Cervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US.We examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance.CIN3 cases varied substantially by size (1–10 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results.We demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion. The natural history of human papillomavirus (HPV) leading to invasive cervical cancer is well established Reporting CIN3 as a single outcome based on the worst histological diagnosis on the cervix Detailed mapping of the LEEP segments that covers the surface of the entire cervix provides an opportunity to identify and characterize clinical subgroups of CIN3 cases. Thus, we performed a detailed characterization of LEEP specimens to understand predictors of CIN3 heterogeneity and to evaluate the relationship of CIN3 heterogeneity with screening test results in a large population-based study of women with abnormal cervical cancer screening results. Examining the heterogeneity CIN3 cases referred to a colposcopy clinic after abnormal cervical cancer screening may aid in elucidating the biological differences between CIN3 cases, and thereby inform future efforts to reduce unnecessary treatment of CIN3 that are not clinically important.We conducted the analysis in the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED), a large population-based study composed of women referred to the University of Oklahoma Health Sciences Center (OUHSC) for abnormal cervical cancer screening test results. SUCCEED design and methodology, including the details on enrollment, questionnaire data, HPV DNA genotyping, histology, and cytology procedures, have been described in depth elsewhere Participants completed interviewer-administered, standardized questionnaires and provided liquid-based cytology specimens for ThinPrep Pap and HPV genotyping by Linear Array (Roche Diagnostics). OUHSC gynecologists performed colposcopic examination according to routine OUHSC practice. Women were treated by LEEP of the transformation zone, if indicated by ASCCP guidelines Every LEEP specimen was divided into 12 topographically designated sections or segments (a “clockface” depiction of the cervix) for detailed histopathological mapping. According to the SUCCEED study protocol, two segments from each LEEP representing the worst lesion and normal cervical tissue were snap-frozen for molecular studies. The remaining 10 segments were formalin-fixed, paraffin-embedded and analyzed to generate individual histology results for each segment. The study pathologists at OUHSC, masked to HPV genotyping data, determined the histology using CIN terminology. One or more of the following diagnoses were noted for each o'clock segment of the cervix per individual: other, negative/normal, atypical metaplasia, CIN1, CIN2, CIN3, adenocarcinoma in situ, squamous cell carcinoma, and adenocarcinoma. In addition, if the clinician determined that the entire transformation zone or extent of a lesion could not be visualized adequately, endocervical curettage (ECC) and/or a deeper, secondary LEEP (“top hat procedure”), which removes tissue from higher up in the endocervical canal, were performed. Per common practice, the cases were categorized according to the worst diagnosis for each woman based on the diagnosis of the most abnormal LEEP segment, ECC, and/or top hat.During the study period, 975 women were managed by LEEP Figure 1continuous adjacent LEEP segments with CIN3. In addition, we dichotomized the CIN3 lesion size as a “small” versus “large” CIN3 lesion, defined by a cut-off of <3 CIN3 segments versus 3+ CIN3 segments. Similar trends were observed with different cut-points .For our analysis, we defined the diagnosis of each o'clock segment of the cervix by the most severe diagnosis, if more than one diagnosis was noted for an individual LEEP segment. We focused our analysis on the following worst outcomes for each LEEP segment: normal, CIN1, CIN2, and CIN3. A priori, we defined subgroups of CIN3 cases by the presence or absence of CIN1 and CIN2 in conjunction with the presence of CIN3 in any of the LEEP segments. Accordingly, we categorized the CIN3 cases into four subgroups: solitary CIN3, CIN3+CIN2, CIN3+CIN2+CIN1, and CIN3+CIN1. As a sensitivity analysis, we further categorized solitary CIN3 as “true” solitary CIN3 cases by excluding cases with CIN1 and/or CIN2 diagnoses in any of CIN3 LEEP segments. We also a priori defined the size of CIN3 by the number of LEEP segments with CIN3 among all LEEP segments analyzed and the number of 2 test. In addition, we tested for trend across ordered groups using the nptrend command, a nonparametric test that is an extension of the Wilcoxon rank-sum test. We also calculated the percent detection of small versus large CIN3 lesions by the diagnostic tests. For all analyses, P-values of ≤0.05 were considered statistically significant. All tests of statistical significance were two-tailed. Analyses were performed using Stata 11.1 .First, we performed a case-case comparison of the four CIN3 subgroups for selected known risk factors of cervical cancer: age at LEEP, length span of sexual activity , parity, OC use, lifetime number of sexual partners, smoking, Pap test history (number in past five years), HPV genotypes  HPV type infections, and presence of HPV16 infections). We also performed a case-case comparison for diagnostic factors based on the following cervical cancer screening tests: cytology results prior to LEEP, biopsy results at LEEP visit, and colposcopic impression results at LEEP visit. In addition to the categorical variables of these screening test results, we dichotomized the screening test results to distinguish low grade from high grade diagnoses: biopsy histology of CIN3+ compared with CIN2 or less, cytology of high-grade squamous intraepithelial lesions (HSIL) or worse (HSIL+) compared with less than HSIL, colposcopic impression of CIN3+ compared with CIN2 or less. Second, we evaluated the differences between categories of CIN3 lesion size with the same factors. We tested for differences between categorical variables and CIN3 subgroups and lesion size using the Pearson χcontinuous CIN3 size (data not shown). In addition, we also observed a variable range in number of CIN2 (1–10 segments) and CIN1 (1–6 segments) present as concomitant lesions among the CIN3 cases (data not shown).Three hundred and nine women were identified as CIN3 cases according to the worst LEEP diagnosis Table 1.We summarized all 309 women included in the analysis in a single circular histogram Figure 3The majority of these women  had CIN lesions of lower grades in other LEEP segments in addition to the CIN3. About half of the heterogeneous lesions were CIN3 cases with CIN2 lesions (n = 116), followed by CIN3 without additional lesions (n = 79) and CIN3+CIN2+CIN1 (n = 79), as well as a small percentage of CIN3+CIN1 (n = 35) Table 2.The distributions of clinical and pathological characteristics examined and presented in CIN3 size was widely distributed in the study population and ranged from 87 women (28%) with one CIN3 segment involved to 55 (18%) women with CIN3 across five or more CIN3 segments Table 3.continuous CIN3 size (data not shown). Dichotomizing CIN3s by lesion size (<3 vs. 3+), the sensitivity of HSIL cytology at LEEP was 74% for small CIN3s and 86% for large CIN3s  presented with CIN3 lesions in conjunction with lower grades of CIN in different regions on the cervix. We observed a wide range of CIN3 sizes from small focal lesions to extensive CIN3 covering most of the cervix. To better characterize women within the CIN3 diagnostic group, we examined the distribution of risk factors as well as clinical and pathological information from cervical cancer screening tests by CIN3 subgroups and CIN3 lesion size.Some previous studies have suggested that CIN may be more common on the anterior and posterior lips of the cervix than at the lateral angles We also observed that compared to CIN3 cases with CIN2, solitary CIN3 and CIN3+CIN1 cases were more likely to have a larger sized CIN3 lesion. Our data corroborate the model that high-grade pre-cancer grows out from a small lesion possibly surrounded by low grade lesions, such that either CIN3 expands while the CIN1 regresses We sought to understand how clinical and pathological characteristics are related to CIN3 lesion size. The CIN3 lesions observed by McCredie and colleagues in their retrospective study of women with untreated CIN3 who progressed to cancer were large We did not observe associations between other risk factors previously reported to be associated with HPV infection and progression  and CIN3 size, suggesting that these factors are not paramount at the later stages of CIN3 natural history. However, we noted an insignificant trend of less OC use in women with larger CIN3s. We recently observed in the same population that contraceptive methods requiring doctor visits such as OCs are associated with more Pap tests in the previous five years, which could explain this observation (data not shown).In addition, we examined the effect of CIN3 size on cervical cancer screening results and found that larger CIN3 lesions were more likely to be diagnosed as HSIL+ at time of LEEP visit. Similarly, larger CIN3 size was more common among more severe colposcopic impression at time of LEEP visit and with a higher percentage of a CIN3+ biopsy result at the colposcopy visit, demonstrating that larger CIN3 cases are easier to detect by colposcopy. These finding highlight a current dilemma in cervical cancer screening: new screening tests such as HPV DNA detection are more sensitive than the current gold standard of following up cytology with colposcopy and biopsy The main strengths of our study are the large population-based sample of CIN3s and the detailed mapping of LEEP specimens, which allowed for a thorough evaluation of the heterogeneous manifestations of CIN3 cases. While examination of the 12 LEEP segments allowed us to study lesion size in unprecedented detail, a finer resolution would have provided more accuracy since multiple histologic diagnoses could be found even within a LEEP segment. Although the cross-sectional design of our study may be viewed as a limitation, it is not possible to follow CIN3 prospectively. Furthermore, this design permitted the accrual of large numbers of women into the study for studying CIN3 cases with detailed mapping of disease in LEEP specimens. In addition, our analysis was not based on panel adjudication of histology results, but on the community histology diagnosis by a single experienced pathologist.In summary, our data showed that women with CIN3 lesions without concomitant CIN2 or CIN1 lesions were more likely to be older, have longer sexual activity span, and have fewer high risk HPV infections and that larger CIN3 lesions were more common among women infrequently screened, with HSIL or worse cytology, and CIN3 or worse impression in colposcopy. Interestingly, we also observed that in our population, HPV16 positivity was not associated with larger CIN3 lesion size. Although our and others' data suggest that CIN3 lesion size is an important indicator of risk of invasion, lesion size can only be determined post-treatment. We show that HSIL cytology and CIN3 impression in colposcopy with CIN3 biopsy results point to larger CIN3s that most likely have a higher risk of invasion compared to small incipient lesions. While the findings from this study are important, they are not sufficient to establish which CIN3 should be treated. We are now conducting detailed molecular analyses of cervical lesions, using microdissection to permit the molecular evaluation of CIN3 heterogeneity to identify better risk markers for management of CIN3.


RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations – predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation  suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease.  GUCY2D (LCA1) RPE65 (LCA2) SPATA7 (LCA3) AIPL1 (LCA4) LCA5 (LCA5) RPGRIP1 (LCA6) CRX (LCA7) CRB1 (LCA8) NMNAT1 (LCA9) CEP290 (LCA10) IMPDH1 (LCA11) RD3 (LCA12) RDH12 (LCA13) LRAT (LCA14) TULP1 (LCA15) KCNJ13 (LCA16) IQCB1MERTKCRXIMPDH1 mutations Leber congenital amaurosis  is a group of retinal dystrophies, which represents the most common cause of blindness in childhood (10–18%) GUCY2D, RPE65, CRB1, TULP1, CRX, RPGRIP1, AIPL1) CEP290, IQCB1) The implication of LCA genes in retinal dystrophies of later-onset is not uncommon , North-America (The United States and Canada) and China were considered in this study including 574 LCA probands who have not had disease-causing mutations identified in the known LCA genes, 96 probands affected with autosomal recessive early-onset severe retinal dystrophies (EOSRD), 150 with autosomal recessive retinitis pigmentosa (RP) and 32 with other retinal dystrophies. The ethnicity of patients and diagnoses are shown in RD3 mutations independently. Patients of all cohorts but one were screened by direct sequencing using intronic primers designed to amplify the two RD3 coding exons and intron-exon boundaries. The 313 LCA probands of the University of Iowa Carver College cohort were screened by single strand conformation using as controls 151 normal individuals. PCR, direct sequencing and SSCP conditions, as well as primer sequences are available on request.Samples were collected and screened for A variant was predicted to be pathogenic when it showed familial cosegregation with the disease and absence in control individuals and when it was predicted to be damaging by Align DGVD, Polyphen-2, SIFT, SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder available through the Alamut Interpretation Software 2.0.RD3 gene. Microsatellite markers distributed over a 12.6 Megabases (Mb) interval spanning the RD3 locus on chromosome 1q32.2-q41 (according to the UCSC Genome Browser GRCh37/hg19 assembly) were used for haplotype analysis. Microsatellite markers included (from telomere to centromere): AFMB347YA5 (D1S2782), AFM281YG1 (D1S471), AFM310VB1 (D1S491), AFM108YA3 (D1S205), AFM179XG5 (D1S414) AFMC011YD5 (D1S2810), AFMB342YG1 (D1S2780), AFM203ZB6 (D1S425), AFMA127WB5 (D1S505), AFM297XC1 (D1S2827), AFM058XG9 (D1S2880). Amplified fragments were electrophoresed on an automatic sequencer  and analyzed using the GeneScan Analysis 3.7 and Genotyper softwares. For each marker, the heterozygote frequency and the size range of alleles were either available from Genethon Linkage Map Haplotype analysis was performed using 4/5 families segregating the c.112C>T mutation of the The degree of linkage disequilibrium and the estimation of the age of the mutation were obtained as described previously Three novel mutations were detected in seven apparently unrelated LCA families . Two outAll three mutations segregated with the disease in families and were neither identified in controls nor reported elsewhere.RD3 mutations. Indirect studies at the RD3 locus were performed on 4/5 families indicate that the two Moroccan families and the Turkish family share a small 1 Mb-long common haplotype that differed in the Libanese family . This variant that changes a basic amino acid into an uncharged residue is predicted to be deleterious by the SIFT, Polyphen-2 and AlignGCVD programs. The change was identified neither in 200 control individuals nor in databases listing SNPs. This change was identified in a heterozygous state in an isolated case born to non-consanguineous parents  mouse rod-cone dystrophy 2 (rcd2) collie RD3 in LCA. Yet, the mutation rate obtained in a very large cohort of LCA cases originating worldwide (7/574 LCA cases excluding other LCA genes) and consistent identification of homozygous mutations in consanguineous families , support the very low frequency of heterozygous RD3 carriers in the general population.Until this report, the implication of RD3 encodes an evolutionary conserved basic protein of 195 amino acids that is preferentially expressed in the retina rd3 mouse which express a truncated unstable rd3 protein resulting from the p.R107* mutation, is consistent with a role of the protein in the trafficking of GC2 in rods as well. As a consequence, like the double GC1/GC2 knock-out, the complete inactivation of both rd3 alleles is expected to preclude cGMP synthesis in both rod and cone outer segments and to hinder recovery of the dark state after light stimulation i.e. when eyes open – and the degeneration is almost complete by the age of 2–4 months rd3 frameshift mutation (p.Pro139Alafs*) of the rcd2 collie rcd2 dogs develops almost normally, by the age of 2–2.5 months, the outer segments of both rods and cones are completely lost, with rods degenerating faster than cones rd3 and GC double knock-out mice LCA12 and LCA1. Indeed, the review of the natural history and clinical data of our patients and that of the two original RD3 families LCA1 patients harboring GUGY2D mutations Interestingly, these data which support a common mechanism of photoreceptor degeneration in RD3 mutations identified thus far are expected to truncate the RD3 protein . It is tempting to hypothesize that similar to several other LCA genes, mutations with a milder effect on the protein structure and/or function may be responsible for milder phenotypes; for recent reviews see RD3 mutations. This finding is consistent with the LCA type I-specific involvement of RD3 mutations. Indeed, of the LCA genes, most genes involved in EOSRD and RP account for LCA type II GUCY2DRPGRIP1CEP290RD3 [present study]. Refraction may represent a distinctive feature in LCA type I since patients with GUCY2D mutations consistently have high hypermetropia (>+7) with frequent enophthalmia RPGRIP1, CEP290 or RD3 mutations present with lower or no hyperopia : RD3 mutations and an individual of the general population with no family history of LCA is negligible. Unions between affected persons with LCA and or between LCA and RP patients are not uncommon. The identification of RD3 mutations in one spouse must prompt the search for RD3 mutations, only when the other spouse is affected with LCA type I. Indeed, if the spouse is affected with LCA type II or autosomal recessive RP of later-onset, the genetic counseling can straightaway be reassuring.With respect to genetic counseling, our data that support a dramatically low RD3 carrier frequency in the general population indicate that the genetic risk in the case of the union between an affected person harboring RD3 mutations in large cohorts of patients accurately phenotyped by referent clinicians has major clinical implications. Most LCA genes have demonstrated a preferential or specific retinal expression CEP290 which mutations are either responsible for non-syndromic LCA or a range of syndromic forms of the disease; for review see IQCB1 gene at the NPHP5 locus have been shown to cause a form of Senior-Loken syndrome characterized by a congenital retinal dystrophy associated with a renal dysfunction of highly variable age of onset (1st to 5th decade) RD3, it is worth noting that none of the patients reported here whose ages ranged from 2 to 31 had renal failure, neurological symptoms or intellectual disability. Owing to the retina-specific pattern of expression of RD3, it is likely that the disease may remain restricted to this tissue. In these patients, anxiogenic and expensive extraocular explorations must not be systematically prescribed.The comprehensive survey of a sine qua none condition to maintain living and functional rod and cone cells. Our knowledge regarding the mechanisms of this complex process - which alteration accounts for ∼17% of LCA cases - are incomplete. In particular, most of the molecular players and disease mechanisms of light-driven trafficking between the inner and outer segments of photoreceptors are unknown. Interestingly, although RD3 alterations are quite uncommon, their description in man and animal models unraveled a crucial protein for GC expression and trafficking in photoreceptors. Further studies designed to identify RD3 partners will hopefully contribute to the deciphering of mechanisms for dark-state recovery and will hopefully allow uncovering some of the yet unidentified LCA causing genes.The recovery of the dark state in the retina through GC-mediated increase in cytoplasmic cGMP concentration in photoreceptor outer segments is RPE65−/− Briard dogs has paved the way for the development of gene and drug therapy for a broad range of eye disorders and made large dogs the species of choice for these developments; for review see RD3 mutations are uncommon causes of LCA, the availability of naturally occurring rd3 animal models, among which a large dog (collie), it is likely that gene and drug therapy protocols will be developed to treat patients with RD3 mutations.Finally, it is worth remembering that the success of gene replacement and drug therapy to restore vision in naturally occurring


African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs. Virulent isolates kill domestic pigs within 7–10 days of infection. In chronic cases ASF causes respiratory disorders and in some cases swelling around the leg joints and skin lesions. Domestic pigs can survive infection with less virulent isolates and in doing so can gain immunity to subsequent challenge with related virulent viruses ASF is endemic in many sub-Saharan African countries as well as in Sardinia. In 2007 ASF was introduced into Georgia and from there spread rapidly to neighbouring countries in the Trans Caucasus region, including Southern European Russia Ornithodoros spp. and warthogs in eastern and southern Africa. In many regions the isolates circulating in domestic pigs are genetically more similar.Genotyping of ASFV isolates by partial sequencing of the B646L gene encoding the major capsid protein p72 has identified up to 22 genotypes + T cells since depletion of these cells was shown to abrogate this protection Previous work has shown that pigs are protected from challenge with related virulent isolates following infection with natural low virulence isolates and with virus attenuated by passage in tissue culture or by deletion of genes involved in virulence Despite this early experience in Portugal and Spain, the prospect of developing successful attenuated vaccines have improved as substantial progress has been made in identifying ASFV genes involved in virulence and immune evasion and the complete coding sequences of a number of ASFV isolates are now available 22.1ASFV isolates used in this study have been described previously and included Portuguese isolates of ASFV, OURT88/3  and OURT88/1  2.24 TCID50 of non-virulent ASFV isolate OURT88/3 and boosted intramuscularly 3 weeks later with 104 HAD50 of virulent ASFV isolate of OURT88/1. Pigs were then challenged 3 weeks later with 104 HAD50 of either Benin 97/1 or virulent Uganda 1965 intramuscularly.Pigs used in the first experiment (experiment 1) at IAH Pirbright Laboratory UK were cross-bred pigs, Large White and Landrace, of average weight 20 kg at the first immunisation. For the second experiment specific pathogen free (SPF) Large White pigs were used from Anses, Ploufragan, France, SPF facility and were of 15 kg average weight at the first immunisation (experiment 2). For the third experiment (experiment 3) carried out at Anses Ploufragan, France, Large White pigs were obtained from a local high health status farm and the average weight at the first immunisation was 11 kg. All pigs were maintained at high security facilities throughout the experiment. The first experiment at Pirbright was performed under Home Office licence PPL 70-6369. Experiments at Ploufragan were performed according to the animal welfare experimentation agreement given by the Direction des Services Vétérinaires des Côtes d’Armor (AFSSA registration number B-22-745-1), under the responsibility of Marie-Frédérique Le Potier (agreement number 22-17). Briefly, pigs were intramuscularly inoculated with 102.3ASFV-inoculated pigs were monitored for body temperature and other clinical symptoms and these were recorded and scored according to the clinical scoring system shown in 2.450.Peripheral blood was analysed at different days post-immunisation for the presence of ASFV by quantitative PCR (qPCR) as described previously 2.55 HAD50/ml. Uninfected porcine bone marrow culture supernatants were used as negative control antigen.Development of T cell immune responses to ASFV after immunisation was analysed by IFN-γ ELISPOT and proliferation assays as described previously The development of ASFV specific antibodies was analysed using a competition ASF ELISA kit (INGENASA PPA3 COMPPAC), and the antibody titre was expressed as log 2 dilution of end point which gives 50% competition.33.1Three experiments were carried out in which pigs were immunised with the non-virulent Portuguese OURT88/3 genotype I isolate followed 3 weeks later by the closely related virulent Portuguese isolate OURT88/1 and then challenged 3 weeks later with either the West African genotype I isolate, Benin 97/1, or the genotype X virulent Uganda 1965 isolate. In the first experiment at Pirbright, 3 immunised pigs and 4 non-immune pigs were challenged with Benin 97/1. In the second experiment at Ploufragan, a total of 12 pigs were immunised and challenged with either Benin 97/1 or virulent Uganda 1965. Ten pigs were prepared as non-immune controls and challenged with either Benin 97/1 or virulent Uganda 1965. As a control for weight gain, an extra group of 5 pigs were included in this experiment. In the third experiment at Ploufragan, a group of 7 pigs were inoculated and 6 of these and 6 non-immunised pigs were challenged with Benin 97/1.7 copies of the virus genome/ml; and up to 8.8 HAD50/ml virus), and died or were euthanized for ethical reasons within 7 days of challenge (7 HAD50/mg tissue) . Virus genome was detected at low copy numbers by qPCR in blood samples from an additional 2 pigs but these were negative by HAD assay. Pigs 1819, 1822 and 1841 were terminated for ethical reasons between day 4 and day 6 post boost with OURT88/1 before the potential development of severe ASF symptoms.In the second experiment of the 12 immunised pigs, 5  developed a transient pyrexia  followin50/ml; pig 1845 had a temperature at day 7 of 40.6 °C and the virus genome was detected at 633 copies/ml; and virus at 2 HAD50/ml. The other two pigs challenged with virulent Uganda 1965 isolate showed no clinical signs and no virus was detected in blood by qPCR or HAD assay. Five pigs were challenged with Benin 97/1, two pigs  developed typical ASF , and either died or were terminated within 8 days of challenge  and tissues (virus ∼7 HAD50/mg of tissue) were detected from all lymphoid tissues in all of the non-immune pigs challenged  showed pyrexia from 2 weeks after the first immunisation . This piUnlike the non-immune pigs, immune pigs challenged increased their body weight during the challenge .3.2Lymphocytes from immunised pigs in experiment 1 were collected at various times post-immunisation and IFN-γ ELISPOT and proliferation assays were performed with OURT88/3 or Benin 97/1 as antigen. In all 3 pigs, the numbers of ASFV specific IFN-γ producing cells was rapidly increased after the OURT88/3 inoculation and further increased after the OURT88/1 boost. Both OURT88/3 and Benin 97/1 isolates stimulated lymphocytes from immunised pigs to an approximately equal amount A–C. Low At the termination of the experiment, lymphocytes from these pigs were tested for cross-reactivity stimulated with various ASFV isolates by IFN-γ ELISPOT assays A. ImmuneIn the second experiment B, lymphoA competitive ELISA based on the p72 major capsid protein was used to measure development of anti-ASFV specific antibodies. The results from analysis of sera collected in experiment 2 and 3 are shown in 4In this study we have demonstrated that experimental immunisation of pigs with a non-virulent ASFV genotype I isolate from Portugal, OURT88/3, followed by a boost with a closely related virulent isolate, OURT88/1, can induce protective immunity in European domestic pigs against challenge from two virulent African isolates of ASFV. These included a genotype I isolate from West Africa, Benin 97/1 and a genotype X isolate from Uganda, virulent Uganda 1965. Overall 85.7% and 100% pigs were protected from Benin 97/1 and Uganda 1965 ASFV challenge respectively. More than 78% of pigs challenged with Benin 97/1 and 50% of pigs challenged with Uganda 1965 were completely protected by not showing any sign of disease or development of viraemia.Phylogenetic analysis of the concatenated sequences of 125 genes conserved between 12 complete genome sequences showed that the OURT88/3 and Benin 97/1 sequences are greater than 95% identical across these genes Ornithodoros erraticus ticks in Portugal and described not to cause clinical signs or viraemia ex vivo, with different ASFV isolates, showed various degrees of cross-reactivity and this correlated well with cross-protection induced in vivo. Good cross-reactivity against genotype X isolate virulent Uganda 1965 (ex vivo assay, all of the pigs immunised and challenged with virulent Uganda 1965 virus were protected. No cross-reactivity to genotype XIII isolate Malawi LIL 20/1 was detected and this correlates with the observation that OURT88/3 and OURT88/1 immunised pigs are not protected from Malawi LIL 20/1 challenge . Taken together these data suggest that this ex vivo, IFN-γ ELISPOT assay might be a useful tool to assess vaccine efficacy and/or to assess possibility of ASFV isolate-cross-protection.The ASFV OURT88/3 strain was isolated from nda 1965 A was obsAn anti-ASFV antibody response also developed after OURT88/3 immunisation and was boosted after the OURT88/1 inoculation. The anti-ASFV antibody titre was measured by a p72 competition ELISA, however we could not conclude from these experiments whether the level of antibody developed by our immunisation protocol is either sufficient or necessary for protection.OURT88/3 has been used as a vaccine model to identify what is required for inducing ASFV protective immunity in domestic pigs. The observations of adverse effects of OURT88/3 immunisation in some of the pigs vaccinated in France suggest that further attenuation of this isolate by deleting additional genes or possibly changing the dose or route of vaccination may be useful. Secondly, the results from experiment 2 showed that our current protocol did not induce complete protection in all of the pigs immunised with the virulent OURT88/1 boost. This may be due to the genetic background of the pigs as we have previously demonstrated that cc inbred pigs are also not always protected by OURT88/3 from OURT88/1 challenge


To analyze the genetic pathogenesis and improve the accuracy of the diagnosis of the disease, this study reported the clinical features of 20 patients with Silver Russell Syndrome (SRS) in the Beijing Children's Hospital and detected the chromosome 11p15 imprinting defects in 16 patients of them.20 SRS cases diagnosed in Beijing Children’s Hospital from 2006 to 2011 were studied retrospectively for clinical manifestations, physical signs, laboratory examinations and respond of GH treatment. We compared with 3 different diagnostic criteria and used the methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) method to detect the chromosome 11p15 imprinting defects in 16 patients of them, meanwhile take 10 normal control.We collected 20 SRS patients over a period from 2006 to 2011 with 3 criteria. The concordance is 90%. Include fifteen males and five females, age range 0.08~12.17yr. The most chief complaint is short, 85%. Then, it is asymmetry (5%), and external genital abnormalities (10%). The clinical characteristics with the frequencies accounted for over 80% included small for gestation age(SGA), postnatal growth retardation, craniofacial dysmorphism, asymmetry and super thin of whole body, extremely limbs, fifth finger clinodactyly, BMI<-2SDS and height was obviously lagging behind the bone age. Six of them had used the growth hormone for 3-months to 12 Months. The growth velocity is from 4cm to 10cm/year. In the 16 patients, 6 patients were found hypomethylation in chromosome 11p15 ICR1, the other one with chromosome 11p15 ICR1 hypomethylation and ICR2 hypermethylation may be the result of the maternal chromosome 11p15 uniparental disomy. Another case had duplication of the maternal chromosome 11p15 fragment. Two cases had good effects of GH treatment, one with chromosome 11p15 ICR1 hypomethylation and the other one was normal.The top 3 clinical features in SRS are ① growth retardation include SGA and/or postnatal. ② Malformation include craniofacial dysmorphism, asymmetry of face and/or limbs, fifth finger clinodactyly. ③ Super severe low BMI and height was obviously lagging behind bone age. No laboratory and imagination specificity. Chromosome 11p15 imprinting defect is the major genetic disturbance in SRS, about 50%, and ICR1 hypomethylation is the predominant molecular alteration. The MS-MLPA is a technique for detecting all chromosome 11p15 imprinting defects of SRS. It is useful for the study of the genetic aspects of SRS. The relation between the respond of GH treatment and genetic changes is uncertainty.


We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The CaThese results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells. We have previously shown significant alterations in endogenous levels of both glutamic and gamma-aminobutyric acids in particular brain structures after the extremely traumatic water immersion restraint stress (WIRS) experience in rats The hippocampal DG has a unique ability to generate new neurons throughout the entire life of an individual, often referred to as adult neurogenesis Myosin VI (Myo6) is an actin-based molecule responsible for the transport of intracellular cargos including proteins, vesicles and organelles trans retinoic acid (ATRA) as a model for neural progenitor cells These previous studies led us to propose a possible role of Myo6 in mechanisms underlying the aforementioned suppression of BrdU incorporation into the hippocampal DG enriched with neural progenitor cells, in mice with traumatic stress. Therefore, the present series of experiments was intended to examine the role of Myo6 in mediating proliferation for self-replication and differentiation into progeny lineages using murine embryonal carcinoma P19 cells endowed to proliferate and differentiate into neuronal and astroglial lineages in the presence of all-2 and 95% air at 37°C with a medium change every 2 days.Mouse embryonal carcinoma P19 cells  were cultured in alpha minimal essential medium (αMEM)  supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL). P19 cells are able to differentiate into a neural lineage in the presence of ATRA  as described previously 5 cells/cm2, followed by culture in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO BRL) with 10% FBS for 24 h and subsequent stable transfection with pSI-Myo6, pSI-GFP and pcDNA3.1 vectors, or pSI, pSI-GFP and pcDNA3.1 vectors, using 2 µg of DNA and Lipofectamine and Plus reagent  in 0.5 ml of Opti-MEM (Invitrogen) as described previously http://blast.ncbi.nlm.nih.gov) analysis revealed a 96% homology between porcine and murine Myo6 proteins.The full-length coding region of porcine Myo6, which was kindly donated by Dr. Tama Hasson , was inserted into pSI vector using the Ligation High reagent as described previously Reverse transcription-polymerase chain reaction (RT-PCR) analysis was conducted as described previously Western blotting was done by using antibodies against Myo6 , Doublecortin , Synapsin I , neuronal nuclei (NeuN) (Merck Ltd.), microtubules-associated protein-2 (MAP2) (Sigma), glial fibrillary acidic protein (GFAP) (Sigma), Sox2 (Santa Cruz Biotechnology) and β-tubulin (Sigma) as described previously Five different visual fields each in parallel experiments were chosen at random from each culture well with undifferentiated P19 cells cultured with ATRA for a period of up to 4 days, and examined under a phase contrast micrograph in a blinded fashion, followed by calculation of areas of round aggregates composed of clustered proliferating cells for summation using Scion Image β 4.02 software  as described previously Stable transfectant cells were cultured in the presence of ATRA for 4 days, followed by re-plating on dishes previously coated with poly-L-lysine for further culture for 2 h and subsequent double staining for DNA with 10 µg/ml Hoechst33342 (Sigma) and 5 µg/ml propidium iodide (PI) (Sigma). Cells were observed under a confocal microscope . The number of cells stained with these dyes was individually counted in five different visual fields selected at random in a blinded fashion in order to calculate the percentages of dead cells stained with the membrane-impermeable dye PI out of the total cells stained with the membrane-permeable dye Hoechst33342. Cellular viability was also quantified by the activity of lactate dehydrogenase (LDH) released into the culture medium by an enzyme-coupled color development method as descried previously 5 cells/ml in αMEM supplemented with 5% FBS and 0.5 µM ATRA, followed by culture for 4 days under floating conditions favorable for proliferation. These floating cells were harvested and trypsinized, followed by plating onto dishes previously coated with poly-L-lysine at a density of 2×105 cells/ml in αMEM supplemented with 10% FBS. They were subsequently cultured for an additional period up to 6 days in the absence of ATRA to induce spontaneous differentiation as shown with murine fetal cortical progenitors 2/95% air at 37°C.Pluripotent P19 cells have been shown to differentiate into neuronal and astroglial lineages after commitment in the presence of ATRA as described previously 5′-ACGCGT (MluI site) GCAAGAACCCTCACTGGC-3′ and the reverse primer 5′-CTCGAG (XhoI site) AGGCTGCCGGGCTGCGGGCG-3′ from the mouse genome. The Myo6 (-1086 to+210) promoter fragment was cloned into the promoterless pGL-3 basic vector , to create the recombinant plasmid –1086/+210 Myo6-Luc.A reporter plasmid with the Myo6 promoter was prepared as follows. The mouse Myo6 promoter was obtained by cloning using the forward primer 5 cells/ml in plates (φ100 mm) previously coated with 0.2% agarose, and cultured in αMEM supplemented with 10% FBS and 0.5 µM ATRA. Twenty-four hours after transfection of the reporter plasmid, the cells were exposed to 1 µM A23187 (Sigma) for 6 h, followed by a medium change, and then cultured for an additional 24 or 48 h. The cells were then lysed for determination of luciferase activity using specific substrates in a luminometer according to the manufacturer’s protocol. Transfection efficiency was normalized against the activity of pure Renilla luciferase Pluripotent P19 cells were transiently transfected with a reporter plasmid containing the promoter region of Myo6 (−1086 bp to +210 bp) along with the internal control vector pRL-SV40 (Promega) using the Lipofectamine 2000 reagent. Transfected cells were plated at 1×10t-test or one-way analysis of variance (ANOVA) with the Bonferroni/Dunnett post hoc test.Quantitative data are expressed as the mean ± S.E. and the statistical significance was determined by the two-tailed Student’s In our previous study To evaluate the mechanism underlying the possible deterioration in proliferative activity, each stable transfectant clone was screened for mRNAs for several cell cycle regulators of the Cyclin family An attempt was thus made to determine whether stable overexpression of Myo6 leads to altered expression of a variety of basic helix-loop-helix (bHLH) genes responsible for the positive and negative regulation of proliferation for self-renewal and/or neuronal differentiation in undifferentiated neural stem cells P19 cells were cultured in the presence of ATRA for 4 days under floating conditions to induce clustered aggregate formation due to self-replication, followed by immunostaining for the neural progenitor cell marker nestin. Cells clustered in round aggregates were immunoreactive for nestin and also exhibited a marked increase in MTT reduction, both indices of cellular proliferation We next examined the effects of Myo6 knockdown on proliferation and differentiation of pluripotent P19 cells using RNA interference techniques. Cells were transfected with Myo6 siRNA, followed by culture with ATRA for 2 days under floating conditions and subsequent determination of endogenous levels of Myo6. A drastic decrease in endogenous Myo6 protein levels in cells transfected with 2 different Myo6 siRNA oligonucleotides compared with those with a control oligonucleotide was invariably seen on Western blotting . QuantifWe then examined the expression of Sox2 in P19 cells treated with Myo6 siRNA for 2 or 4 days in the presence of ATRA under floating conditions. Western blotting analysis clearly revealed a marked, statistically significant increase in endogenous Sox2 protein levels in P19 cells transfected with Myo6 siRNA for 2 and 4 days compared to those exposed to control siRNA  Figure . Cells wTo test the selectivity of the correlation between Myo6 expression and cellular proliferation, the MTT assay was used to determine whether Myo6 siRNA affects cellular proliferation in cultured proliferating cells other than pluripotent P19 cells. In C6 glioma cells cultured for 2 days, marked expression of Myo6 protein was seen in a manner sensitive to knockdown by siRNA . AlthougWe have previously shown a more than twofold increase in Myo6 mRNA expression is induced in P19 cells exposed to the calcium ionophore A23187 The essential importance of the present findings is that proliferation was markedly suppressed in pluripotent P19 cells stably overexpressing the motor protein Myo6 without induction of cell death. Conversely, the current findings that proliferation was significantly promoted in P19 cells by decreased endogenous Myo6 levels due to siRNA transfectionargue in favor of the idea that Myo6 negatively regulates self-replication in pluripotent P19 cells endowed to either proliferate for self-renewal or differentiate into neuronal and astroglial lineages. Taking into consideration the unresponsiveness of different cell cycle regulators and bHLH proneural genes other than Hes5, it is conceivable that downregulation of Hes5 expression is at least in part responsible for the inhibition of proliferation in stable Myo6 transfectants. In mice defective for the activator bHLH factor Mash1, for example, severe loss of neuronal precursors together with disappearance of the Notch signaling target Hes5 is seen in the ventral telencephalon normally enriched for Mash1 during neurogenesis However, the reason why Myo6 overexpression inhibited cellular proliferation without affecting the mRNA levels of several cyclins in P19 cells is still not clear. A future comprehensive analysis should be conducted on mRNA expression by all cyclin family members and relevant kinases to evaluatethe possible involvement of particular cyclin family members in the underlying mechanisms other than those tested here. Although the activator type bHLH factor NeuroD1 plays a pivotal role in the survival and neuronal differentiation of neural stem cells during adult neurogenesis Nevertheless, the exact signaling pathway from stable overexpression of Myo6 to preferential downregulation of Hes5 amongst different repressor and activator bHLH proneural genes examined is not clarified so far. One possible but hitherto unproven speculation is that intracellular Myo6 overexpression promotes the migration of particular cargo proteins and/or organelles required to suppress the transactivation of the Hes5 gene toward the nucleus. For instance, the mammalian target of rapamycin (mTOR) signals are known to be essential for maintenance of neural progenitor status through a mechanism associated with upregulation of Hes5 and Pax6 expression in pluripotent P19 cells cultured with ATRA sv/sv mice defective for Myo6, a significant deficit is seen in internalization of a particular ionotropic subtype of non-N-methyl-D-aspartate receptor (NMDAR) after stimulation In hippocampal neurons from Although Myo6 is highly expressed in cultured astrocytes expressing GFAP rather than in cultured neurons expressing NeuN It thus appears that the motor protein Myo6 selectively inhibits cellular proliferation required for self-replication through a mechanism relevant to downregulation of the repressor bHLH proneural factor Hes5 in undifferentiated P19 cells. Further evaluation of Myo6 expression will be undoubtedly beneficial for the future discovery and development of drugs useful for the prophylaxis, therapy and treatment of a variety of abnormalities and malfunctions seen in patients suffering from different neurodegenerative and neuropsychiatric diseases.


BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China.Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations  accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations  accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated.A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.In this study, our findings suggest that  BRCA mutation spectrum in Chinese populations are limited BRCA1BRCA1/2 mutational screening using conventional full gene sequencing BRCA1 and BRCA2 mutation screening is rarely reported. In additional to BRCA mutation spectrum, identification of founder mutations in various ethnic groups is also important to improve genetic screening and cancer risk assessment because it makes a more specific approach for molecular testing that targets the founder allele possible, thus resulting in reduced cost of genetic testing and faster turnaround time. The high frequency of founder mutations in a given population provides a large patient cohort not only for robust information regarding penetrance but also accurate assessment of the effectiveness of preventive measures.The incidence of breast cancer in Asia has rapidly increased over the past 10 years and is one of the highest in Hong Kong population BRCA1 and BRCA2 genes using conventional full gene DNA sequencing and identified a recurrent mutation c.3109C>T BRCA1 and BRCA2 mutations in a group of 651 Chinese probands (inclusive of the 119 probands) from Southern China using full gene sequencing and full high resolution DNA melting (HRM) analysis. Our results also prompted us to investigate the usefulness of rapid HRM for screening of the BRCA1 and BRCA2 founder mutations in Chinese population.The Hong Kong Special Administrative Region of the People’s Republic of China is an advantageous location to conduct such studies related to hereditary cancers in the Chinese population where 95% of the population is comprised of Chinese The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants involved in this study. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority West Cluster and other contributing hospitals, Hong Kong.www.HRBCP.org) from 1 March 2007 to 28 Feb 2011, were recruited. This group of 651 probands contained all 119 probands from our previous report BRCA mutations; or (6) they were ovarian cancer patients with a family history of breast cancer. The distribution of the 651 patients into the 6 categories was shown in BRCA counseling and testing, and were consented for genetic testing and blood and tumor collection. Patients who tested positive for a BRCA mutation were asked to help recruit their first-degree relatives, who were also offered testing. Written informed consent was obtained from all participants involved in this study. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority West Cluster and other contributing hospitals, Hong Kong.A total of 651 clinically high-risk breast and/or ovarian cancer patients (probands), referred to the Hong Kong Hereditary and High Risk Breast Cancer Programme  according to manufacturer instructions. Mutation analysis was performed by direct DNA sequencing of all coding exons of BRCA1 and BRCA2 and partial flanking intronic sequences. PCR conditions and primer sequences are available upon request BRCA1 and BRCA2, 41 PCR reactions for BRCA1 and 63 PCR reactions for BRCA2 were developed in our HRM run per each patient sample. Thus, samples were amplified in 384-well plates. LightCycler 480 High Resolution Master kit  was used for HRM analysis in patient samples in LightCycler 480 system (Roche). In brief, each reaction was performed in a final volume of 10 µl containing 20 ng of DNA, 0.25 µM of each primer (forward or reverse) and 1x LightCycler 480 HRM Master mix (Roche). The PCR profile was pre-activation at 95°C for 10 min, followed by 45 cycles of 95°C for 10 sec, a touchdown from 65–53°C for 30 sec at 2.5°C/sec and 72°C for 20 sec. At the end of the PCR cycles, PCR products was denatured at 95°C for 1 min and rapidly cooled to 40°C for 1 min. HRM analyses were performed from 60°C through to 98°C at a temperature gradient of 1°C/sec, acquiring 25 data points per °C. Each sample was run in duplicates for analysis. The analytical methods have been applied previously to our mutation scanning BRCA1 and BRCA2 genes were listed in To cover all exons of BRCA1 and BRCA2 germline mutations from unrelated families were genotyped for allele sharing indicative of a common ancestor. Thus, haplotype analysis was conducted at 6 microsatellite polymorphic loci D17S791, D17S855, D17S1323, D17S1322, D17S1335 and D17S1185 of the BRCA1 gene and at 6 loci D13S289, D13S1699, D13S1698, D13S171, D13S1695 and D13S260 of the BRCA2 gene. Primer sequences of all microsatellite polymorphic markers were listed in BRCA1 on chromosome 17q21.2–17q21.31 and a ∼2.5 Mb region encompassing BRCA2 on chromosome 13q12.3–13q13.1. Fluorescently end-labeled primers were used to amplify the microsatellite polymorphic regions. Size fractioning of PCR products were performed on a 3130xl Genetic Analyzer (Applied Biosystems) using the GeneScan 500 LIZ Size Standard and analyzed using the GeneMapper v3.7 software (Applied Biosystems). Haplotype estimations were performed using software program PHASE.Individuals with identical 2 analyses describe any difference in BRCA mutations carriers among all Chinese female patients. The significance of age and BRCA status of patients was determined by Wilcoxon test. Fisher's exact test was used in the analysis of categorical data where expected counts are less than 5. Linear-by-Linear association was used in the analysis of ordinal data. SPSS for Windows Release 16.0  was used to analyze the data; statistical significance and marginal statistical significance were set at P<0.05 and P<0.1 respectively.P values from χThis study included 651 probands , comprised of 611 breast cancer patients,17 ovarian cancer patients and the remaining 23 patients with both breast and ovarian patients. The mean age at diagnosis of breast cancer was 43 years old (range 18–82) and that of ovarian cancer was 43 years old (range 19–64). All probands were from Chinese ancestry and over 90% were from Guangdong province of Southern China.BRCA1 and BRCA2 of a total of 451 probands out of the 651 patients were conducted. Based on our sequencing results, 69  deleterious BRCA gene mutations were identified. Of the 69 deleterious mutations, 29  were in BRCA1 and 40  in BRCA2. There was no significant difference in the age of breast cancer diagnosis between BRCA carriers and non-BRCA carriers . Although the mean age of breast cancer diagnosis of BRCA2 mutation carriers (mean age 45.7) are slightly higher than that of BRCA1 mutation carriers (mean age 40.4), the difference was not significant . The spectra of all deleterious mutations identified are illustrated in BRCA1 and 17 were in BRCA2. Most of the novel deleterious mutations cause sequence frame-shift, leading to early termination of translation for protein products. In this study, we identified 4 recurrent BRCA1 mutations  accounted for 34.5% (10/29) of all BRCA1 mutations and 4 recurrent BRCA2 mutations  accounted for 40% (16/40) of all BRCA2 mutations. In addition, among our cohort of the 33 male probands, 7  deleterious mutations were found. Intriguingly, all 7 male probands carried BRCA2 deleterious mutations only.Extensive sequence analysis of all coding exons in BRCA1 and BRCA2 gene sequencing, we also developed full HRM assays for rapid screening of BRCA1 and BRCA2 mutations. In our developed full gene HRM assays, there are 38 BRCA1 and 63 BRCA2 HRM reactions per patients were established. In order to validate the testing performance of our in-house developed HRM assays, 8 probands with known BRCA1 deleterious mutations and 12 probands with known BRCA2 deleterious mutation from the 451 patients were analyzed. Those known BRCA mutations were previously identified by our full gene sequencing. In this first validation set, all 24 deleterious mutations and variants identified by sequencing were also detected by our in-house developed HRM assays. Thus, the detection rate for variant detection was 100% when compared to sequencing.Apart from the development of full BRCA1 deleterious mutations and 2 patients with different BRCA2 deleterious mutations. The detailed breakdown of all mutations and variants in the 24 patients was shown in BRCA1 and 1512 BRCA2 amplicons were analyzed (BRCA1 and BRCA2 were 1.2% (11/912) and 1.6% (24/1512) respectively. The calculated sensitivity and specificity for BRCA1 mutation and variant detection was 100% and 98.6% respectively while the sensitivity and specificity for BRCA2 mutation and variant detection was 100% and 98.3% respectively  and 4 recurrent BRCA2 mutations  were identified in our Chinese cohort . For BRCA2, the genotypes of all cases were examined at 6 polymorphic markers (BRCA1 mutation (c.981_982delAT) and 3 recurrent BRCA2 mutations  shared the same haplotype suggesting that these 4 putative founder mutations are derived from a common ancestor (BRCA2 mutation (c.3109C>T) BRCA2 mutations and so far is the highest proportion found in our cohort. For the only BRCA1 founder mutation confirmed, it accounted for 6.9% (2/29) all BRCA1 mutations in this cohort while all the BRCA2 founder mutations accounted for 35% (14/40) of all BRCA2 mutations. Characteristics of all 451 probands with or without BRCA mutations were shown in BRCA mutations have higher frequency of the family history of ovarian cancer than those without BRCA mutations. Furthermore, probands with BRCA mutations have greater average number of family member with breast cancer than those without BRCA mutations.In this study, a total of 4 recurrent e cohort . To dete markers . Haplotyancestor . For theBRCA2 founder mutation (c.7436_7805del370) is not easily detected by HRM. Thus, a panel of 5 HRM assays for the remaining founders was developed (BRCA2 founder mutation (c.3109C>T) from the 200 patients. Taken all together, the overall frequency of BRCA2 (c.3109C>T) founder mutation observed was 1.7% (11/651) among 651 Chinese patients and accounted for 26.8% (11/41) of all BRCA2 mutations in our Southern Chinese cohort. The total of 3 recurrent BRCA2 mutations then accounted for 36.6% (15/41) of all BRCA2 mutations.As some recurrent mutations were confirmed to be founder mutations in the Southern China population, we then rapidly developed HRM screening assays targeting each founder or recurrent mutations so as to further screen our Chinese population. Due to the 370 bp deletion, eveloped  and 2 anBRCA mutations to high-risk families of Chinese population from Southern China. Increasing cancer rates are of great concern to these Asian countries because these countries often have limited health and medical care resources and infrastructures to meet the needs of these patients. Thus, it is important to obtain a better understanding of the causes of breast cancer among Asians and other populations so as to improve prevention and cancer risk assessment efficiently worldwide. To our knowledge, this is one of the larger Chinese cohort studies comprising of 651 probands and comprehensive full BRCA1 and BRCA2 gene sequencing on 451 probands was employed for mutation screening analysis. Our findings revealed that the proportion of BRCA mutation is15.3% in a defined high risk group of Chinese population while Caucasian cohorts are estimated to be 5–13% In this study, we report the contributions of BRCA2 mutations (58%), similar to that reported in some Asian studies BRCA2 founder mutations in our cohort was higher when compared to that of the BRCA1 founder mutations. Of note, a similar pattern of a predominance of BRCA2 mutations compared to BRCA1 was recently observed in a study of high-risk Asian-Americans Unlike the Caucasian population, we found that a relative predominance of BRCA mutations which have not been published in the BIC Database of NIH. This proportion is relatively high in our Chinese cohort. Reports have found a high frequency of variants in different ethnic populations BRCA testing have not be widely performed, especially in Chinese population In this study, we identified 29 novel deleterious BRCA1 and three BRCA2 putative founder mutations in our Southern Chinese cohort. Interestingly, we also observed that half of the putative founder mutations (3 of 6) have not been published in the BIC Database , their founder effects are required to be confirmed by larger sample size of unrelated probands. The BRCA1 and BRCA2 recurring mutations that did not share a common haplotype could also be attributable to factors such as age of mutation such that associated alleles are no longer in linkage disequilibrium with microsatellite marker. Founder mutations not only provide population-specific genetic risk assessment, but are also useful in the study of penetrance of BRCA mutation in a specific population Notably, we discovered three Database . Since tBRCA1 c.981_982delAT and BRCA2 c.3109C>T are one of the most common mutations found in Asian countries such as China, Korea and Singapore BRCA1 c.5496_5506del11insA, BRCA1 c.390C>A, BRCA2 c.7480C>T and BRCA2 c.3109C>T, are unique mutations that were not found in either other Asian or even Caucasian populations according to our database search from BIC and HGVS. On the other hands, one recurrent mutation BRCA2 c.2808_2811delACAA was frequently observed in other ethnic populations such as Caucasian, African American, Hispanic and Australia.Based on our recurrent mutations identified in this study, some of them such as BRCA1 and BRCA2 founder mutations detectable by conventional DNA sequencing were also detectable by HRM, giving a high sensitivity for the latter method. The true sensitivity remains to be determined in a larger cohort including unselected Chinese women and men.The discovery of this founder mutation may provide a cost-effective option to rapidly screen a population. Our finding of complete concordance between conventional sequencing data and the HRM output in patients DNA suggests that the HRM technology is ready for use in diagnostic setting. Furthermore, there are several advantages of using HRM over other mutation screening methods: (i) Recent reports showed that the sensitivity and specificity of HRM is better than that of denaturing high-performance liquid chromatography (DHPLC) which is the current gold standard of scanning methods BRCA mutation analysis in a large Southern Chinese cohort and four founder mutations were identified. We then rapidly developed HRM mutation screening assays for those recurrent or founder mutations. The only BRCA1 confirmed founder mutations account for 6.9% of all identified BRCA1 mutations, whereas BRCA2 founder mutations account for 37.5% of all BRCA2 mutations. Our findings indicate that both BRCA1 and BRCA2 mutations account for a substantial proportion of hereditary breast/ovarian cancer in the Southern Chinese population. Identification of a founder mutation and knowledge of its prevalence in the Southern Chinese population provides important information both to genetic counseling and cancer risk assessment, as well as to the development of a cost-effective screening strategy.In conclusion, we conducted an extensive Table S1Distribution of the patients in this study according to the recruitment criteria.(DOC)Click here for additional data file.Table S2Sequences of high resolution melting (HRM) primers for BRCA1 and BRCA2 genes.(DOC)Click here for additional data file.Table S3Sequences of PCR primers for microsatellite polymorphic markers.(DOC)Click here for additional data file.Table S4The breakdown of mutations and variants of BRCA1 and BRCA2 in the 24 patients in the blind validation.(DOC)Click here for additional data file.Table S5Genotype of carriers with BRCA1 or BRCA2 founder mutations and family members without mutations.(DOC)Click here for additional data file.


Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. Relaxin family peptide receptor (RXFP) 1 and RXFP2 are class A G-protein coupled receptors (GPCR). They are therefore members of the largest gene family in humans  by an unA similar mechanism of action of the LDLa module by the two receptors is implied by the degree of sequence similarity they share. The LDLa modules of RXFP1 and RXFP2 share 60% sequence similarity Figure , while rThe present study aims to further investigate this seeming contradiction, and establish why an ectodomain-swapped chimera of RXFP1 with RXFP2 can signal, while an equivalent LDLa-swapped chimera cannot. Upon scrutiny of the constructs made by Kern et al., it would appear that the point chosen to swap the LDLa modules was 27 residues C-terminally to the final cysteine (Cys40) necessary for LDLa structural integrity for RXFP1. Importantly, the equivalent region of RXFP2 which was replaced in the chimera is seven amino acids shorter Figure , meaningl-glutamine and 1% penicillin/streptomycin (referred to as complete DMEM) in incubators (Thermo scientific) maintained at 37°C, with 5% CO2 and 85% humidity.Recombinant human gene-2 relaxin (H2 relaxin) peptide was kindly provided by Corthera and human INSL3 was chemically synthesized as previously described . Human e2, 0.5 μl of Taq (Bioline Velocity) and distilled, de-ionized water to make up to a total of 50 μl. PCR conditions were 2 min at 98°C, then 30 cycles of 98°C (30 s), 58°C (30 s), 72°C (15 s) followed by a 7 min extension at 72°C. The LRR/TM regions of each construct were similarly amplified  at 4°C overnight or at room temperature for 3 h. The vectors were then transformed into DH5α E. coli cells using the heat shock method and resultant colonies were picked and grown before isolating the plasmid DNA using a PureLink® Quick Plasmid Miniprep Kit. The entire insert of successful clones were sequenced to ensure the desired end product was correct, with no unintentional mutations incorporated.RXFP1, RXFP2, and chimeric constructs were cloned into a pcDNA3.1™/Zeo+ AmpR mammalian expression vector  which contained an N-terminal FLAG tag and a bovine prolactin signal sequence . The chi6 cells, and grown overnight. The following day the cells were washed and resuspended in 1% FBS in phosphate buffered saline (PBS) (Gibco) (FBS/PBS) medium. Alexa 647 labeled anti-FLAG antibody (Invitrogen) was added at a 1:1000 dilution in FBS/PBS and cells were incubated on ice for 30 min, then passed through a filter and sorted using FACS (Becton Dickinson FACS Aria III). Only the top 10% of cells with fluorescence levels significantly higher than the background fluorescence in non-transfected HEK293T control cells were collected. The sorted cells were grown in complete DMEM in the presence of 200 μg/ml Zeocin until confluent. HEK293T cells stably expressing WT RXFP1 were used as a positive control in the FACS sort.Transient transfections were performed using lipofectAMINE™ 2000 (Invitrogen) according to the manufacturer’s instructions. For competition binding assays chimeric receptors were selected for semi-stable expression and compared with cells stably expressing RXFP1 or RXFP2 . Semi-stThe presence of the chimeric receptors at the surface of cells was assessed in triplicate, exploiting the FLAG epitope on their N-termini using the method described previously . Briefly50 values were subjected to one-way ANOVA and uncorrected Fisher’s LSD comparison test.Competition binding assays were performed on whole cells as described previously, using Europium labeled INSL3 (Eu-INSL3)  and Euro50 and maximum response (Emax) values were subjected to one-way ANOVA and uncorrected Fisher’s LSD comparison test.Cells were assayed for cAMP signaling by co-transfection of receptors with a pCRE β-galactosidase  reporter construct as previously described . Cells wp < 0.01) higher expression than RXFP1 (141.2 ± 11.3% of RXFP1 expression)  from WT RXFP1 (pEC50 10.89 ± 0.03) . The replacement of the RXFP1 TM domain with that of RXFP2 in RXFP212 restored the H2 relaxin stimulated efficacy to 99.9 ± 5.3% Forskolin response   and 8.22 ± 0.17 for RXFP121 (p < 0.05)  did not differ significantly from RXFP2.RXFP122 has the TM and LRR domains from RXFP2 and the LDLa module from RXFP1. RXFP121 has the LDLa module and the TM region from RXFP1 and the LRRs from RXFP2 Figure ; Table 22+ binding (The activation mechanisms of RXFP1 and RXFP2 represent a unique paradigm in GPCR functioning, since these are the only two mammalian GPCRs that possess an LDLa module. When found in the LDLR and related receptors, these modules are typically involved in protein–protein interactions, and are thus involved in a variety of interactions both with peptides and other molecules . The evi binding , 20, the binding . Both Ty binding . However50 values and unfortunately there was not sufficient Eu-labeled ligand to perform saturation binding to obtain Kd values. However, we have previously demonstrated that RXFP1 and RXFP2 receptors lacking the LDLa module bind ligand normally (50 values are unlikely to be related to the LDLa module swaps.While Kern et al. only made the RXFP1 chimera with the LDLa module of RXFP2 attached, we sought to characterize both this and the equivalent RXFP2 construct with the LDLa from RXFP1. We hypothesized that such chimeras would be informative in relation to common and distinct mechanisms of activation by the LDLa module. Additionally, we explored the concept that the LDLa is exerting its effect by interacting with the TM domain of the receptor in a specific manner by creating LDLa chimeras with matched TM domains in RXFP121 and RXFP212. The chimeric receptors were all expressed at the cell surface indicating they were folded correctly and able to be trafficked to the cell surface. The RXFP211 chimera had significantly higher cell surface expression as previously demonstrated for the chimera produced by Kern et al. (normally . Hence t50 was similar to what we have previously demonstrated when the key RXFP1 LDLa residue Leu7 is mutated to Lysine as it is in the RXFP2 LDLa module (Emax values may reflect the mismatch of the LDLa with the TM domain resulting in the RXFP2 LDLa module acting like a partial agonist on the RXFP1 receptor. Notably, we have seen similar decreases in Emax with multiple mutations of the RXFP1 LDLa module (Emax values compared to RXFP1 (As anticipated the chimeric receptors did show differences in ligand-mediated cAMP activity compared to the WT receptors. This was most obvious for the RXFP1 chimeras where H2 relaxin demonstrated both decreased potency as well as decreased % maximum Forskolin activity on RXFP211. The shift in pECa module . The deca module , 9. Furtto RXFP1 . The simto RXFP1 , 24. HenIt should also be pointed out that it is highly unlikely that changes in potency or efficacy in the receptor chimeras is related to decreased efficiency of homodimerization of the receptors. Our previous studies on dimerization of RXFP1  and RXFPTaken together, this study has shown that the LDLa modules of RXFP1 and RXFP2, which are unique among GPCRs, behave in a comparable fashion to one another, and that their mechanism of action must therefore be closely related. Additionally it is clear that the length of the linker region between the LRRs and the LDLa module is important for RXFP1 function. This information can be used to further elucidate a model of activation, as we gradually clarify the myriad of different elements that come into play upon binding and activation of these complex receptors. Given that relaxin has been implicated in various pathologies including cancer  and thatThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purkinje neurons express, in high abundance, a glutamate gated chloride channel commonly known as the Excitatory Amino Acid Transporter subtype 4 (EAAT4). EAAT4 belongs to the family of glutamate transporters, which in mammalian nervous system is responsible for clearing synaptic glutamate . StudiesThe role of the chloride channel in glutamate transporters is only known for the EAAT subtype 5 (EAAT5). On rod bipolar cell axon terminals, EAAT5 activation by glutamate results in membrane hyperpolarization, which consequently inhibits terminal glutamate release . WhetherThe model comprises a single compartment with uniform distribution of AMPA receptors and EAAT4. The EAAT4 model is based on a 16-state kinetic model of EAAT2 . AMPA re


Bedside percutaneous dilatational tracheostomy (PDT) is a safe procedure with an acute complication rate of 10 to 15%. Our hypothesis was that having an experienced person performing or supervising the procedure results in extremely low complications with PDT. We formed a tracheostomy team which always included at least a consultant or specialist experienced (at least 25 procedures) in performing the procedure.A retrospective chart review of all patients who had PDT in a multidisciplinary adult medical surgical ICU during November 2008 to December 2010. The patients' demographics, indications for intubation and PDT, early and late complications, date weaned off the ventilator, date of decannulation, discharge from ICU and hospital, and outcome of these patients in the hospital were noted.n = 14 (24%) followed by severe head injury n = 13(23%) and cerebrovascular accident n = 8 (14%). The commonest indication for tracheostomy was airway protection n = 40 (73%) followed by prolonged mechanical ventilation n = 25 (45%). The median duration of intubation before PDT was 11 days (IQ 8 to 18). The median time elapsed between tracheostomy and weaning of ventilator was 1 day (IQ 1 to 3). However, the median time to decannulation was 37 day (IQ 10 to 136). Acute complication of paratracheal insertion occurred in n = 1 (1.8%) patient. No deaths were reported related to the procedure. However, n = 13 (22.8%) patients died during the hospital stay. No procedure was converted to surgical tracheostomy. The median duration between tracheostomy and discharge from ICU was 12 days (IQ 5 to 21). Chronic complication of subglottic stenosis occurred in n = 1 (1.8%) patient.Out of a total of 2,364 admission 57 patients underwent PDT, all with bronchoscopic guidance by an intensivist experienced in PDT (>25 procedures); there were 45 (78.9%) males and 12 (21%) females with the median age of 42 (range 18 to 90) years. The most common admission diagnosis was cardiac arrest PDT is an extremely safe procedure when performed by an experienced intensivist under bronchoscopic guidance. Our low complication rate is due to careful screening and selection of patients and being performed or supervised by an experienced intensivist under direct vision.


In Cambodia, the highly pathogenic avian influenza (HPAI) A subtype H5N1 virus was detected for the first time in January 2004. From 2004 to 2010, there have been 26 outbreaks in poultry and 10 human cases reported in the country but the origin of these epizootics remains unclear.The phylogenetic relationships among the H5N1 strains were reconstructed by neighbour-joining and Bayesians methods. We analyzed the sequences of all 8 genomic segments of 40 H5N1 Cambodian viruses together with sequences from over 100 isolates from Southeast Asia including Vietnam, Thailand and Laos.All viruses isolated in Cambodia since 2004 belong to clade 1, genotype Z. Based on phylogenetic relationships, HPAI H5N1 virus was probably introduced from Thailand in 2004. In 2005 and 2006, several sublineages emerged in Cambodia and were probably the result of multiple introductions of H5N1 virus from Vietnam where similar strains were detected before outbreaks occurred in Cambodia. Interestingly, in 2006, we observed a north to south spread of the virus following a main road. A new sublineage appeared in summer 2006. Since then, all viruses isolated in Cambodia and South Vietnam clustered into this group, suggesting that this sublineage became endemic in the Southern Indochina peninsula. Other clades which have been imported to neighboring countries by migratory birds  have not been detected in Cambodia.Cambodia is essentially a poultry-importing country. The first poultry deaths were observed in semi-industrial farms that imported broiler and layer parental stocks from a sister company in Thailand, where concomitant outbreaks occurred. Then, multiple introductions of H5N1 viruses most likely occurred through illegal trading in poultry from Vietnam. Our data suggest that the clade 1 H5N1 virus is spreading essentially through poultry trading, particularly along road transportation routes. The role of the migratory birds appeared to be at most limited to local or regional transmission. The mechanisms described here would explain the maintenance and extension of the H5N1 virus within the Cambodia-South Vietnam regions for the last 6 years. This also highlights the persistent risk of H5N1 virus transmission in humans in the region while the new H1N1 pandemic virus that affects millions of humans is also frequently detected in pigs and shows a dangerous propensity to recombine.


Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls . ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway. ALS is a late-onset, rapidly progressive and devastating neurodegenerative disorder, which is generally associated with selective degeneration of both upper and lower motor neurons (MNs) in the brain, brainstem and spinal cord. Ten per cent of all ALS cases are inherited and referred to as familial ALS (fALS); the remaining 90% are sporadic (sALS) et al. demonstrated that intermediate-length polyQ expansions are associated with neither Alzheimer’s nor idiopathic Parkinson’s diseases, but with ALS and the Parkinson-plus entity progressive supranuclear palsy et al. as well as Gispert et al. reported that among several other polyQ neurodegenerative disease proteins, only ATXN2 is associated with ALS risk In addition to rare mutations and common SNPs, a recent publication reported that ATXN2 dysfunction influences the TDP-43-dependent toxicity seen in ALS and that the intermediate-length expansions to 27–33 triplets in the ATXN2 polyglutamine (polyQ) region act as ALS risk factors in 4.7% of North American patients 8CAA(CAG)4CAA(CAG)8 sequence; expansion of this domain to a size ≥34 triplets with a pure CAG sequence primarily causes autosomal dominant SCA2 et al. to be interrupted by at least one CAA triplet et al. identified ATXN2 expansions in 40 ALS patients to be always interrupted by CAA triplets, and defined a haplotype of two ATXN2 SNPs (rs695871 and rs695872) in common between most cases with 3 CAA interruptions and another haplotype in common between most cases with 1–3 CAA interruptions ATXN2 usually contains a repeat structure with 22 or 23 triplets coding for glutamine and the (CAG)This study now aims to investigate the association of the ATXN2 chromosomal region with ALS risk in the Turkish population, considering not only the polyQ repeats, but also common SNPs and haplotype patterns.The Ethics Committee of Boğaziçi University approved the use of patient samples for this study. Written informed consent forms were obtained from all patients. Control samples were collected anonymously.A total of 236 Turkish ALS patients (158 sALS and 78 fALS) matching El Escorial Criteria 5′- GGG CCC CTC ACC ATG TCG -3′ and the FAM labeled reverse primer 5′−/56-FAM/CGG GCT TGC GGA CAT TGG -3′. PCR cycles included 5 min. at 95°C, 30 cycles  and 5 min. at 72°C. Repeat sizes were determined by GeneScan Analysis  and evaluated independently by two authors (SL and ÖÖ). The reproducibility of the GeneScan analyses was validated via repeating 25 samples among 236 ALS cases and 45 samples out of 420 controls, corresponding to ∼10% of both cohorts. A SCA2 positive individual with 41 ATXN2 repeat expansion size was used as an internal control in both PCR amplifications and GeneScan analyses. ALS patients with an ATXN2 expansion were further subjected to DNA sequencing, to assess the presence of CAA interruptions .The ATXN2 triplet repeat was amplified from DNA samples of patients and healthy controls, using polymerase chain reaction (PCR) with the forward primer Fisher’s exact test was applied to evaluate the genetic association of ATXN2 expansion sizes with ALS risk, under both the allelic and genotypic models.http://pngu.mgh.harvard.edu/purcell/plink/) http://www.broad.mit.edu/haploview/haploview) In our independent GWA study (unpublished data) performed earlier, we investigated 733,202 SNPs in 116 out of the above 158 Turkish sALS patients and 109 age- and sex- matched neurologically healthy individuals, using the Illumina HumanOmniExpress SNP array. To examine the association of ATXN2 locus variants with ALS risk in the Turkish cohort under study, we extracted 250 kb genotype data, comprising the ATXN2 locus and the surrounding 50 kb (25 kb from 5′ and 3′ ends) using PLINK software  had a significant p-value of 0.0057 and correlated with an increased ALS risk (OR: 2.23). This risk haplotype was observed in heterozygous state in both Turkish ALS patients with ATXN2 expansions, which were part of the GWA genotyping study. Using permutation analysis, which eliminates false positive data after multiple testing more effectively than Bonferroni corrections We investigated a 250 kb region on chromosome 12q in 116 out of the above 158 Turkish sALS patients, including two of the four ALS patients with ATXN2 expansions. Ten of 28 SNPs were excluded due to low HWE and MAF scores. In single marker analysis, none of the SNPs within the ATXN2 gene by itself showed any significant association with ALS risk, but a trend towards association was observed for the SNP rs2239194 within the SH2B3 gene (p = 0.063) . On the The first analysis of Turkish ALS patients regarding ATXN2 confirms its role as a risk factor. More importantly, this study identifies a common risk haplotype for ALS, containing the ATXN2 and its neighbouring SH2B3 gene.Saccharomyces cerevisiae via Drosophila melanogaster to Homo sapiensThe initial observation that ataxin-2 acts as a modifier protein of TDP-43 overexpression toxicity as a model of ALS risk was consistent from In our study, novel evidence indicates that ALS risk is impacted by a 15-SNP haplotype block in linkage disequilibrium across the genes ATXN2 and its downstream neighbour SH2B3. Haplotype association became more significant when SNPs from both the ATXN2 and SH2B3 genes were included; this suggests a role of the SH2B3 gene in ALS risk. The SH2B3 protein, also known as LNK, is a member of the SH2B (1–3) adaptor protein family. They all contain Src Homology 2 (SH2) domains, pleckstrin motifs and proline-rich regions. Thus, they can bind to phosphatidylinositol-lipid containing membranes and to the phosphorylated tyrosine residues, e.g. of receptor tyrosine kinases, modulating the signal transduction that controls proliferation and growth. They exert strong effects in hematopoiesis This first analysis of Turkish ALS patients on ATXN2, not only confirmed its role as a risk factor in rare cases with intermediate polyQ expansions, but also revealed novel evidence that SNPs across the ATXN2/SH2B3 genomic locus may modulate risk in a substantial fraction of ALS patients. These data need to be validated in large and independent populations. In the light of these findings, our results implicate a genetic  interaction between ATXN2 and SH2B3 genes, therefore we propose that it will be useful to investigate genetic variations in this genomic region of ALS patients.Table S1Data on Turkish ALS patients with previously identified mutations.(DOC)Click here for additional data file.Table S2Association Analysis of 18 SNPs across 250 kb at the ATXN2 Locus.(DOC)Click here for additional data file.