Oxidative stress (OxS) has recently been linked with osteoporosis; however, we do not know the influence of OxS as an independent risk factor for this disease.®. Osteoporosis was considered when subjects had a BMD of 2.5 standard deviations or more below the mean value for young adults.We conducted a case-control study in 94 subjects ≥60 years of age, 50 healthy and 44 with osteoporosis. We measured total antioxidant status, plasma lipid peroxides, antioxidant activity of superoxide dismutase and glutathione peroxidase (GPx), and calculated the SOD/GPx ratio. Bone mineral density was obtained at the peripheral DXA in calcaneus using a portable Norland Apollo Densitometerp < 0.01); in addition, the SOD/GPx ratio was significantly higher in the group of individuals with osteoporosis (p < 0.05). In logistic regression analysis, we found OxS to be an independent risk factor for osteoporosis .GPx antioxidant activity was significantly lower in the group of subjects with osteoporosis in comparison with the group of healthy subjects (Our findings suggest that OxS is an independent risk factor for osteoporosis linked to increase of SOD/GPx ratio. Oxidative stress (OxS) is a biochemical disequilibrium propitiated by excessive production of free radicals (FR) and reactive oxygen species (ROS), which provoke oxidative damage to biomolecules and which cannot be counteracted by antioxidative systems. This biochemical alteration has been linked with aging and more of 100 chronic-degenerative diseases, among which osteoporosis is found .Thus, epidemiologic studies on osteoporosis should consider OxS, in addition to risk factors linked with lifestyles, hormonal changes, and aging -5. It haSimilarly, experimental studies have shown a diminution in antioxidant activity in patients with osteoporosis ,10. In tIn an exploratory study, our investigation group found a negative correlation between BMD and total antioxidant status (TAS) linked with serum levels low in glutathione peroxidase (GPx) ,13; notwWe carried out a case-control study in a convenience sample of 94 subjects ≥ 60 years of age, 50 healthy, 20 men and 30 women (mean age 67.9 ± 6.5 years) and 44 with osteoporosis, 19 men and 25 women (mean age 69.7 ± 7.3 years). The subjects were community-dwelling Mestizo Mexican elderly living in Mexico City for 10 years or more. Informative brochures were distributed in the community specifying the objectives of the study and admission criteria.All the women had intact uterus and the mean age in which their menopause began is the same between the two study groups. None of the subjects studied had been taking antioxidant supplementation , hormone replacement therapy or antiosteoporotic medication for at least 6 months prior to the study, none had acute or chronic diseases, or was receiving prescription medications.Both groups were well-nourished, Mini Nutritional Assessment (MNA) score was > 23.5, and caloric intake was between 2,000 and 2500 kcal per day, and the alimentation had the nutrients requirements  between Recommended Dietary Allowance (RDA) measured by 24-h dietary recalls and serum albumin > 35 g/L ,15.The subjects agreed to participate in the study after giving their informed consent. The Ethics Committee of the Universidad Nacional Autónoma de México (UNAM) Zaragoza Campus approved the research protocol for this study.The following anthropometric measurements were obtained: weight, height, body mass index (BMI). It was considered as overweight when BMI ≥ 27 .® scale  was used, calibrated before each weight measurement. Height was obtained with an aluminum cursor stadiometer graduated in millimeters. The subject was barefoot, back, and head in contact with the stadiometer in Frankfurt horizontal plane. BMI was calculated by dividing weight (in kilograms) by height (in square meters).Weight was measured while the subject was wearing underwear and a clinical smock and in a fasted state (after evacuation). A Torino®. Osteoporosis was considered when subjects had a BMD of 2.5 standard deviations (SD) or more below the mean value for young adults  . The intra- and inter-assay variation coefficients were less 5% in all the determinations.The within-run precision for the markers were as follows: LPO by TBARS assay 4.6%; erythrocyte SOD 3.8%; GPx and TAS with Randox Laboratories 6% and 4.3%, respectively. We calculated SOD/GPx ratio.th percentile of young healthy subjects. A stress score [SS] ranging from 1 to 6 was used, representing the severity of biomarkers modifications; a score 1 was given to each value higher or lower than the cut-off. We categorize subjects as follows according to their scale in SS: with OxS if SS was > 3, and without OxS if SS was 0–3  ≥ 27) has been reported as a protective factor for osteoporosis , which wThere must be considered as limitations of the study that BMD was not measured with gold standard" , it was used instead peripheral-calcaneus DXA; nevertheless, several studies have shown that this method has a 90% sensitivity and 90% specificity for osteoporosis at the hip or spine ,43. BesiThe most relevant finding of the present study comprised having found evidence through multivariate analysis that OxS is an independent risk factor for osteoporosis, supporting the hypothesis that links OxS with the etiology and physiopathology of this disease. Therefore, the development of controlled clinical assays could be justified for evaluating the usefulness of diets and/or antioxidant supplements in complementary or preventive management of osteoporosis.The author(s) declare that they have no competing interests.MASR participated in the design of the study and biochemical analysis, MRR participated in the design of the study and biochemical analysis. ECM recruited participants, participated in the subject interviews, coded transcripts, and anthropometric measures. VMM conceived and designed the study, drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Continuous-time Markov models allow flexible, parametrically succinct descriptions of sequence divergence. Non-reversible forms of these models are more biologically realistic but are challenging to develop. The instantaneous rate matrices defined for these models are typically transformed into substitution probability matrices using a matrix exponentiation algorithm that employs eigendecomposition, but this algorithm has characteristic vulnerabilities that lead to significant errors when a rate matrix possesses certain 'pathological' properties. Here we tested whether pathological rate matrices exist in nature, and consider the suitability of different algorithms to their computation.We used concatenated protein coding gene alignments from microbial genomes, primate genomes and independent intron alignments from primate genomes. The Taylor series expansion and eigendecomposition matrix exponentiation algorithms were compared to the less widely employed, but more robust, Padé with scaling and squaring algorithm for nucleotide, dinucleotide, codon and trinucleotide rate matrices. Pathological dinucleotide and trinucleotide matrices were evident in the microbial data set, affecting the eigendecomposition and Taylor algorithms respectively. Even using a conservative estimate of matrix error , both Taylor and eigendecomposition algorithms exhibited substantial error rates: ~100% of all exonic trinucleotide matrices were pathological to the Taylor algorithm while ~10% of codon positions 1 and 2 dinucleotide matrices and intronic trinucleotide matrices, and ~30% of codon matrices were pathological to eigendecomposition. The majority of Taylor algorithm errors derived from occurrence of multiple unobserved states. A small number of negative probabilities were detected from the Padé algorithm on trinucleotide matrices that were attributable to machine precision. Although the Padé algorithm does not facilitate caching of intermediate results, it was up to 3× faster than eigendecomposition on the same matrices.Development of robust software for computing non-reversible dinucleotide, codon and higher evolutionary models requires implementation of the Padé with scaling and squaring algorithm. P(t), U is the matrix of eigenvectors and σmax(Q) and σmin(Q) are the maximal and minimal singular values [Q respectively.where r values  of Q resThe PyCogent ,20 builtHS contributed to implementation of the matrix error measurements and interpretation; VBY contributed to implementation of constrained optimisation method, study design and interpretation; SE contributed to study design; RDK contributed to study design, microbial genome data sampling and interpretation; GAH contributed to study design, implementation of constrained optimisation, data sampling and interpretation.A pathological rate matrix example. An analytical example of a matrix pathological to eigendecomposition.Click here for file


Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure.Fusobacteria (23 – 40% of reads), Firmicutes (14 – 28% of reads) and Bacteroidetes (31 – 34% of reads) being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering.Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5%) diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases. Inflammatory bowel diseases have been associated with changes in the “healthy” gut microbiome of humans Firmicutes, Bacteroidetes, and FusobacteriaCompanion animals suffer from many of the same maladies as humans; however, knowledge of the gut microbiome in dogs is much less complete than that in humans. Several large clone libraries have been generated from human fecal samples and colonic biopsies, showing dominance of Firmicutes and Bacteroidetes phyla Our objectives in this experiment were to characterize the phylogeny of the canine hindgut microbiome using barcoded 454-pyrosequencing, and assess the phylogenetic changes induced by dietary fiber.Estimated metabolizable energy intake among dogs was not different between the C and BP diets . We were able to obtain 17,828 high quality sequence reads from 5 samples of animals fed the C diet  and Fusobacteria (40 to 24%) was lower (P<0.05) when dogs were fed the BP diet, whereas Firmicutes were increased  by the BP diet  in this phylum, regardless of diet, but still increased  when dogs were switched to the BP diet. The increase in Clostridia was complemented by a decline in Erysipelotrichi, which were reduced by half . Actinobacteria and Fusobacteria also changed significantly in their total representation by dietary treatment  and subsequently to a phylum within Bacteria (≥89.2%). The difference between raw sequences and bacteria-assigned sequences was due mostly to short (<59 nt) reads, and a few low-confidence assignments. In the individually sequenced samples, the percentage of sequences assigned to  BP diet . With th samples  showed a samples  or a sta samples  may indireatment ; howeverComparison of the individual samples using UniFrac PCA  showed aFirmicutes  When evaluating pooled DNA samples based on a 2000-sequence subset, our data suggest an overall number of OTU 96%,  that is The pooled DNA samples appeared to have similar diversity compared to individual samples based on the 2000-sequence subset analysis. Based on full-information analysis, however, diversity estimates for the pooled samples were markedly higher. Nevertheless, the apparent similarity among individual samples and the pooled DNA samples by diet in UniFrac analysis (discussed below) may indicate that the full-information pooled samples accurately reflect the overall community structure as affected by diet. Therefore, pooling DNA samples may give representative results for total community structure in an experiment or experimental treatment, recognizing that pooled data clearly lacks information on individual diversity as was previously noted by Brulc et al. Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, and Tenericutes. However, Spirochaetes and Tenericutes were not detected in every sample. All of these phyla have been reported in humans Tenericutes. Tenericutes have been identified in dogs, but in the small intestine Fusobacteria. Whereas Firmicutes and Bacteroidetes typically account for 75% or more of the microbial composition in humans, chicks, and macaques, the dog hindgut appears to be co-dominated by Firmicutes, Bacteroidetes, and Fusobacteria. This observation is supported by previously published data Firmicutes, 29% Fusobacteria, and 30% Bacteroidetes in canine colon contents. Of the less dominant phyla that were present in the current study, the fraction of Proteobacteria appeared to be higher than in previously published work (∼7% here vs 1.4%) Whereas diet did not seem to have a major effect on the number of OTU observed in our experiment, the abundance of certain phyla was significantly affected. We detected 7 bacterial phyla, including Fusobacteria appeared to be dominant among them, most notably on the C diet. The addition of fiber to the diet did not greatly alter Bacteroidetes, but significantly shifted the Firmicutes:Fusobacteria ratio in favor of Firmicutes, possibly due to diet selection for complex fermentative activity. This contrasted with earlier findings where dogs fed a fiber-enriched diet (5% soyhulls and 5% beet pulp) did not have different DGGE banding patterns compared to dogs fed a low-fiber diet Fusobacteria was due mainly to lower overall sequence counts, as this phylum was almost exclusively represented by the genus Fusobacterium. The dynamics in the Firmicutes were more complex, with changes at the class level  was tripled  within the Firmicutes when dogs were fed the BP diet. A similar effect was noted on the phylogenetic tree  should be carefully interpreted. At lower taxon levels, classification becomes increasingly less reliable with relatively short sequences, and the resolution of our dataset decreased rapidly beyond the higher level taxa. This was the main reason why we were unable to classify most sequences beyond genus level. This loss of resolution at lower levels illustrates that although our dataset is large, it is not yet comprehensive. Choosing different primers spanning a longer or different section of the 16S gene might improve the depth with which the gut microbiome can be evaluated. Moreover, improved chemistry for 454-pyrosequencing now allows for longer sequence reads (∼400 nt) which should improve robustness of classification based on single reads.Of the three largest phyla ss level  but alsoThe clustering by diet according to UniFrac is striking, taking into account that all samples came from essentially the same environmental conditions, yet a clear separation by diet exists. Previously, in obese mice, UniFrac analysis was able to separate groups by diet fed  UniFrac can separate different gut environments efficiently by species such as macaque, human, and mouse Fusobacteria gave ambiguous results, which was likely due to the low diversity within this phylum (>99.9% Fusobacterium). For the same reason, combined with the low number of sequences classified in the phylum, Actinobacteria (who were significantly changed) were not evaluated in this manner. The key phylum that probably was responsible for the distinct clustering by diet was Firmicutes, which had a large presence and was highly diverse. This hypothesis was supported by the distinct UniFrac clustering by diet when just sequences from the Firmicutes were analyzed  purpose-bred dogs  were used in a crossover experimental design. None of the dogs used were obese. Animals were housed under environmentally controlled conditions  at the Small Animal Clinic of the University of Illinois College of Veterinary Medicine.The experimental diets  were forThe experiment used a crossover design with two 14-d periods. Dogs were randomly assigned to one of the two experimental diets prior to the first period, and subsequently received the second diet in period two, so that each animal served as its own control. Dogs were fed once daily and 300 g of the assigned diet was offered. This amount was sufficient to meet the metabolizable energy needs of the heaviest dog based on NRC recommendations Immediately after feces were voided, they were collected, weighed, aliquoted into cryogenic vials  and then flash-frozen in liquid nitrogen. After the samples were thoroughly frozen, they were stored at −80°C until DNA extraction.DNA was extracted using a modification of the method of Yu and Morrison Amplification of the variable region 3 of the bacterial 16S rDNA gene was utilized to assess gut microbial diversity. Primers used to amplify the V3 region (341F and 534R) 2+), 4 µL dNTP mix , 10 pmol each of the forward and reverse primer, 1 µL (∼50 ng) of extracted DNA, and was brought to 50 µL using sterile water. To minimize PCR bias (where highly represented sequences are amplified faster than those that are rare), we used 20 PCR cycles and obtained sufficient quantities of product. The PCR conditions were as follows: initial denaturing, 94°C for 5 min followed by 20 cycles of 30 s at 94°C (denaturing), 30 s at 69°C , 30 s at 72°C (extension), and after cycling was complete, 7 min at 72°C to extend unfinished product. After PCR, the resulting product was checked for size and purity on an agarose-EtBr gel and then prepared for 454-pyrosequencing according to manufacturer instructions with the deviation that the PCR product was cleaned using a Qiagen silica column  instead of sizing beads. In addition to individual DNA samples, two pooled (by diet) DNA samples were prepared, such that each individual sample contributed an equal amount of DNA. These pooled samples were then amplified and sequenced to assess the effect of sample pooling on sequencing results. Sequencing of the PCR products was performed at the W. M. Keck Center for Biotechnology at the University of Illinois using a 454 Genome Sequencer FLX . After sequencing was completed, all reads were scored for quality and any poor quality reads and primer dimers were removed. All sequences that passed quality control are provided by sample in Initial PCR was performed in triplicate for each sample in a total reaction volume of 50 µL. Each 50 µL reaction mixture contained 1.25 U Takara Ex Taq DNA polymerase, 5 µL 10X Ex Taq buffer  Figure S1Phylogenetic tree constructed from sequences from one dog (G). The tree displays phylogenetic relationships between the microbiome structures when the dog was fed two different diets. Sequences marked red are from the control diet, sequences in black are from the beet pulp-supplemented diet.(0.08 MB PDF)Click here for additional data file.Sequence Data S1Sequences derived from dogs fed the Control diet, individual animals (5) and a pooled DNA sample.(0.73 MB ZIP)Click here for additional data file.Sequence Data S2Sequences derived from dogs fed the Beet pulp diet, individual animals (5) and a pooled DNA sample.(0.61 MB ZIP)Click here for additional data file.Sequence Data S3Representative sequences selected from dog G data used to construct the tree in (0.01 MB ZIP)Click here for additional data file.


Pheochromocytoma is a chromaffin cell catecholamine-secreting tumour originating from the adrenal medulla.We report here an unusual presentation of pheochromocytoma with secondary amenorrhoea and its resolution after medical treatment with the alpha adrenergic blocker, Phenoxybenzamine.Pheochromocytoma is a rare cause of secondary amenorrhoea. Pheochromocytomas are chromaffin cell catecholamine-secreting tumours originating from the adrenal medulla. They may present in a number of ways from an incidental finding to that of the classical triad of episodic headache, tachycardia and sweating due to hypersecretion of catecholamines resulting in episodic hypertension. However2.A 35 year white Caucasian nulliparous female presented to the fertility clinic with 18 months history of secondary amenorrhoea that was ascribed to post pill amenorrhoea. She had a regular menstrual cycle since the age of 15. At the age of 18 she started taking the contraceptive pill and she had a regular withdrawal bleeding every 28 days, but amenorrhoea ensued following its cessation at the age of 33. There was also no history of eating disorder or recent weight loss, no previous medical history and there was no family history of relevance including autoimmune disease. She was a non smoker, who drank less than 10 units of alcohol a week. Physical examination revealed a lady with normal blood pressure of 128/78 mmHg, weight of 60 kg, height of 173 cm giving her a Body Mass Index of 20 kg/mHer investigations in the fertility clinic revealed iron deficiency anaemia, Hb 9.7 g/dl, microcytosis and a low ferritin of 4 ug/L (15–30 Units). She had luteal phase FSH, LH, oestradiol and progesterone of 7.9 iu/L (0–5), 3.0 i.u/L (2–9), <70 pmol/L (150–1000) and 1 nmol/L, respectively. Her prolactin was 220 mU/L (0–550) and testosterone was 1.3 nmol/L (0–4.1) while the free androgen index was 3 (0–8) and SHBG was 42 nmol/L. Her liver enzymes, electrolytes, urea and creatinine were all within the normal range. Her ultrasound of the pelvis revealed normal ovaries and normal endometrial thickness and hysterosalpingography showed patent fallopian tubes. Her iron deficiency anaemia was corrected by ferrous sulphate treatment. Induction of ovulation with clomiphene 150 mg for 5 days and two trials with gonadotrophins neither achieved pregnancy nor ovulation, with a complete failure of oocytes response. Meanwhile, she underwent investigations for the initial iron deficiency anaemia with gastroscopy and colonoscopy CT. The latter revealed an incidental left adrenal mass of 3 cm in diameter that did not show features typical of an adenoma. Two urinary 24 hour urinary fractionated catecholamines showed an excess secretion of norepinephrine at 1567 and 1159 nmol/L (NR 70–550) with normal epinephrine and dopamine secretion. She was treated initially with an escalating dose of phenoxybenzamine starting at 10 mg twice daily before adding propranolol slow release 80 mg daily prior to undergoing surgery. Two weeks after starting the phenoxybenzamine, she reported resumption of regular menstrual periods after 24 months of secondary amenorrhoea. Definitive treatment in the form of laparoscopic left adrenalectomy confirmed the presence of an encapsulated pheochromocytoma that was completely excised.++ and phosphoinositide pathways[This unusual presentation of pheochromocytoma has not been reported before. One possibility is that ovarian granulsa luteal cells in the human and animal have alpha and beta adrenergic receptors. It was shown recently that activation of alpha adrenergic receptors causes inhibition of LH and HCG mediated progesterone release through  pathways although pathways in the oA less likely possibility is the interaction of norepinephrine at the level of the hypothalamus. There is animal evidence that GnRH release and LH pulsatilty are modulated by extrahypothalamic norepinephrine . The medThis case report illustrates a patient presented with amenorrhoea that was secondary to a pheochromocytoma, a rare differential diagnosis for patients presenting with secondary amenorrhoea.++: ionized calcium.Hb: haemoglobin; FSH: follicular stimulating hormone; SHBG: sex hormone binding globulin; LH: leutinizing hormone; NR: normal range; HCG: human chorionic gonadotropin; GnRH: gonadotropin releasing hormone; CThe authors declare that they have no competing interests.SLA conceived the study, coordination and helped to draft the manuscript. AW reviewed the clinical records, wrote and submitted the case for publication. All authors read and approved the final manuscript.Written informed consent was obtained from the patient. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Infected persons had slept in an infested cabin. Rickettsia prowazekii. Analysis of blood and tissue from 14 southern flying squirrels trapped in the woodlands around the cabin indicated that 71% were infected with R. prowazekii. Education and control measures to exclude flying squirrels from housing are essential to reduce the likelihood of sylvatic epidemic typhus.In February 2006, a diagnosis of sylvatic epidemic typhus in a counselor at a wilderness camp in Pennsylvania prompted a retrospective investigation. From January 2004 through January 2006, 3 more cases were identified. All had been counselors at the camp and had experienced febrile illness with myalgia, chills, and sweats; 2 had been hospitalized. All patients had slept in the same cabin and reported having seen and heard flying squirrels inside the wall adjacent to their bed. Serum from each patient had evidence of infection with  Rickettsia prowazekii. It is associated with a cycle of infection involving flying squirrels and their ectoparasites and secondary transmission to humans. Illness in humans is characterized by fever, myalgia, severe headache, and rash. Historically, classic louse-borne epidemic typhus, caused by the same organism, has caused large epidemics where conditions were favorable for person-to-person spread of body lice.Sylvatic epidemic typhus, hereafter referred to as sylvatic typhus, is a rare but potentially lethal zoonotic exanthematous disease caused by Glaucomys volans, was found to be naturally infected with R. prowazekii  identified a case of sylvatic typhus in a 31-year-old counselor at a therapeutic wilderness camp for troubled youth in Pennsylvania (case-patient 1). A review of the Pennsylvania electronic disease surveillance system found that in December 2004 another counselor from the same camp (case-patient 2) had become ill with fever, chills, and sweats and that serologic testing at a local laboratory had been suggestive of sylvatic typhus. During a 25-year period (1976−2001), only 2 other cases of sylvatic typhus associated with flying squirrels had been reported in Pennsylvania (>38°C) plus >1 of the following: headache, myalgia, confusion, rash, or photophobia, with illness onset prior to March 1, 2006, in a staff member or student at the camp. A confirmed case of sylvatic typhus was defined as a case that met the definition of a suspected case plus had immunoglobulin (Ig) M and/or IgG reactive with whole-cell R. prowazekii antigen at a dilution >1:64, according to immunofluorescence antibody (IFA) assay performed at CDC  were assessed to identify illness suggestive of sylvatic typhus occurring during their residence. Records of students discharged from the program before February 2006 were not available for review. Data analyses were performed by using Epi Info 2000 (We defined a suspected case of sylvatic typhus as fever (During the winter (October–March), staff and students had lived in 1 of 6 cabins (cabins A–F); during the rest of the year, they had slept in tents . All 6 cR. prowazekii by IFA. Serologic testing was performed at CDC with an anti−flying squirrel fluorescein-isothiocyanate conjugate prepared in-house. Samples were first screened at 1:32 dilution; any reactive samples were titered to end point. Necropsies were performed and tissues were collected, frozen on dry ice, and kept at –70°C before further testing. DNA was prepared from whole blood and heart tissues by using QIAamp DNA Mini Kit  according to manufacturer’s instructions. PCRs to amplify a fragment of 17-kDa protein antigen were carried out in a total volume of 25 μL by using Taq Master Mix (QIAGEN) in a Gradient Mastercycler . Details of the PCR setup and primer sequences are described elsewhere (Blood was obtained from trapped flying squirrels for detection of IgG (heavy plus light chain) polyvalent antibodies against R. prowazekii infection were classified as non–case-patients. Two persons who had suspected cases but did not undergo serologic testing could not be further classified and were grouped with other survey-only respondents for the purpose of analysis.Of 66 camp staff members on site during the March 2006 investigation, 45 (68%) completed a questionnaire; 33 of the 45 (73%) provided a serum sample, including 17 staff members who met the case definition for suspected sylvatic typhus. Five additional staff members reported having had febrile illness since employment at the camp but declined testing; 2 of them met the definition for suspected sylvatic typhus. Serologic testing confirmed 4 (12%) cases of sylvatic typhus among the 33 tested staff members, including case-patients 1 and 2, who were identified at the start of the investigation . The remOn January 7, 2006, a 31-year-old man from western Pennsylvania became ill with high fever (39.6°C), chills, sweats, headache, and joint and muscle aches. The next day, he was admitted to the hospital. During his hospitalization, he continued to have fever and muscle aches, and laboratory test results were positive for mononucleosis and influenza A. He was discharged on January 17 but continued to have headache and fever. After hearing of a case of sylvatic typhus at the camp where he worked the previous year, he requested testing from his hospital clinician. On January 24, he was reevaluated and serum was obtained for sylvatic typhus testing. He was given oral doxycycline, 100 mg twice a day for 10 days; his signs and symptoms resolved. The man had lived in cabin A and stated that although he had never handled a flying squirrel, he had heard them frequently in the wall next to his bunk (bunk B) .On December 27, 2004, a 39-year-old man from southwestern Pennsylvania had fever (40.3°C), chills, sweats, headache, and muscle and joint pain. He later became confused and was admitted to the hospital on January 4, 2005. At the time of admission, he was febrile and dehydrated. He was treated with azithromycin and ciprofloxacin but did not improve. When he told clinicians that he worked at a wilderness camp and had been bitten by fleas, he was administered a regimen of intravenous doxycycline for 3 days. His fever subsequently resolved; he was discharged from the hospital in early January and given oral doxycycline, 100 mg twice a day for 7 days. He visited his primary care provider in February 2005. IgG and IgM tests performed at a commercial laboratory on serum collected on February 1, 2005, showed qualitatively positive results for typhus group–specific antibodies. The man had lived in cabin A at the time of his illness. He reported having seen a flying squirrel 1 time in the main living area of the cabin and seeing squirrels in the walls next to his bunk. He also reported feeding the squirrels through wide gaps in the wall boards next to his bunk (bunk B) and being bitten by insects he recognized as fleas in his bunk at least 1 time the previous year.In January 2004, a 26-year-old man from Pennsylvania had high fever (39.4°C), chills, sweats, muscle aches, nausea, and vomiting. The man did not seek medical attention and recovered within a week. He worked as a counselor at the camp in Pennsylvania and, at the time of his illness, was living in cabin A. He reported that he had seen animals inside gaps in the wall next to his bunk (bunk B) and had seen a flying squirrel in the main living area of the cabin on 1 occasion.In late December 2005, a 26-year-old man from Pennsylvania had high fever (39.8°C), severe headache, muscle and joint pain, chills, sweats, and photophobia. He did not seek medical attention and recovered after 2 weeks. He worked as a counselor at the camp and, at the time of his illness, lived in cabin A. He reported having seen and heard animals inside the wall next to his bunk (bunk B) many times and attempting to seal gaps in the wall with foam caulking spray. Although he had never touched a flying squirrel, he had seen one traversing a roof truss inside cabin A, had handled nesting material, and had been bitten by insects he recognized as fleas while living in the cabin before his illness.The median age of all 45 questionnaire respondents was 34 years (range 21–69 years); 97% were white, and 84% were male. The median length of employment at the camp was 2 years (range 1 month–31 years). Selected characteristics reported are summarized in The 4 confirmed case-patients had lived in cabin A for a significantly longer cumulative period (median 17 months) than non–case-patients . Bunk B had been slept in by all 4 case-patients but by only 9 of 28 non–case-patients . From January 2004 through January 2006, 8 (18%) staff members lived primarily in cabin A, and other staff members occasionally slept in cabin A.R. prowazekii infection had negative results.Although the students living in cabin A were not tested for typhus, a review of their medical records indicated that none had sought medical care for a febrile illness at any time during their enrollment. Of 43 students whose records were reviewed, 7 (16%) had experienced febrile illness compatible with sylvatic typhus since their arrival at the camp. These students resided in cabins D, E, and F. All 6 (86%) who were tested for evidence of At the time of the March 2006 investigation, counselors and students were living in 5 of 6 cabins located on a 1,500 acre tract of land in southwestern Pennsylvania . The cabWhen examined, the cabins at sites A, C, D, and F had evidence of deer mice and flying squirrel infestation, including nesting material protruding from the walls and ceiling and damaged or chewed insulation in walls adjacent to counselor bunk A (cabin C), counselor bunk B (cabin A), and student bunks (cabin D). A large opening providing access to the exterior of the cabin was present on the wall above the counselor bunk B in cabin A. In April 2006, camp staff removed the interior wooden planks from the wall adjacent to bunk B in cabin A and confirmed the presence of flying squirrel nesting materials and extensive damage to insulation inside the wall.R. prowazekii, 6 with end-point titers from 32 to 1,024 (R. prowazekii DNA was detected in specimens (2 hearts and 3 whole blood samples) from 5 (36%) squirrels. Of the 14 squirrels, 3 showed both serologic and molecular evidence of infection and 12 hosted fleas or lice (data not shown).Trapping efforts obtained 14 southern flying squirrels from several outdoor sites throughout the camp, including the site where cabin A was located and a field site adjacent to a state park ; Table 3to 1,024 . These 8R. prowazekii infection indicates that the pathogen is well established among these squirrels.Our investigation found that transmission of sylvatic typhus to humans occurred during 3 consecutive winters at the camp. All 4 cases among counselors were epidemiologically linked to cabin A. All had slept in a specific bunk, bunk B, next to a wall that had evidence of flying squirrel infestation. Other counselors sleeping in the same cabin but without direct exposure to bunk B had no evidence of infection. The finding that 10 (71%) of 14 flying squirrels collected at multiple sites showed evidence of R. prowazekii is transmitted from flying squirrels to humans is not well understood. The lack of detectable antibodies to R. prowazekii in household members of documented sylvatic typhus case-patients  for PCR analysis may provide options for earlier diagnosis, but these assays are not widely available. Serologic confirmation of infection is based on 4-fold IgG titer increases between acute and convalescent samples; both IgM and IgG can persist for years after infection with Because southern flying squirrels are distributed throughout the eastern United States, other woodland settings frequented by humans during winter may present a similar risk for sylvatic typhus. Parks and campgrounds that maintain rental cabins should be aware of risks associated with flying squirrels and take steps to exclude these animals from structures occupied by humans by sealing openings at attic vents and around roof joists with heavy-gauge screen or similar products. In addition, use of repellents may keep arthropods from biting humans, and premise sprays may be useful for reducing arthropods in building structures. Squirrel removal without concomitant arthropod control is not recommended because the presence of potentially infected external parasites may increase the risk for disease transmission to humans.


MCPH1 and ASPM contribute to variations in human brain size and its correlates. We measured general mental ability, head circumference and social intelligence in 644 Canadian adults . The gene polymorphisms were assessed from buccal DNA; mental ability by Wonderlic Personnel Test and Multidimensional Aptitude Battery; head circumference by stretchless tape; and social intelligence by prosocial attitude questionnaires. Although all measures were construct valid and the allele frequencies showed expected population differences, no relationship was found between the genes and any of the criteria. Among Caucasian 18–25 year olds, for example, the two mental ability tests correlated with each other , with head circumference  and with prosocial attitudes .We test the hypothesis that polymorphisms of the brain regulator genes  Microcephalin (MCPH1) on chromosome 8p23 and abnormal spindle-like microcephaly associated (ASPM) on chromosome 1q31, attracted much attention when reported to be (i) associated with autosomal recessive primary microcephaly, (ii) positively accelerated in molecular evolutionary rate through the simian line leading to Homo sapiens, and (iii) under recent positive selection in modern humans  allele, was estimated to have arisen approximately 37 000 years ago (95% CI: 60 000–14 000 years ago), about the time symbolic behaviour became widespread in Europe, while the favoured D allele for ASPM was estimated to have arisen approximately 5800 years ago (95% CI: 14 000–500 years ago), about the time cities developed in the Near East. The D alleles for both MCPH1 and ASPM now exist in high frequency. Both genes were hypothesized to confer a selective advantage such as increased brain size and general mental ability (GMA).Two newly discovered genes, r=0.20 with external head circumference and r=0.40 with MRI measured brain volume . They were paid Canadian $40 (approx. US $32) for a 2-h testing session. Between February and April 2005, 405 people were recruited from a university subject pool and by local advertisements; another 239 were later recruited from an employment agency.K Scale  were coded: AA for the homozygous non-D allele, AD for the heterozygous alleles and DD for the homozygous D allele.One of the authors (Trudy Ann Bons) administered the tests in small groups. Participants completed questions on handedness, demographic background and social intelligence, then the Wonderlic (12 min) and the 10 MAB subtests (7 min each). Finally, they provided two DNA buccal swabs, one from inside each cheek. The DNA was genotyped according to standard procedures as published previously. Three genotypes of the MCPH1 had an overall frequency of 84% and was in Hardy–Weinberg equilibrium among all population groups and among Caucasians was more often in homozygote than heterozygote form. Similarly, the D allele of ASPM had an overall frequency of 32% and was in Hardy–Weinberg equilibrium among all population groups and among Caucasians was more often in heterozygote and less often in homozygote form. These are similar to the initial reports by Null results were found across all ways of examining the 644 participants, despite evidence showing the measures were construct valid. The allele frequencies, means and s.d.s of the measures are shown in r=0.78, N=476, p<0.001) and, as an aggregate g factor, with head circumference , Altruism  and Mini-K . The two prosocial attitudes scales correlated with each other , but not with head circumference.  Regardless of sample, a principal components analysis of the Wonderlic and the 10 MAB subtests showed loadings of 0.69 or higher on the first factor. Each individual's g factor score was also calculated based on the first component as this factor is the one most likely to show a genetic effect . Neither eliminating the heterozygotic AD category nor combining it with the DD category showed the AA versus DD comparisons to be significant (FS<1.00). Nor did the effect emerge when examining the relation in a variety of other ways including combining the two genes, or all the samples.To eliminate the confounding effect of population stratification, our main analysis was limited to Caucasians, which are by far the largest of our constituent samples. ASPM and MCPH1 and individual differences in head circumference, GMA or social intelligence. The absence of evidence was noted even in the analysis of the full sample (N=644) despite stratification effects due to ethnicity that would have biased the results in favour of finding a genetic effect . Null findings can arise due to procedural problems, but in this study, the variables showed construct validity and expected measurement properties. Moreover, the null results join one other based on 120 MRI-measured brain volumes  provides only 95% power to exclude an association that accounts for 2% of the variance. Given that genetic effect sizes turn out to be extremely small, typically 0.1%, and contribute interchangeably and additively (


Bunyaviridae) is due to its ability to adapt to changing conditions through intramolecular genetic changes and segment reassortment. Vertical transmission of LACV in mosquitoes increases the potential for segment reassortment. Studies were conducted to determine if segment reassortment was occurring in naturally infected Aedes triseriatus from Wisconsin and Minnesota in 2000, 2004, 2006 and 2007. Mosquito eggs were collected from various sites in Wisconsin and Minnesota. They were reared in the laboratory and adults were tested for LACV antigen by immunofluorescence assay. RNA was isolated from the abdomen of infected mosquitoes and portions of the small (S), medium (M) and large (L) viral genome segments were amplified by RT-PCR and sequenced. Overall, the viral sequences from 40 infected mosquitoes and 5 virus isolates were analyzed. Phylogenetic and linkage disequilibrium analyses revealed that approximately 25% of infected mosquitoes and viruses contained reassorted genome segments, suggesting that LACV segment reassortment is frequent in nature.The evolutionary success of La Crosse virus (LACV, family  Bunyaviridae, genus Orthobunyavirus) emerged as a significant human pathogen in the upper Midwestern United States, and it is now the most common cause of pediatric arboviral encephalitis in the U.S [Aedes triseriatus and small mammals . Aedes triseriatus develop a life-long infection, and infected females can transovarially transmit (TOT) the virus to their progeny [In the 1970s, La Crosse virus , medium (M), and small (S). The L segment encodes the RNA-dependent RNA polymerase , the M sBunyaviridae is attributed in part to their ability to adapt to varying conditions through genetic drift (intramolecular genetic changes) and genetic shift (segment reassortment).LACV exhibits considerable evolutionary potential in nature. There are distinct geographic genotypes of the virus in different areas of the United States -14, and Genetic drift occurs during genome replication and can result in viral diversity and altered fitness . RNA virAe. triseriatus and the newly generated viruses can be transmitted. The potential for segment reassortment increases when a transovarially-infected mosquito takes a blood meal from a viremic host [Laboratory studies have demonstrated the occurrence of genetic shift (segment reassortment) in mosquitoes that have become dually infected by ingesting viruses of two different LACV genotypes, either simultaneously or within two days of each other . LACV remic host . These mmic host . Segmentmic host -22.Evidence has also been presented for reassortment between LACV genotypes in nature. For example, the genomes of 23 isolates of LACV were analyzed by oligonucleotide fingerprinting and categorized in terms of the degree of their RNA sequence relatedness . One genOrthobunyaviruses and in other Bunyaviridae genera. Ngari virus is a newly emerged reassortant virus associated with severe disease epidemics in Africa [Orthobunyavirus that was first detected in Malaysia [Orthobunyaviruses also reassort [Hantavirus) have been detected in rodents in nature [Nairovirus) have also been detected [Genome segment reassortment has also been demonstrated among other n Africa . SequencMalaysia . Group Creassort . Phylogen nature  and reasdetected .Bunyaviridae family, the epidemiologic consequences of these evolutionary events are poorly understood. In this study molecular epidemiological techniques were used to investigate the evolutionary and reassortment potential of LACV in field-infected mosquitoes from the upper Midwest of the United States.Although genome reassortment appears to occur frequently in the A total of 6,791 mosquitoes collected as eggs at 151 study sites in Wisconsin, Minnesota, and Iowa  was actually 3.24 times larger than the diversity among synonymous substitutions (πs). The location and amino acid replacements are listed in Table The numbers of sequences analyzed and the number of segregating sites in each segment are shown in Table The program Tipdate  estimateThe haplotype grouping system was determined through a conservative phylogenetic analysis. The system identified three S haplotypes based on seven polymorphic sites, five of which were nonsynonymous mutations. The three haplotypes identified in the M segment were based on twelve polymorphic sites, seven of which were nonsynonymous. For the L segment, two haplotypes were identified based on thirteen polymorphic sites, twelve of which were nonsynonymous substitutions Figure .Ae. triseriatus  the genome sequence obtained by direct amplification of the viral RNA from the mosquito is the dominant viral sequence in the mosquito as well as in infectious virus and 2) that the estimation of reassortment frequency was not confounded by potential RNA quasispecies in the mosquitoes. Estimating the frequency of reassortment of LACV in nature would be improved by analysis of plaque-purified viruses isolated from the mosquitoes, preferably from their saliva or ovaries, which are the epidemiologically significant organs of transmission.In this regard, we were unsuccessful in isolating LACV from most field mosquitoes. The reasons for this are unknown; however, there are several potential explanations for this. Eggs were collected in the field and stored in a hot warehouse for variable periods of time awaiting shipment to Colorado. As soon as the eggs reached AIDL, they were placed in the insectary, hatched and reared. Environmental factors in the collection and shipping process could contribute to loss of virus titer. An additional complication could have been the isolation method. In previous studies, virus was isolated by inoculation of samples into suckling mouse brains. Cell culture assays are likely not as sensitive. Low virus titer, titer loss during processing, and insensitive isolation methods, likely contributed to the inability to isolate virus from mosquitoes.Ae. triseriatus in the field. LACV reassortants could be more virulent and could have altered vector species and vertebrate host ranges. New viruses could create new arbovirus cycles with potentially significant epidemiological consequences [Ae. triseriatus and chipmunks and tree squirrels. If a new virus established a transmission cycle that involved a mosquito species that fed more aggressively on humans, increased human infections could occur. If a new reassortant virus was more virulent or exhibited different tissue tropisms, infections could become clinically significant in both adults and children. For example, a new reassortant virus could replicate more efficiently in humans, resulting in greater viremia titers and more efficient infection of the central nervous system. Determination of the evolutionary potential of LACV through genetic shift may permit prediction of the epidemiologic consequences of these events.There are important public health implications of reassortment in LACV-infected equences . For exaThese studies illustrate the significant evolutionary and epidemic potential of viruses in the family Bunyaviridae. Viruses in this family have contributed inordinately to the list of newly emerged viruses , and theAedes triseriatus eggs were collected from five oviposition traps in each of 151 sites in Minnesota (n = 37), Wisconsin (n = 108) and Iowa (n = 6). Sites were established in areas where LACV encephalitis cases occurred or areas that contained clusters of people judged by the La Crosse County Public Health Department to be at risk for infection . Mosquito eggs that had entered diapause in fall 2000 were collected in the spring of 2001. Mosquito eggs were also collected between mid-June and August of 2004, 2006 and 2007. Eggs were collected in Crawford, La Crosse, Monroe, Vernon, Lafayette and Iowa counties in Wisconsin; Winona, Houston, and Grant counties in Minnesota; and Clayton and Allamakee counties in Iowa . A 1978 LACV (78V-8853) was isolated from an The LACV-positive mosquitoes were triturated with a pellet pestle (Fisher Scientific) in a 1.5 ml microcentrifuge tube containing 1 ml of minimum essential medium (MEM) (Gibco), 2% fetal bovine serum, 200 μg/ml penicillin/streptomycin, 200 μg/ml fungicide, 7.1 mM sodium bicarbonate, and 1× nonessential amino acids. The homogenate was centrifuged for 10 minutes at 500 × g to form a pellet.2. Supernatant from the centrifuged mosquito homogenate (0.2 ml) was added to one well in a six-well plate, incubated at 37°C for one hour. Following the incubation, 5 ml of medium were added to each well.Cell monolayers of Vero cells were grown in six-well plates at 37°C in an atmosphere of 5% CO-1 to 10-6 and 200 μl of each virus dilution was added to individual wells and incubated at 37°C for 1 hour. The virus inoculum was removed and 5 ml of overlay was added to the well. After six days of incubation at 37°C in 5% CO2, 200 μl of the detection solution, methylthiazolyldiphenyl-tetrazolium bromide (MTT) (5 mg/ml in PBS), was added to each well. The plates were incubated overnight and visible plaques were picked and placed in 1 ml of MEM with 0.2% FBS for 1 hr at 37°C. An aliquot of the medium from the wells was added to Vero cells, and the presence of virus confirmed by detection of cytopathic effect.The virus isolates from 2006 and 2007 were plaque purified using monolayers of Vero cells in six-well plates . Virus iThe posterior half of each mosquito abdomen was individually homogenized in 500 μl of Trizol , using a pellet pestle , and then total RNA was extracted according to manufacturer's instructions.The medium and cells from wells with plaque purified virus were removed and placed in a 15 ml conical tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was removed and the cell pellet was resuspended in 500 μl of Trizol . Total RNA was extracted according to manufacturer's instructions.RNA from the 1960 and 1978 LACV isolates was prepared by infection of C6/36 cell cultures at a multiplicity of infection of 0.01. Three days post-infection, cells were scraped into the medium, centrifuged and cell pellets were resuspended in 500 μl of Trizol for RNA extraction.Taq DNA polymerase  according to manufacturer's instructions. The primers specific for the S segment  amplified a 462 nucleotide region (nucleotides 144 to 604) of the nucleocapsid and NSs genes. This region was selected because it was the most variable region of the published S sequences. The S segment is 984 nucleotides in length, so the amplified region encompasses almost half the entire segment. The primers specific for the M segment  amplified a highly variable 411 nucleotide region in the 5' half of the G1 gene (nucleotides 1585 to 1995) [Ae. triseriatus ribosomal protein RpL34 mRNA were used as a positive control. PCR was performed as follows: 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute followed by a final extension at 72°C for 8 minutes.Portions of the LACV S, M, and L RNA segments were transcribed to cDNA using Superscript II reverse transcriptase  and amplified by PCR using Ex to 1995) . The priE. coli cells . Cells were grown on LB agar containing ampicillin (50 μg/ml) and kanamycin (50 μg/ml). Colonies were screened for inserts by PCR amplification using the original primers and positive products were purified using QIAquick spin columns . Three to five cDNA clones per segment from each mosquito were sequenced in both directions using the ABI PRISM dye terminator cycle sequencing kit  and the ABI 310 DNA automated sequencer at Macromolecular Resources, CSU. A 415 nucleotide region of the S segment (nucleotides 190–604), a 358 nucleotide region of the M segment (nucleotides 1637–1994), and a 447 nucleotide region of the L segment (nucleotides 179–625) were sequenced.PCR products were separated by electrophoresis in 1% agarose gels with TAE buffer, visualized with ethidium bromide, excised and extracted using the Powerprep Express Gel Extraction kit  according to manufacturer's instructions. PCR products were inserted into the pCR4-TOPO cloning vector  and resulting plasmids were used to transform competent TOP10 Genetic haplotypes were established for each of the three segments through maximum parsimony analysis, sequence identity matrix, neighbor joining distance matrix, and ratio of synonymous to nonsynonymous substitutions.s) and replacement substitutions (πs). The rate of molecular evolution (substitutions/site/year) was estimated using the program TipDate [For each segment, the computer program DnaSP 4.5  estimate TipDate . TipdateLinkage disequilibrium is a measure of the degree to which substitutions in a segment occur independently of one another. Substitutions that occur together in a segment at a rate predicted by their independent frequencies are in linkage equilibrium. Substitutions that occur more or less often than expected by random chance are considered to be in linkage disequilibrium. Linkage disequilibrium also tests whether sampling a portion of a genome segment is representative of the whole segment. Linkage equilibrium is detected when different parts of a segment are evolving independently and sequencing a portion of the segment may not provide a representative sample of the whole.i, Mj, and Lk appear in the same mosquito, where for example, Si is a unique sequence of the segment.A linkage disequilibrium analysis was also performed to determine if entire segments assort randomly, thereby suggesting segment reassortment. Segment are in disequilibrium when some combinations of segments occur together in a mosquito more or less often than would be predicted by their independent frequencies. The first step is to determine the number of times segment, Sijk is the number of times haplotype i, j, and k are expected to occur in a mosquito.Ei is the frequency of Si in the mosquito population and N is the number of mosquitoes. Linkage disequilibrium (Dijk) was then estimated.where pijk was determined [A Hill and Robertson correlation coefficient Rtermined .ijk2) was used as a metric of disequilibrium because it ranges from zero (linkage equilibrium) to one (linkage disequilibrium). Linkage disequilibrium patterns among all polymorphic sites were plotted on a heat map using the LDheatmap program in R [2Link) and the corresponding level of significance were calculated for each combination of haplotypes to test the hypothesis that the individual haplotype combinations are in linkage equilibrium.The squared correlation coefficient  4.0b10 package . The phyThe authors declare that they have no competing interests.SR and BrB carried out virological and molecular characterization of viruses. SR and WB conducted phylogenetic analyses and statistical analyses. DG identified field collection sites and managed field collections and logistics. CB, BaB, and WB conceived, designed, and managed the study. All authors participated in the preparation of the MS and approve its submission to Virology Journal.


SRY, DAZ, CDY1, DBY, UTY and AZFa, b and c specific STSs. Real Time PCR and FISH demonstrated tandem duplication/multiplication of the SRY and DAZ genes. At sequence level, the SRY was normal in 8 TS patients while the remaining 7 showed either absence of this gene or known and novel mutations within and outside of the HMG box. SNV/SFV analysis showed normal four copies of the DAZ genes in these 8 patients. All the TS patients showed aplastic uterus with no ovaries and no symptom of gonadoblastoma. Present study demonstrates new types of polymorphisms indicating that no two TS patients have identical genotype-phenotype. Thus, a comprehensive analysis of more number of samples is warranted to uncover consensus on the loci affected, to be able to use them as potential diagnostic markers.Presence of the human Y-chromosome in females with Turner Syndrome (TS) enhances the risk of development of gonadoblastoma besides causing several other phenotypic abnormalities. In the present study, we have analyzed the Y chromosome in 15 clinically diagnosed Turner Syndrome (TS) patients and detected high level of mosaicisms ranging from 45,XO:46,XY = 100:0% in 4; 45,XO:46,XY:46XX = 4:94:2 in 8; and 45,XO:46,XY:46XX = 50:30:20 cells in 3 TS patients, unlike previous reports showing 5–8% cells with Y- material. Also, no ring, marker or di-centric Y was observed in any of the cases. Of the two TS patients having intact Y chromosome in >85% cells, one was exceptionally tall. Both the patients were positive for  Turner Syndrome (TS), the common genetic abnormalities affecting ∼1 in 1500–2000 live female births SRY and DAZ genes in addition to known and novel SRY mutations within and 5′/3′ regions of the HMG box.Present study was conducted to investigate molecular alterations in the Y-linked loci in 15 clinically diagnosed TS patients. We detected large scale Y chromosome mosaicism ranging from pure 45/XO conceptus to ∼90% cells positive for an intact Y chromosome and XXX, XYY, XXY constitutions. Further, several Turners patients showed tandemly arranged multiple copies of the SRY, 46A6 (DAZ) and 336F2 (gr/gr AZFc amplicons). Patients showed two extreme karyotypes, ones with >85% cells harboring Y chromosome (AT1 and AT15) and others with almost negligible presence of the Y chromosome   . Marker,and AT7)  but no sand AT7) . Pure XOand AT7) .DAZ gene, HERV sequences, AZF boundaries and other crucial genes were found to be intact except few randomly scattered microdeletions  values observed were unexpected. In case of single copy SRY and 4 copy DAZ genes in a normal male, ΔCt values observed are 1 and −1, respectively SRY and DAZ gene were >1 in case of TS patients  did not inherit all the 16 copies instead the same was the result of multiple rounds of tandem duplications. Although no direct evidence is available, these observations suggest that non-disjunction of the Y chromosome and duplications of the linked genes are two independent events. As mentioned earlier, total number of genes/loci affected in case of TS is not known. In addition, involvement of the autosomal genes, their possible up-, down- regulation or genetic imprinting remains allusive in such patients. It would therefore be of relevance if expression level of Y linked genes and possibly autosomal ones are assessed in TS with or without cytogenetically detectable Y chromosome or its mosaicism. Of all the autosomal genes, those that are candidates for testicular functions and hormonal profiles may prove to be attractive targets. Similarly, in-depth mutational analysis of all the candidate genes involved in testicular functions in addition to Y linked loci would also prove to be equally informative. Information on this line is envisaged to be of relevance not only for molecular diagnosis of TS but also for prenatal prognosis and management of clinical cases on routine basis.Turner Syndrome (TS) is globally acknowledged and well defined genetic anomaly, postulated to be an effect of the absence of genes located on the second sex chromosome. However, it is not clear as to how many loci/genes are affected in TS nor do we know whether the abnormal genes/loci are cause or effects. Similarly, there is no information on the type of Y linked genes/loci affected in somatic tissue and gonad of a Turner Patient. Primary focus of the present study was to analyze the fate of the human Y chromosome and its linked loci/genes in TS patients with respect to their possible copy number variation and new type of mutations. This study was undertaken with an ultimate view to uncover consensus changes in the genes/loci to use the resultant information for molecular diagnosis. Extreme mosaicism in terms of the presence or absence of Y chromosome with almost similar hormonal profile and characteristic Turners' features was found to be most astonishing. This suggests that genes involved in control and regulation of hormonal profiles were not affected. Clinically, most accepted karyotype of TS is 45/XO. However, we detected >85% cells harboring intact Y chromosome  in some phenotypically females TS patients. Despite such a large percentage of Y bearing cells, these patients did not represent Swyer syndrome . We hypothesize that in addition to sex chromosomes, there may be several other factors including autosomal genes, responsible for TS phenotype. Other significant part of this study was the absence of dicentrics, marker or ring Y chromosomes in any of the 15 TS patients analyzed though these features were reported to be common in Turner mosaics ectively . Based oPresent study is an attempt to analyze status of the human Y chromosome in patients with Turner Syndrome. Clinical Turner symptoms were common both in females with negligible presence of Y  and the ones carrying Y in >85% cells . We also infer that in addition to +/− mosaicism of the Y chromosome, Turners may harbor copy number polymorphism of several Y linked genes and possibly that of autosomes. Notwithstanding a great deal of information available in the literature, it is still not proven whether the Y mosaicism observed is a cause or consequence of Turner Syndrome.Blood samples from all the TS patients were collected from J.N. Medical College, Aligarh Muslim University, Aligarh, India, with the informed consent of patients following strictly the guidelines of the Institute's Ethical and Biosafety Committees. All the patients, except two, were phenotypically females with short stature and webbed neck. TS patients had shield like chest, reproductive sterility and primary amenorrhea . DNA isoMajority of the Turner patients were illiterate, therefore, their oral consent were obtained. This was facilitated by a recognized local clinician who was also known to them. From the literate people, written consents were obtained following which Institute's Ethical Committee accorded its due clearance. After Institute's Ethical Clearance, no additional approval regarding a particular gene sequence/gene variant was required for publication of the data.Level of FSH, TSH, LH and PRL was estimated taking 100 µl serum obtained from each sample by radioimmunoassay using commercial kits from Bhaba Atomic Research Center, Bombay according to supplier's instructions.SRY, CDY, DAZ, XKRY, DBY, UTY, CDY2 genes and several STS markers. The amplified product of SRY gene from the TS patients was sequenced for its mutational analysis. The DAZ genes were assessed for the presence of sequence family variants SRY fragments were also sequenced in multiples by cloning into pGEMT-easy vector (Promega).PCR was performed to amplify AZFa region was also assessed for possible provirus mediated recombination leading to duplication or deletion Structural analysis of crucial Y regions was done by selected STSs employing single and multiplex PCRs. The absence of a particular STS was confirmed by repeating the PCR reactions thrice followed by Southern hybridization using amplified PCR product as probe from the normal male. The SRY and DAZ were chosen as candidate genes for their copy number status. Copy number of was calculated using Real Time PCR and TaqMan/SYBR green assays following procedures described earlier SRY (Cat 32-191007) DNA FISH probe for SRY/CEP X was purchased from VYSIS . FISH probes for DAZ included Cosmids 18E8 for 5′ DAZ, Probe A; 46A6 for 3′DAZ, probe B and 63C9 containing exons 2 through 11, Probe C www.geneservice.co.uk/home) and BAC from Children's Hospital Oakland Research Institute (CHORI). Details of the clones are given in Approximately, 400 µl of whole blood was cultured for chromosome preparation following standard protocols Figure S1Details of the FISH probes used for the DAZ genes are listed in the table(11.64 MB TIF)Click here for additional data file.Figure S2Nucleotide sequence polymorphism of the SRY gene in Turners AT1 and AT15.(14.71 MB TIF)Click here for additional data file.


The purpose of this study is to fabricate the polyethylene glycol matrix tablet by mold technique. Indomethacin and hydroxypropylmethylcellulose were used as model drug and polymer, respectively, in PEG matrix system. The physical and drug release characteristics of developed matrix tablet were studied. This inert carrier system comprising 7:3 polyethylene glycol 4000: polyethylene glycol 400 could effectively enhance the solubility of indomethacin and an addition of hydroxypropylmethylcellulose could sustain the drug release. Scanning electron microscope photomicrograph indicated the drug diffusion outward through the porous network of this developed matrix tablet into the dissolution fluid. Least square fitting the experimental dissolution data to the mathematical expressions  indicated the drug release kinetics primarily as Fickian diffusion. Both the enhancement of drug dissolution and the prolongation of the drug release could be achieved for aqueous insoluble drug such as, indomethacin, by using polyethylene glycol-hydroxypropylmethylcellulose matrix system prepared with melting and mold technique. Typically, a tablet dosage form is prepared by compression method. Some special tablet such as orodispersible tablet could be prepared by freeze drying. Melt extrusion has been recently reported as the procedure to produce the drug matrix system for enhancing or retarding the drug release. The main excipient employed for this system should be molten at elevated temperature and could set up as solid matter at room temperature.Polyethylene glycols (PEGs) are widely used in a variety of pharmaceutical formulations including parenteral, topical, ophthalmic, oral and rectal preparations. PolyethIndomethacin is non-steroidal antiinflammatory agent with antipyretic and analgesic properties. It is a nonselective inhibitor of cyclooxygenase (COX) 1 and 2, enzymes that participate in prostaglandin synthesis from arachidonic acid. It has been used in the symptomatic management of painful and inflammatory conditions. It is used in musculoskeletal and joint disorders including ankylosing spondylitis, osteoarthritis, rheumatoid arthritis and acute gouty arthritis. Usual initial dose by mouth in musculoskeletal and joint disorder is 25 mg two or three times daily with food. To alleviate night pain and morning stiffness, 100 mg may be administered by mouth, or rectally as a suppository. In acute gouty arthritis a suggested dose is 50 mg three times daily and in dysmenorrheal up to 75 mg daily has been suggested. Indomethacin is practically insoluble in water, soluble 1 in 50 of ethanol, 1 in 30 of chloroform, and 1 in 40 to 45 of ether, and soluble in acetone. BecauseHydrophilic matrix has become extremely popular in controlling the release rate of drugs from solid dosage forms. When a matrix containing a swellable glassy polymer comes into contact with a solvent, a progressive alteration from the glassy to the rubbery state leads to a swelling process. For matrix system, drug is often released by diffusion, because a sort of receding drug boundary comes to exist within the system. HydroxyIt is interesting to create the system comprising both the drug dissolution enhancement together with the sustained drug release of that dissolved drug in form of matrix system. Therefore, in this study, indomethacin solid dispersion tablets were prepared with melting and mold method using 7:3 PEG4000:PEG 400 as inert carrier and HPMC as hydrophilic polymer to obtain the model formulation having functions of both the drug solubility enhancement and the extended release properties.HPMC type K 15 M, lot No. PH26012N31,  and indomethacin  were used as received. Polyethylene glycol 4000 (lot no. 504907) and polyethylene glycol 400 (lot no. PO76049) were purchased from P. C. Drug Center Co., Ltd., Bangkok, Thailand. Hydrochloric acid  was used as received. Potassium dihydrogen orthophosphate (lot no. E23W60), di-sodium hydrogen orthophosphate (lot no. 405300), sodium chloride (lot no. AF 407256), sodium hydroxide (lot no. AF 310204) were purchased from Ajax Finechem, Canning Vale, Australia.Tablets containing 75 mg indomethacin and different amount of HPMC using PEG4000:PEG400 (7:3) as carrier were prepared with the melting and mold technique. The tablet was prepared by melting PEG 4000 on the water bath and mixed with PEG 400, drug and polymer, respectively, and then the mixtures were poured into the stainless steel mold with diameter of 12 mm.The hardness of the tablets was determined using a hardness tester . The tablet thickness and diameter were measured using a thickness tester . A test of drug release was undertaken in 900 ml phosphate buffer pH 6.2 using a dissolution apparatus  with the basket method at 100 rpm. Samples were collected at specific time intervals and assayed by a UV/Vis spectrophotometer  at a wavelength of 323 nm. During the drug release studies, the tablets were observed for physical integrity. Drug release from these tablets was compared to capsule containing 75 mg indomethacin powder.2) was used to indicate the degree of curve fitting. Goodness-of-fit was also evaluated using the model selection criterion (MSC)[msc=h{∑i=1nwi(Yobs i−Y¯obs)2∑i=1nwi2}−2pn,Least square fitting the experimental dissolution data (cumulative drug release>5% and up to 80%) to the mathematical equations  was carried out using a nonlinear computer programme, Scientist for Windows, version 2.1 . The coefficient of determination (rion (MSC), given aobsi and Ycali are observed and calculated values of the i-th point, respectively, and wi is the weight that applies to the i-th point, n is number of points and p is number of parameters.where Yet al[The swelling behavior of the tablets was investigated through textural analysis. Tablets were placed in the dissolution vessels under condition identical to that described above for dissolution testing. The hydrated tablets were removed at predetermined intervals, and subjected to textural profiling to determine a total work of probe penetration into the entire matrix. All measurements were carried out in triplicate for each time point and tablets were discarded. Textural analysis was performed using a TA.XT2i texture analyzer  equipped with a 5 kg load cell and Texture Expert software. The force displacement-time profiles associated with the penetration of a 3 mm round-tipped steel probe into the swollen matrices were monitored at a data acquisition rate of 200 points per second. Probe approached the sample at pretest speed of 1.0 mm/s. Once a trigger force of 0.005N was detected (at contact of the probe with tablet) the probe was advanced into the sample at a test speed of 0.5 mm/s until the maximum force of 40 N was reached as described by Baumgartner et al.The DSC thermograms of drug, PEG 4000, PEG 400, HPMC, tablet containing 7:3 PEG 4000:PEG 400 without or containing 25% HPMC were obtained using differential scanning calorimetry (DSC) . The experiment was done in non-hermetically sealed aluminium pans; the heating rate was 10 °/min and using nitrogen as purge gas (20 ml/min). Samples of approximately 5 mg were weighed into aluminium pans. The heating range was 25-300° and -30-70° with reverse run.The surface topography of the prepared matrix tablet was determined using scanning electron microscope (SEM) . Samples after dissolution test were freeze dried for 24 h to dry the samples and to avoid the collapse of porous structures. The samples were stuck on a metal stub using carbon double adhesive and sputter-coated with gold before test. Micrographs were taken with a scanning electron microscope operated at an accelerating voltage of 20 KeV.System comprising 70:30 PEG4000:PEG400 was chosen as carrier for tablet since the tablet prepared by mold technique with this system could be easily remove from the mold and had suitable hardness for tablet dosage form. Hardness of tablet containing different amount of HPMC was increased as the amount of polymers was increased . HPMC so12The dissolution of indomethacin from tablets containing different amount HPMC is shown in 14et al[et al[Tablet containing 5% HPMC was faster than tablets containing 10%, 15%, 20%, 25% HPMC and capsule, respectively. High swelling capacity and gel formation of HPMC could retard the release of dissolved drug from the matrix. Relationship between drug release rate and polymer concentration was previously reported by Fu et al. As also al[et al, water i al[et al. It was  al[et al. Drug re al[et al.Dry tablet (before dissolution test) containing 25% HPMC exhibited higher total work penetration than tablet after dissolution test at different time intervals figs. . Total w20Differential scanning calorimetry (DSC) was conducted to indicate the molecular dispersion of indomethacin into carrier matrix. Indomethacin and system containing 7:3 PEG4000:PEG400 exhibited the endothermic peak at 161.4° and 48°  whereas 625Micrographs of indomethacin powder are shown in Most likely, the morphological change was due to a leaching out of the water soluble polymers, PEG, HPMC, and the release of the indomethacin. This morphological characteristic change of other matrix has been previously claimed. Porositin vitro release data, the curve fitting of drug dissolution profiles to various kinetic models were carried out to describe the release kinetics. The coefficient of determination (r2) was used to indicate the degree of curve fitting. Goodness-of-fit was also evaluated using MSC. The zero order describes the systems where the drug release rate is independent of its concentration[To analyze the entration. The firentration. Higuchientration.2 from curve fitting to power law equation was in range 0.9881 to 0.9973 and msc was in range 4.01 to 4.71  values for almost formula are shown in n could indicate the release mechanism such as Fickian diffusion, case II transport, or anomalous transport. In the present study  the limits considered were n = 0.45  and n = 0.89 . Values of n between 0.45 and 0.89 can be regarded as indicators of both phenomena (transport corresponding to coupled drug diffusion in the hydrated matrix and polymer relaxation), commonly called anomalous non-Fickian transport[n values bigger than 1.0 would allow for a more desirable incremental release. To achieve delayed release pattern that was therapeutically useful, n values were expected to be bigger and k values to be smaller with a prerequisite of acceptable lag time[The exponent transport. The k v lag time.n values of tablet containing different amount of HPMC indicated that the release mechanism of these tablets were close to anomalous transport. Therefore, mechanism and kinetics of drug release are dependent on the solubility of the active moiety and the swelling and erosion properties of the polymer, with water soluble drugs being released predominantly by diffusion with a limited contribution from matrix erosion and anomalous diffusion resulting from the relaxation of the macromolecular polymer chains[From curve fitting to power law equation, the er chains. Therefo


Sedentarism, exercise and physically active lifestyles modify it. For that reason, this study evaluates the associations between RER during sub-maximum exercise and other well established fitness indicators , during maximum exercise test and during three different sub-maximum exercises at fixed workload: below, within or above the lactate threshold.The RER, O2 uptake, lower blood lactate concentrations and lower RER values than those in untrained men at the three similar relative workloads. Even though with these differences in RER, a strong association (p < 0.05) of RER during sub-maximum exercise with the other well established fitness indicators was observed, and both maximum O2 uptake and lactate threshold determined more than 57% of its variance (p < 0.05).Endurance trained men presented higher OThese data demonstrate that RER measurement under sub-maximum exercise conditions was well correlated with other established physical fitness indicators, despite training condition. Furthermore, the results suggest that RER could help obtain an easy approach of fitness status under low exercise intensity and could be utilized in subjects with reduced exercise tolerance. Relevant anthropometric characteristics of them are presented in 2 uptake (VO2) and CO2 production (VCO2) were continuously recorded and blood lactate was assayed (see below). From fourth to sixth session, subjects performed at random three different SEFW, during which the described parameters were recorded.The evaluations were done between 08:00 and 11:00 h in 6 sessions, after 10–12 h overnight fasting and 8–9 h of sleep. Subjects were further instructed to refrain from any sort of heavy physical labour study. The tests were performed in a room with environmental temperatures kept between 22 and 25 ºC. At the first session anthropometrical assessments, diet studies and a practice session on bicycle ergometer were performed. On the second and third sessions, the subjects completed a maximum exercise test during which heart rate, workload (in watts), OTwo expert anthropometrists, using an anthropometric kit  and following the International Society of Advancement in Kinanthropometry (ISAK) technique as described by Norton and Olds , performThe food intake was assessed for three sequential days (one during the weekend) by the 24-hour dietary record method. All dietary records were checked out through interviews. We asked the subjects to keep their diet habits during the study. The dietary records were analyzed with the Diet Balancer software, version 1.4c . This computer software has a section for Mexican foods, and the defaulting foods were added using food tables from National Institute of Medical Science and Nutrition, Salvador Zubirán, Mexico .2, and RER, the percentages of O2, CO2 in inspired and expired air as well as minute pulmonary ventilation were measured with a gas analyzer . The system was calibrated before and during each test by using certificated gas mixtures of known concentrations . A 3-L syringe  verified the flow of gases. The environmental barometric pressure was measured by a fortin type mercurial barometer , and the temperature and relative humidity by a mason type hygrometer . During exercise, expired gases were analyzed with the breath-by-breath system (facemask system). The exercise tests were carried out on an electronic bicycle ergometer .In order to determine VO2max was calculated from the highest VO2 value, attained by each participant during two maximum exercise tests . The initial workload and subsequent increments (in watts) were established 24 h before the test, according to the aerobic capacity of each subject, in such way that the total time of the test ranged between 8 and 12 min. The maximum exercise test was initiated with a workload adjusted between 50 and 75 W, and then a ramp protocol was started with increments of 25–30 W per min in trained participants, and of 15–25 W in untrained participants.VO2 vs. lactate) was used to determine LT, which was detected before a curvilinear increase in plasma lactate concentration was observed  1.5 mM below the LT, b) ±1 mM within the LT and c) 2.5 mM above the LT, found in the preceding maximum exercise tests. The individual breaking point on the blood lactate concentration .Results in tables are presented as mean ± standard deviation (SD) and in figures as mean ± standard error (SEM). Differences between groups were determined by unpaired student-t test. The degree of association among variables was assessed by multiple correlation analysis. The independence of associated variables on RER was evaluated with multiple regression analysis and the best model was selected by allpossible regressions procedure. Statistical significance was accepted when α level of p < 0.05. Statistical analyses were conducted with SAS System software, version 8.0.2max, workloadmax and lactate threshold (measured as watts and VO2) were significantly different in trained subjects compared to untrained  association of RER with fitness variables , and together with workload at LT (in watts) were the determinant variables for the changes in RER at above LT (the model explained the variance in 87%)  was the unique variable for determining changes in RER at below LT (the model explained the variance in 68%). From the same analysis VO in 87%) .2, and lower blood lactate concentrations than untrained subjects. The differences in relative intensity at below LT and within LT may not be statistically significant due to the subject response variability, but the differences are large enough to be physiologically significant. The lower RER values mean that during submaximal exercise, trained subjects oxidized a greater proportion of lipids at higher workload than untrained subjects (2 did not show any significant change. Our results are in agree with those of 2peak), lactate increase while the pH decreases. However, increases on blood lactate concentration during exercise could occur without decreases in blood pH . Furthermore, results of multiple regression analysis showed that the aerobic fitness indicators VO2max and lactate threshold determine between 57% and 87% of RER variance during exercise. The association of the RER with variables of physical fitness, like anthropometric characteristics, VO2max and lactate threshold, has not been clearly established. Goedecke et al.  were associated with RER in both groups despite of training condition; in addition, the aerobic fitness (VO2max and lactate threshold) determine more than 57% of RER variance during exercise.Trained subjects showed higher oxidative metabolism and lower RER values than the untrained subjects, even when exercising at similar relative submaximal workloads. The physical fitness indicators (body fat, heart rate, VOSince RER provides information about physical fitness, RER measurement could be considered in the evaluation of physical activity intervention programs; especially in people with a limited capacity for physical activity, because its assessment applied under submaximal exercise conditions and at low intensity is health, safety, easy and reliable.2max.The RER could be considered as an additional fitness indicator at low-moderate intensity and being independent of LT and VOAt similar relative submaximal exercise intensities, endurance trained subjects oxidized a greater proportion of lipids than untrained male.In physically active healthy men, the physical fitness parameters are strongly associated with the RER during exercise.The main obstacle of this study was to maintain working all our subjects more than 4 min above of the LT. For this reason, the high-work intensity has not to be far away the LT.To realize more research looking for associations of RER with other fitness indicators and heath parameters in general and special populations.


Parental consanguinity is a risk factor for congenital heart disease (CHD) worldwide, suggesting that a recessive inheritance model may contribute substantially to CHD. In Bangalore, India, uncle-niece and first cousin marriages are common, presenting the opportunity for an international study involving consanguinity mapping of structural CHD. We sought to explore the recessive model of CHD by conducting a genome-wide linkage analysis utilizing high-density oligonucleotide microarrays and enrolling 83 CHD probands born to unaffected consanguineous parents.HOMEZ gene, a ubiquitously expressed transcription factor containing leucine zipper as well as zinc finger motifs. Re-sequencing of HOMEZ exons did not reveal causative mutations in Indian probands. In addition, genotyping of the linked allele (G) in 325 U.S. CHD cases revealed neither genotypic nor allele frequency differences in varied CHD cases compared to 605 non-CHD controls.In this linkage scan involving single nucleotide polymorphism (SNP) markers, the threshold for genome-wide statistical significance was set at the standard log-of-odds (LOD) score threshold of 3.3, corresponding to 1995∶1 odds in favor of linkage. We identified a maximal single-point LOD score of 3.76 (5754∶1 odds) implicating linkage of CHD with the major allele (G) of rs1055061 on chromosome 14 in the HOMEZ may harbor recessive mutations leading to CHD in the Indian population. Further research involving large multinational cohorts of patients with specific subtypes of CHD is needed to attempt replication of the observed linkage peak on chromosome 14. In addition, we anticipate that a targeted re-sequencing approach may complement linkage analysis in future studies of recessive mutation detection in CHD.Despite the statistical power of the consanguinity mapping approach, no single gene of major effect could be convincingly identified in a clinically heterogeneous sample of Indian CHD cases born to consanguineous parents. However, we are unable to exclude the possibility that noncoding regions of  EVC or EVC2PDA1 as a candidate locus for patent ductus arteriosus in an Iranian population Congenital heart disease (CHD) affects approximately one percent of newborn infants The consanguinity mapping approach has not yet been widely applied to nonsyndromic CHD, in part because parental consanguinity is uncommon in places where research efforts have historically been most intense. However, interest is growing in the statistical power of this approach for genetic investigations of other complex phenotypes, such as autism, as demonstrated by the Homozygosity Mapping Collaborative for Autism CHD presents major public health challenges in India, due to the large annual number of births in the Indian population and the limited resources for specialized care of children with CHD HOMEZ on chromosome 14 at 22,814,772. The LOD score was 3.76 (5754∶1 odds), and the optimal theta value was 0.05. The genotype frequencies of the probands were GG = 0.83, GA = 0.13 and AA = 0.04. The second SNP, rs12433225, yielded a LOD score of 3.65 (4467∶1 odds) and maps to chromosome 14 at 25,596,730, more than 250 kb away from any known gene in either direction. As a supplemental analysis, we also conducted a TDT test of association using WASP, and obtained a significant p-value of 0.0495 for rs1055061 with the major allele (G) overtransmitted and a non-significant p-value of 0.8185 for rs12433225.The phenotypic distributions of the cohorts are described in Each of the linked SNPs was examined in the context of autozygosity of the probands. SNP rs1055061 resided in a region of autozygosity in 13 patients, while SNP rs12433225 localized to a region of autozygosity in 14 patients. Analysis of 39 other SNPs randomly selected from the Affymetrix platform resulted in a mean of 6.6 probands  with autozygosity surrounding the region of interest. Additionally, the minimum region of autozygosity shared by the 13 patients surrounding rs1055061 is Chr14:21,071,230–24,393,433, and the minimum region of autozygosity shared by the 14 patients surrounding rs12433225 is Chr14:24,952,386–25,774,000.HOMEZ in 54 probands. On sequence analysis of the two coding exons, four previously known SNPs We re-sequenced the coding regions of HOMEZ SNP rs1055061 was also associated with CHD in an American population, we genotyped cases and controls from this population to assess allele frequency differences using an ABI TaqMan assay. Frequencies were 282 GG, 42 GA, and 1 AA in 325 patients with CHD and 548 GG, 57 GA and no AA in 605 control patients. The difference in the frequency of the GG genotype in cases versus controls was not statistically significant by Chi-squared analysis (P = 0.074). Secondary analysis of an allele frequency difference between cases and controls was not statistically significant by Chi squared analysis (P = 0.062).The population in our linkage analysis was comprised of children of consanguineous Indian parents. To determine if the We have presented statistically significant linkage evidence for a potential CHD susceptibility locus on chromosome 14 in a South Indian population selected for consanguinity. However, the linkage finding was not robust in a genetic association follow-up study of CHD in the general United States population. Utilizing the extensive genotyping information in oligonucleotide arrays, linkage analysis revealed two markers with LOD scores above 3.3. Because approximately 2.8 Mb separates these markers, a distance far in excess of typical linkage disequilibrium values in the Indian population HOMEZ, a vertebrate specific homeobox gene with an interesting structural organization. This transcription factor harbors three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The protein is ubiquitously expressed in human tissues, and has been further studied in a murine developmental model HOMEZ have not been described Of the two markers, only rs1055061 is intragenic, causing a non-synonymous amino acid substitution. This SNP lies within The rs1055061 SNP identified by linkage induces an amino acid substitution from the polar, positively charged arginine to the polar, but neutral glutamine. The allele with the higher population frequency was overtransmitted in the TDT analysis, whereas one might expect a rare recessive allele to confer susceptibility to an uncommon phenotype such as CHD. Linkage analysis resulted in a theta value of 0.05, indicating that a causative mutation most likely lies within 50 kb of this SNP. Additionally, an increased number of probands exhibited a region of autozygosity surrounding each of the linked SNPs, supporting our hypothesis of autosomal recessive inheritance due to parental consanguinity. We do acknowledge that the LOD score of 3.76 indicates a 1∶5754 odds of this finding being due to chance alone, but argue that the convergence of multiple methods of inquiry support rs1055061 as a true finding.HOMEZ in a subset of the Indian probands with the hypothesis that a deleterious mutation in linkage disequilibrium with rs1055061 would be discovered. We detected previously reported SNPs and three novel sequence variations. None of the novel variations were found to be homozygous, as would be expected for causative mutations in offspring of a consanguineous relationship. Thus, we were unable to identify any causative homozygous mutations in the coding regions of HOMEZ.We thus proceeded with sequence analysis of the coding regions of We also studied the rs1055061 SNP in an American cohort of patients affected with various types of CHD and compared them to population controls. No significant difference was detected in the genotype or allele distribution between cases and controls, suggesting that our linkage finding in South Indians does not extend to genetic association in an American population of mixed European ancestry.Although this study did not reveal definitive evidence of causal mutations leading to autosomal recessive CHD, a number of future directions and refinements are suggested. A limitation in the application of consanguinity mapping to CHD is that the fraction of this heterogeneous group of diseases attributable to recessive inheritance and the approximate number of loci involved is not known. Ascertaining consanguineous families with multiple affected children (suggesting recessive inheritance) would theoretically reduce genetic heterogeneity and increase power for linkage. If only multiplex pedigrees are enrolled, the proportion of probands with sporadic or autosomal dominant forms of CHD will be minimized.a priori which forms of CHD share a common genetic etiology. This may have resulted in diffusion of the linkage signal because of both clinical and genetic heterogeneity. Enriching the study population for predefined, specific phenotypic classes would likely decrease genetic heterogeneity in the study population.We did not restrict our analysis to specific CHD phenotypes because it is impossible to select Recent work in massively parallel whole exomic Written informed consent was provided by the parents of all children assenting to participate in the study, using consent documents approved by the ethics board of Narayana Hrudayalaya Heart Hospital. Dr. Sunita Maheshwari, Principal Investigator at Narayana Hrudayalaya Heart Hospital, and Dr. Amit Misri, co-investigator, obtained consent from participating families. Genotyping was performed on de-identified samples without knowledge of any personal identifiers under IRB approval from Yale University, Washington University in St. Louis, and Vanderbilt University.We enrolled children affected with congenital heart disease and born to consanguineous parents in order to identify candidate regions for CHD by linkage analysis. The families were ascertained in a cardiology clinic at Narayana Hrudayalaya in Bangalore, India, from 07/2004 to 07/2007. The inclusion criteria were the presence of structural congenital heart disease and parental consanguinity. Exclusion criteria were isolated conduction system defect, cardiomyopathy, or extracardiac syndromic features. The patients had an echocardiogram performed in the clinic and interpreted by pediatric cardiologists. In all, 83 probands were recruited from 81 families. Probands were not known to be related to each other with the exception of one family that enrolled three affected siblings. Both parents were recruited in 71/81 families, and one parent in 10/81 families. The phenotypic distribution of the probands' cardiac lesions is described in A replication cohort of 325 probands affected with congenital heart disease was ascertained at Vanderbilt University Medical Center via therapeutic clinical trials of oral Genomic DNA for probands 1–26; 39–50 and relatives was isolated from blood spots on Whatman filter paper using GenSolve blood spot DNA recovery kit according to the manufacturer's protocol . DNA from probands 27–38 was isolated from peripheral blood samples using Gentra PureGene Blood Kit . For probands 51–83 and relatives, saliva was collected with Oragene kits, and DNA was extracted per manufacturer's protocol . We performed genome wide scans utilizing Affymetrix SNP 5 microarrays for probands 1–30 and relatives with genotyping reactions performed at Precision Biomarker Resources . Probands 39–83 and relatives were genotyped on Affymetrix SNP 6.0 microarrays, and genotyping reactions were performed according to the manufacturer's instructions . Genotypes were generated using the BRLMM-P for Affymetrix SNP 5 arrays and the Birdseed V-2 algorithm for Affymetrix SNP 6.0 arrays as released in Genotyping console v3.0.1 . The SNP 5 arrays had an average QC call rate of 94.5% with SD of 4.8%. The SNP 6 arrays had an average QC call rate of 91.2% with SD of 5.6%. The genotype data was exported as .chp files and further analyzed with Merlin,v1.1.2 Assessment of regions of autozygosity were conducted in Partek software, V6.4 Copyright 2009  with the algorithms for loss of heterozygosity (LOH). Unpaired analysis parameters included max probability 0.99, genomic decay 0, genotype error 0.02, and default frequency 0.3. All genotype calls from the probands were used to generate array specific baselines. Random markers were selected from the Affymetrix SNP5 & SNP6 platforms using a random number generator. Each result from the LOH analysis overlapping the genomic location of interest was visually inspected to determine true autozygosity.HOMEZ with bidirectional sequencing in a subset of Indian probands (n = 54). Four primer sets covering the coding exons and their flanking regions were designed with Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Fragment amplification was performed under standard PCR conditions. In brief, 50 ng of DNA was used in the reaction; PCR program started with 94°C for 5 min, 35 cycles of 94°C, 57°C and 72°C, a final extension cycle of 72°C for 6 minutes and stored at 4°C. PCR products were purified using Exo-SAP (Fermentas) and sequenced with the ABI 3730xl DNA Analyzer at the DNA Sequencing Facility, Vanderbilt University.We carried out a mutation screening of For the replication cohort, a proprietary TaqMan genotyping assay for rs1055061 was obtained from Applied Biosystems (Assay ID: C___2484687_10). After lyophilizing 10 ng genomic DNA per sample, the genotyping assay and TaqMan Universal PCR Master Mix (Applied Biosystems 4371355) were then prepared and run as per manufacturer's protocol. Applied Biosystems SDS® 2.3 was used to analyze the results of the genotyping assay. Genotypes were assigned without knowledge of the affected status of the samples.