

-RSB-

Ataxia-telangiectasia

-LRB-

AT

-RRB-

ARATMWhite

blood

cellsMutations

causing

AT

in

homozygotes

confer

susceptibility

to

BC

in

heterozygotes

where

women

with

ATM

mutations

have

a

risk

of

BC

and

of

these

women

will

develop

the

disease

-LSB-

-RSB-

GC

has

been

reported

in

association

with

the

syndrome

-LSB-

-RSB-

There

is

evidence

of

excess

risks

of

GC

in

heterozygotes

-LRB-

RR

CI

to

-RRB-

-LSB-

-RSB-

Xeroderma

-LRB-

XPB

-RRB-

-LRB-

XPD

-RRB-

-LRB-

XPE

-RRB-

-LRB-

XPF

-RRB-

-LRB-

XPG

-RRB-

POLH

-LRB-

XP-V

-RRB-

SkinEyesBC

and

GC

have

both

independently

been

reported

with

the

syndrome

-LSB-

-RSB-

Werner

SyndromeARWRNConnective

tissue

SkinThyroidGC

has

been

reported

in

association

with

the

syndrome

-LSB-

-RSB-

There

are

no

reports

of

BC

in

association

with

Werner

syndrome

Although

there

is

evidence

supporting

WRN

as

a

low-penetrance

familial

BC

susceptibility

gene

where

patients

harboring

both

WRN

or

MspI

variants

had

an

increased

BC

risk

-LRB-

OR

CI

-RRB-

-LSB-

-RSB-

AD

autosomal

dominant

AR

autosomal

recessive

With

the

recent

demonstration

of

a

mutation

in

a

family

ascertained

through

an

index

case

of

LBC

and

in

view

of

the

additional

new

mutation

in

a

predominantly

breast

cancer

family

that

we

have

described

here

the

evidence

for

establishing

LBC

as

part

of

the

HDGC

syndrome

is

strong

There

now

is

a

need

for

establishing

the

prevalence

of

mutations

in

LBC

families

to

avoid

the

ascertainment

bias

generated

from

only

looking

at

cases

from

families

identified

because

of

their

family

history

of

GC

It

is

not

currently

known

what

the

risk

of

GC

is

in

these

families

which

present

predominantly

as

having

a

susceptibility

to

breast

cancer

and

therefore

identification

of

as

a

true

susceptibility

gene

for

LBC

could

result

in

screening

and

effective

risk

reduction

strategies

for

selected

breast

cancer

families

and

further

studies

examining

their

risk

for

gastric

and

other

cancers

Most

hereditary

cancer

syndromes

are

associated

with

cancer

risk

involving

multiple

organs

Here

we

have

discussed

germline

mutations

and

the

risks

with

regard

to

DGC

and

LBC

however

as

the

recognised

spectrum

of

related

cancers

broadens

more

affected

families

will

be

identified

and

successfully

managed

with

regard

to

avoidance

of

specific

cancer

risks

Longer

life

expectancy

in

individuals

with

penetrant

mutations

could

potentially

lead

to

the

development

of

different

later

onset

disease

as

yet

to

be

identified

in

these

kindreds

This

represents

a

particular

challenge

in

hereditary

cancer

practice

as

the

clinical

community

tends

to

be

segregated

into

organ

specific

specialties

where

as

the

cancer

risks

and

the

risk

reduction

strategies

for

germline

mutation

carriers

require

a

variety

of

expertise

The

medical

needs

of

the

HDGC

families

are

therefore

best

served

through

an

engaged

multidisciplinary

team

O B I I O O O B I I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

diffuse gastric cancer
cdh1 mutation
lobular breast cancer
screening
prophylactic total gastrectomy

hereditary diffuse gastric cancer (hdgc)