De novo reciprocal translocation t(5;6)(q13;q34) in cattle: cytogenetic and molecular characterization.

The cytogenetic analysis of a phenotypically normal bull from the Marchigiana breed revealed the presence of an abnormal karyotype due to the presence of a very long chromosome. This finding, identified in all the metaphases observed, was associated with the 2n = 60, XY karyotype, suggesting the presence of a reciprocal translocation. RBG- banding analyses identified a de novo reciprocal translocation involving BTA5 and BTA6, t(5;6)(q13;q34), while FISH analyses using cattle-specific BACs as probes enabled the confirmation and narrowed down the breakpoint regions. Array-CGH analysis also established that neither deletions nor duplications were present in the regions including the breakpoints, nor were they present elsewhere in the genome, confirming the balanced state of the translocation.

Physical mapping of 20 unmapped fragments of the btau_4.0 genome assembly in cattle, sheep and river buffalo.

The recent advances in sequencing technology and bioinformatics have revolutionized genomic research, making the decoding of the genome an easier task. Genome sequences are currently available for many species, including cattle, sheep and river buffalo. The available reference genomes are very accurate, and they represent the best possible order of loci at this time. In cattle, despite the great accuracy achieved, a part of the genome has been sequenced but not yet assembled: these genome fragments are called unmapped fragments. In the present study, 20 unmapped fragments belonging to the Btau_4.0 reference genome have been mapped by FISH in cattle (Bos taurus, 2n = 60), sheep (Ovis aries, 2n = 54) and river buffalo (Bubalus bubalis, 2n = 50). Our results confirm the accuracy of the available reference genome, though there are some discrepancies between the expected localization and the observed localization. Moreover, the available data in the literature regarding genomic homologies between cattle, sheep and river buffalo are confirmed. Finally, the results presented here suggest that FISH was, and still is, a useful technology to validate the data produced by genome sequencing programs.

Analysis of radical cystectomy and urinary diversion complications with the Clavien classification system in an Italian real life cohort.

INTRODUCTION: Standardized methods of reporting complications after radical cystectomy (RC) and urinary diversions (UD) are necessary to evaluate the morbidity associated with this operation to evaluate the modified Clavien classification system (CCS) in grading perioperative complications of RC and UD in a real life cohort of patients with bladder cancer. MATERIALS AND METHODS: A consecutive series of patients treated with RC and UD from April 2011 to March 2012 at 19 centers in Italy was evaluated. Complications were recorded according to the modified CCS. Results were presented as complication rates per grade. Univariate and binary logistic regression analysis were used for statistical analysis. RESULTS AND LIMITATIONS: 467 patients were enrolled. Median age was 70 years (range 35-89). UD consisted in orthotopic neobladder in 112 patients, ileal conduit in 217 patients and cutaneous ureterostomy in 138 patients. 415 complications were observed in 302 patients and were classified as Clavien type I (109 patients) or II (220 patients); Clavien type IIIa (45 patients), IIIb (22 patients); IV (11 patients) and V (8 patients). Patients with cutaneous ureterostomy presented a lower rate (8%) of CCS type ≥IIIa (p = 0.03). A longer operative time was an independent risk factor of CCS ≥III (OR: 1.005; CI: 1.002-1.007 per minute; p = 0.0001). CONCLUSIONS: In our study, RC is associated with a significant morbidity (65%) and a reduced mortality (1.7%) when compared to previous experiences. The modified CCS represents an easily applicable tool to classify the complications of RC and UD in a more objective and detailed way.

Cytogenetic elaboration of a novel reciprocal translocation in sheep.

Reciprocal translocations represent one of the most common structural chromosomal rearrangements observed in both humans and domestic animals. In these translocations, the balanced forms are most frequent but may remain undetected because the carriers show a normal phenotype. For this reason, routine cytogenetic analysis of domestic animals should necessarily rely on banded karyotypes. In fact, during a screening analysis, carried out on phenotypically normal young sheep (Ovis aries, OAR, 2n = 54) from Laticauda-Comisana hybrids, a new structural rearrangement was detected. Two abnormal acrocentric chromosomes (the smallest and the largest one) were found in all metaphases of this carrier animal, suggesting the presence of a reciprocal translocation (rcp). CBA and RBA banding were performed in order to characterize the translocation, and FISH with chromosome-specific BAC probes and telomere probes was applied to confirm the cytogenetic data. The translocation was classified as rcp(4q;12q)(q13;q25).

Reciprocal translocations in cattle: frequency estimation.

Chromosomal anomalies, like Robertsonian and reciprocal translocations, represent a big problem in cattle breeding as their presence induces, in the carrier subjects, a well-documented fertility reduction. In cattle, reciprocal translocations (RCPs, a chromosome abnormality caused by an exchange of material between non-homologous chromosomes) are considered rare as to date only 19 reciprocal translocations have been described. In cattle, it is common knowledge that the Robertsonian translocations represent the most common cytogenetic anomalies, and this is probably due to the existence of the endemic 1;29 Robertsonian translocation. However, these considerations are based on data obtained using techniques that are unable to identify all reciprocal translocations, and thus, their frequency is clearly underestimated. The purpose of this work is to provide a first realistic estimate of the impact of RCPs in the cattle population studied, trying to eliminate the factors that have caused an underestimation of their frequency so far. We performed this work using a mathematical as well as a simulation approach and, as biological data, we considered the cytogenetic results obtained in the last 15 years. The results obtained show that only 16% of reciprocal translocations can be detected using simple Giemsa techniques, and consequently, they could be present in no <0.14% of cattle subjects, a frequency five times higher than that shown by de novo Robertsonian translocations. This data is useful to open a debate about the need to introduce a more efficient method to identify RCP in cattle.

Molecular characterization of Xp chromosome deletion in a fertile cow.

A young cow of the Marchigiana breed (central Italy) with normal body conformation and external genitalia underwent routine cytogenetic analyses prior to its use for reproduction. After normal chromosome staining, only one X chromosome was observed with a normal diploid number (2n = 60) in all 200 studied cells. Subsequent cytogenetic analyses by using both CBA- and RBA-banding techniques evidenced that almost all the p arms of the other X chromosome was lacking. Detailed FISH-mapping analyses with BAC covering this Xp arm region demonstrated that this large chromosome region was deleted. RBA-banding showed that the deleted X was late replicating. CGH array analysis evidenced that deletion involves the Xp arm from the telomere to around 39.5 Mb, referring to the BosTau6 cattle genome assembly. This abnormality deletes about 40 Mb of the X chromosome sequence, but, despite the large number of genes deleted, none of them are programmed to escape from inactivation. This can explain the normal phenotype of the female which is actually pregnant. Finally, we evidenced, by analysis of an SNP mapped to the deleted region (SNP rs29024121), that the only normal (e.g. nondeleted) X chromosome present derives from the father. Hence, the deletion has a maternal origin.

XX SRY-negative true hermaphrodism in two dogs: clinical, morphological, genetic and cytogenetic studies.

This work aimed at giving a deeper insight into peculiar cases of intersexuality occurring in dogs and known as XX true hermaphrodism due to the existence of both testicular and ovarian tissue in one or both gonads in the presence of an XX chromosome constitution. Clinical, histological and genetic approaches were used in the study of an 8-month-old Cocker Spaniel dog and a 3-year-old mixed-breed Pitbull, both showing a female phenotype, clitoromegaly and male behavior. A normal female karyotype (2n = 78,XX) was noticed, and polymerase chain reaction failed to detect SRY in genomic DNA obtained from peripheral blood lymphocytes of both dogs. The reproductive tract was removed by standard ovariohysterectomy and processed for histology. Thereafter, a normal female phenotype was reconstructed by vaginoplasty. Histological examination revealed bilateral ovotestis in both cases: the gonads showed immature testicular parenchyma containing seminiferous tubules, Sertoli and Leydig cells, but no signs of spermatogenesis, together with differently developed ovarian follicles containing oocytes. In the ovotestes, steroidogenesis was detected by P450c17-immunoreactivity in Leydig cells as well as in theca cells, whereas no MIS-immunoreactivity was shown by the Sertoli cells. Genital tracts of Wolffian and Müllerian origin co-existed in both subjects. Both dogs belong to the very rare cases in which testicular tissue develops in the absence of the key gene, SRY. Up to date very few genetic events have been associated with this abnormal sexual differentiation: SOX9 over-expression and RSPO1 mutation. Nevertheless, neither of them has been found in these dogs.

Molecular mechanisms of sexual development.

Gonadal cellular organization is very similar in all vertebrates, though different processes can trigger bipotential gonads to develop into either testes or ovaries. While mammals and birds, apart from some exceptions, show genetic sex determination (GSD), other animals, like turtles and crocodiles, express temperature-dependent sex determination. In some groups of animals, GSD can also be overridden by hormone or temperature influences, indicating how fragile this system can be. This review aims to explain the fundamental molecular mechanisms involved in mammalian GSD, mainly referring to mouse as a major model. Conceivably, other mammals might show a molecular mechanism different from the commonly investigated murine species.

Characterization of Elastofibroma Dorsi with (18)FDG PET/CT: a retrospective study.

UNLABELLED: Elastofibroma dorsi has been described in the literature as an unusual tumor or pseudotumor. However, autopsies and imaging studies have revealed that it is a non-negligible finding. PURPOSE: The aim of this study has been to illustrate and become familiar with this type of lesion in order to prevent misdiagnosis. MATERIALS AND METHODS: From 3 December 2008 to 5 January 2010, 1,751 patients were evaluated with (18)FDG-PET/CT. Of these, 29 cases of elastofibroma dorsi were recorded as an incidental finding. A retrospective and descriptive analysis was performed on this study series. RESULTS: The study showed a prevalence of 1.66%. Out of the 29 findings, 22 (75.86%) were females and 7 (24.14%) males. Seventeen (58.62%) cases were bilateral, 12 (41.38%) unilateral and the SUVmax ranged from 1.4 to 3.2. These lesions were reported as soft tissue density images with mild or moderate diffuse metabolic activity. CONCLUSION: The elastofibroma dorsi is a relatively common finding in PET/CT that should be known in order to avoid making wrong diagnoses.

A new and unusual reciprocal translocation in cattle: rcp(11;25)(q11;q14-21).

A new and unusual reciprocal translocation was detected in a heifer of the Agerolese cattle breed during a routine cytogenetic screening carried out on 13 animals (2 males and 11 females) kept at the ConSDABI Conservation Center in Benevento (Southern Italy). The 13 animals investigated had a normal karyotype except for a 1-year-old female, which carried one autosome smaller than the smallest normal bovine autosomes. This small autosome showed very little C-banding in comparison to the other autosomes, while another medium-sized autosome showed 2 distinct and prominent C-bands. RBA-banding and karyotype analysis revealed that these 2 chromosomes were the result of a reciprocal translocation between chromosomes 11 and 25. FISH analysis with BAC142G06 mapping to the proximal (subcentromeric) region of both BTA25 and der11, BAC513H08 (ELN) mapping to BTA25q22dist and der25, and BAC533C11 mapping to the proximal region of BTA11 and der11 confirmed the localization of the breakpoints on band q11 (centromere) of chromosome 11 and q14-21 of chromosome 25. Ag-NOR and sequential RBA/Ag-NOR techniques detected the presence of NORs on both BTA11 and BTA25 and both der11 and der25. To our knowledge, this is the first report of a reciprocal translocation event in cattle with the breakpoint located in the centromeric region.

FISH mapping in cattle (Bos taurus L.) is not yet out of fashion.

Physical mapping of genes by fluorescence in situ hybridization (FISH) seems to be out of fashion in species whose assembled genome sequences are available. However, in this work we evidence the existence of errors in gene location in the Btau_4.0 assembly. We show that DFNA5 and CHCHD6 genes are located on BTA4 and BTA22, respectively, instead of BTA10 and BTA3, as displayed by Btau_4.0. This report emphasizes the need to verify the data on physical localization of genes in the cattle genome (at least by taking into account comparative data reported in available papers) and the need to improve the cattle genome assembly. Our results indicate that FISH mapping in cattle is still useful.

Chromosomal assignment of R-spondin genes in the donkey (Equus asinus, 2n = 62).

R-spondins constitute a recently discovered small family of growth factors, and the evidence of their role in several developmental pathways is growing fast. In this work we describe the chromosomal location of the four RSPO genes in the donkey. Using horse BACs, we localized RSPO1 on EAS 5q23, RSPO2 on EAS 12q13, RSPO3 on EAS 24q26, and RSPO4 on EAS 15p13. Moreover, RSPO2, RSPO3, and RSPO4 are the first genes mapped on donkey chromosomes 12, 24, and 15, respectively.

Cytogenetic and genetic studies in a hypospadic horse (Equus caballus, 2n = 64).

A 4-year-old male horse of Friesian breed with normal body conformation, development and libido, and showing an evident ventral penis deviation with hypospadias, underwent both cytogenetic and genetic investigation. Although the karyotype showed normal male arrangement (2n = 64,XY), one telomere of horse (ECA) chromosome 1 was shorter than both the other one and those of a normal horse (control), as revealed by CBA- and RBA-banding, and by Ag-NOR and FISH-mapping techniques using telomere PNA probes. Genetic investigation of the SRY and MAMLD1 coding sequences revealed a normal SRY sequence and a mutation in the MAMLD1 gene sequence: a homozygous change (C>A) was found, leading to the synthesis of an isoleucine, instead of a leucine. Although it is difficult to find a strict correlation between hypospadias and the genetic defects revealed by this investigation, this study is the first to be performed in a hypospadic horse using both cytogenetic and genetic investigation.

X trisomy in a sterile mare.

This report concerns the cytogenetic analysis, using both C-banding and fluorescence in situ hybridisation techniques, of a sterile mare. Results obtained revealed a 2n = 65, XXX condition with no sign of mosaicism. The work supports the suggestion that X trisomy, rare in horse, causes infertility in mares and is not associated to other clearly visible phenotypic features.

Reciprocal translocation t(4;7)(q14;q28) in cattle: molecular characterization.

Cytogenetic analysis of a phenotypically normal young bull from the Marchigiana breed revealed the presence of an abnormal chromosome. The finding of one oversize chromosome in all metaphases, associated with a 2n = 60, XY karyotype, suggested that a reciprocal translocation had occurred. RBG-banding and FISH analyses, using specific bovine BAC probes, identified a de novo reciprocal translocation t(4;7)(q14;q28). The presence of rcp(4;7) was confirmed by FISH experiments using BTA4 and BTA7 whole chromosome probes. An array-CGH analysis (Agilent 244A) using a bovine custom design was performed to investigate if the translocation was associated with loss or gain of genetic material. The absence of a concomitant deletion or duplication at the break points allowed the balanced state of the translocation to establish. The analysis also revealed the presence of several CNVs throughout the genome. To our knowledge this is the first time the balanced condition of a cattle RCP has been ascertained using the array-CGH approach.

The case of an Sry-negative XX male Pug with an inguinal gonad.

A case of intersexuality in a Pug that was bought as a male in a pet shop is described. The dog was presented at the Veterinary Teaching Hospital, University of Turin, for a reddish mass protruding from the prepuce. The mass had the aspect of an enlarged clitoris, with a caudoventral direction and a dorsal urethral ostium. A gonad was palpable in the left inguinal region. Laparotomy confirmed ultrasound detection of an abdominal uterine structure together with the right gonad. The histology of both gonads was similar, showing an exclusively masculine character, with seminiferous tubules lined only by Sertoli cells; the uterus showed a normal histological structure. Karyological analysis revealed a female karyotype (78,XX), and polymerase chain reaction showed the absence of Sry. The diagnosis was an XX male. The pathogenesis of the XX sex reversal syndrome in dogs is not completely understood, as Sry, the master gene regulating testis differentiation, is not present; to date, no genetic cause has been identified for this phenotypic condition in dogs. This case is unusual because the dog showed an inguinal testis, implying a partial activity of the mechanisms leading to abdominal testis translocation along a gubernaculum and transinguinal migration.

Classical and molecular cytogenetics of disorders of sex development in domestic animals.

The association of abnormal chromosome constitutions and disorders of sex development in domestic animals has been recorded since the beginnings of conventional cytogenetic analysis. Deviated karyotypes consisting of abnormal sex chromosome sets (e.g. aneuploidy) and/or the coexistence of cells with different sex chromosome constitutions (e.g. mosaicism or chimerism) in an individual seem to be the main causes of anomalies of sex determination and sex differentiation. Molecular cytogenetics and genetics have increased our understanding of these pathologies, where human and mouse models have provided a substantial amount of knowledge, leading to the discovery of a number of genes implicated in mammalian sex determination and differentiation. Additionally, other genes, which appeared to be involved in ovary differentiation, have been found by investigations in domestic species such as the goat. In this paper, we present an overview of the biology of mammalian sex development as a scientific background for better understanding the body of knowledge of the clinical cytogenetics of disorders of sex development in domestic animals. An attempt to summarize of what has been described in that particular subject of veterinary medicine for each of the main mammalian domestic species is presented here.

Estudio comparativo entre el 18-FDG PET/CT y la técnica de ganglio centinela para la detección de metástasis axilares en cáncer de mama. Resultados preliminares del protocolo Roffo PET/CT 1 / Comparative study between 18-FDG PET/CT and centinela node technique for the detection of axillary metastases in breast cancer. Preliminary results of Roffo PET/CT 1 protocol

Objetivo: comparar al capacidad del PET y del ganglio centinela (GC) para la detección de metástasis (MTS) axilares. Evaluar la sensibilidad, especificidad, valor de predicción positivo y negativo del PET/CT. Criterios de inclusión: cáncer de mama T1 T2 N0. Pacientes con axilas clínicamente dudosas. Criterios de exclusión: carcinoma ductal in situ, carcinoma inflamatorio, biopsia reciente de mama o axila, embarazo o lactancia, diabetes. Método: el 18-FDG PET/CT se realizó 15 días antes de la cirugía. La técnica de ganglio centinela se efectuó con el método combinado (radiosótopos y azul patente). Material: fueron evaluadas 44 pacientes. Edad promedio 58 años (37-79). T1: 29 pacientes (66%); T2: 15 pacientes (34%). Tamaño tumoral entre 5 y 40mm (promedio 19mm). Axila clínicamente negativa: 38 (86%); axila dudosa 6 (14%). Estadio I: 27 pacientes (61%); estadio II: 17 pacientes (39%). Once pacientes tuvieron tumores no palpables (25%). Nueve pacientes (20%) tuvieron biopsias previas. Resultados: treinta y siete (84%) tumores fueron carcinomas ductales infiltrantes; 5 (11%) lobulillares; y 2 (5%) ductolobulillares. Doce pacientes tuvieron MTS en el GC (27%), de éstas 2 casos eran micrometástasis (17%). En 2 pacientes el PET/CT cambió la estadificación, ya que fue positivo para metástasis óseas y pulmonares. Resultados PET en mama: once negativos (FN: 20%). Tres (33%) eran carcinomas lobulillares infiltrantes; 3 eran T1a (33%). Sensibilidad 79% VPP=100%. Resultados PET en axila: en 36 pacientes el PET fue negativo (82%); 4 de éstas presentaron MTS en el ganglio centinela; 2 de ellas eran micrometástasis. Falso negativo: 33%. Sensibilidad: 66%. En 8 pacientes el PET fue positivo (18%) y el ganglio centinela presentaba MTS en todos los casos. Especificidad: 100%. VPP=100%. En 6 pacientes la axila era dudosa, de éstas sólo en una paciente el PET y el GC fueron positivos.

Aporte de 18-FDG PET/CT en el manejo de los estadios localmente avanzados de cáncer de mama. Resultados preliminares / Contribution of 18-FDG PET/CT in the Management of locally advanced breast cancer. Preliminary results

Objetivos: valorar la respuesta a la quimioterapia neoadyuvante en el cáncer de mama localmente avanzado mediante [18F]-FDG-PET/CT. Evaluar la detección de metástasis a distancia en estadios localmente avanzados mediante [18F]-FDG-PET/CT y su correlación con estudios convencionales. Material y métodos: se realizó [18F]-FDG-PET/CT en 26 pacientes antes de efectuar la biopsia de mama y antes del inicio de los tratamientos. En 23 casos se efectuó un segundo PET/CT luego del primer o segundo ciclo de quimioterapia neoadyuvante. Todas las pacientes recibieron el mismo esquema de quimioterapia con antraciclinas más paclitaxel, realizando tres cursos preoperatorios. Veintiún pacientes fueron sometidas a cirugía. Sobre éstas se compararon los hallazgos del PET/CT respecto de la patología. Se valoró el porcentaje de disminución de la SUV en el PET posquimioterapia versus el previo y se correlacionó con los hallazgos patológicos. Se valoró la detección de metástasis mediante este método y su correlación con estudios convencionales de detección de metástasis. Resultados: en 12 casos se objetivó disminución del SUV superior al 50% y en 5/12 esa disminución fue superior al 80%. En estos 5 casos se correlacionó con respuesta patológica completa (1 caso) o enfermedad residual mínima (4 casos) en la pieza operatoria. En los 7 casos restantes se halló respuesta patológica completa en 1 caso y enfermedad macroscópica en 6 casos. En 8 pacientes se observó una disminución del SUV inferior al 50%. Todas ellas mostraron enfermedad residual macroscópica en las piezas operatorias. En 3/23 pacientes se evidenció progresión de la enfermedad mediante PET/CT, ya sea a nivel local/regional o por progresión de metástasis a distancia. De ellas, 1 paciente fue operada y 2 no operadas. La operada mostró enfermedad residual macroscópica. El valor del SUV inicial se correlacionó con la respuesta.