BRCA1 and BRCA2 mutation testing in Cyprus; a population based study.

This paper presents the largest study in Cyprus evaluating the frequency and distribution of BRCA1/2 mutations in a high risk patient cohort. Deleterious mutations in the BRCA1/2 genes were identified in 68 of the 527 patients tested (13%). It is of interest that a quarter of those tested positive, did not have an extensive family history of breast/ovarian cancer but were diagnosed with early onset breast cancer, ovarian cancer under the age of 60 or triple negative breast cancer. The spectrum of mutations identified in our patient cohort is different compared to other Mediterranean countries. Furthermore, several of the mutations detected are novel and have not been identified in other ethnic populations. This highlights the importance of operating a national reference center for cancer genetic diagnosis which offers services tailored to the needs of the Cypriot population.

Marine and terrestrial foods as a source of brain-selective nutrients for early modern humans in the southwestern Cape, South Africa.

Many attempts have been made to define and reconstruct the most plausible ecological and dietary niche of the earliest members of the human species. While earlier models emphasise big-game hunting in terrestrial, largely savannah environments, more recent scenarios consider the role of marine and aquatic foods as a source of polyunsaturated fatty acids (PUFA) and other brain-selective nutrients. Along the coast of southern Africa, there appears to be an association between the emergence of anatomically modern humans and accumulation of some of the earliest shell middens during the Middle Stone Age (200-40 ka). Fragmentary fossil remains classified as those of anatomically modern humans, along with marine food residues and numerous material cultural indicators of increased social and behavioural complexity have been recovered from coastal sites. In this paper, new information on the nutrient content of marine and terrestrial foods available to early modern humans in the southwestern Cape is presented and compared with existing data on the nutritional value of some wild plant and animal foods in Africa. The results suggest that coastal foraging, particularly the collection of abundant and predictable marine molluscs, would have allowed early modern humans to exploit some of the richest and most accessible sources of protein, micronutrients and longer-chain omega-6 and omega-3 fatty acids. Reliable and accessible sources of omega-3 eicosapentaenoic and docosahexaenoic acid are considerably more restricted in terrestrial foods.

Early treatment with glucocorticoids or cyclophosphamide retains the slit diaphragm proteins nephrin and podocin in experimental lupus nephritis.

Renal podocytes and their slit diaphragms ensure the integrity of renal basement membrane and prevent urinary protein loss. We have previously reported that decreases of the podocyte slit diaphragm proteins nephrin and podocin represent early events in the podocytopathy of lupus nephritis (LN). We asked whether immunosuppressive agents such as glucocorticoids and cyclophosphamide may have direct effects on podocytes. We assessed in New Zealand Black/New Zealand White (NZB/W) F1 LN mice glomerular nephrin and podocin expression and localization by the use of Western blot and immunofluorescence; mRNA levels were measured by real-time polymerase chain reaction (PCR) and renal histology by light and electron microscopy. Early treatment with glucocorticoids and cyclophosphamide halted the histologic alterations associated with LN, preserving podocyte foot processes. Nephrin and podocin protein expression significantly increased in both glucocorticoid and cyclophosphamide groups as early as after three months of therapy. Real-time PCR revealed similar enhancement in nephrin and podocin mRNA levels after three to six months of treatment. This study documents that early treatment in experimental LN with glucocorticoids or cyclophosphamide preserves slit diaphragm proteins in podocytes and halts histological changes of the glomeruli, thus raising the possibility of a direct protective effect of these drugs on podocytes.

Human muscle-derived stem cells. Effectiveness in animal models of faecal incontinence. Research scheduling.

Researchers believe that human muscle-derived cells are able to restore leak-point pressure to normal levels by differentiating into new muscle fibres that prevent anal sphincter muscle atrophy. Laboratory data are needed to identify exactly how these cells work to regenerate muscle. The objective of this study is to test whether stem cells can be employed to treat internal anal sphincter (IAS) injuries in humans; to this end, this work will use a two-step process to study: first, the effectiveness of the treatment in a sample of animals with artificial injuries to the IAS and then to verify the results in a population of selected humans affected by pathology.

Podocyte main slit diaphragm proteins, nephrin and podocin, are affected at early stages of lupus nephritis and correlate with disease histology.

Renal podocytes and their slit diaphragms ensure the integrity of the renal basement membrane that forms the barrier to urinary protein loss. A putative disruption of the slit diaphragm and its main protein components, nephrin and podocin, may be implicated in the pathogenesis of lupus nephritis (LN). We studied the glomerular protein expression of nephrin and podocin in NZB/W LN mice by Western blot and immunofluorescence; mRNA levels were measured by real-time PCR. Human kidney biopsies of class II (n = 5), IV (n = 4), V (n = 7) LN were evaluated for nephrin expression by immunohistochemistry. Glomerular protein expression of nephrin and podocin were significantly reduced in NZB/W LN, starting from the earlier stages (mild mesangial LN) and becoming pronounced at advanced histological forms (focal and diffuse proliferative LN). Nephrin and podocin mRNA levels were substantially decreased in diffuse proliferative disease. Decreased expression of both proteins correlated with electron microscopy findings of distorted slit diaphragms. In patients with LN, nephrin was decreased particularly in diffuse proliferative LN. The main slit diaphragm proteins, nephrin and podocin, are affected from the earlier stages of LN and their expression correlates with disease histology. Our findings suggest a novel role of podocytes and their structures in immune-mediated nephritis.

Is pattern III as evidenced by US color-Doppler useful in predicting thyroid nodule malignancy? Large-scale retrospective analysis.

OBJECTIVE: To evaluate whether pattern III of color Doppler Ultra-sound may identify malignant nodules. MATERIALS AND METHODS: We have retrospectively analyzed data pertaining to 1090 patients of both genders (230 males, 860 females), with an average age of 53 years (min 17 years, max 81 years), who underwent thyroidectomy in Department of Surgical Sciences of Sapienza University of Rome since January 2003 through June 2009. We correlated color-Doppler characteristics and histological features through statistical analysis so as to verify statistical correlation between them. RESULTS: Our study showed that 164/273 (60.1%) patients with malignant disease were associated with vascularization pattern III. Regarding benign disease, 152/268 (56.7%) patients showed a pattern of vascularization 3 at ultrasonography. The statistical analysis was not able to show any correlation between pattern III and malignancy. CONCLUSION: Pattern III cannot be used to predict malignancy with confidence, and FNA is still mandatory to rule out the nature of the nodule.

Thyroid tissue remnants after "total thyroidectomy".

Total Thyroidectomy (TT) is a gold standard for benign bilateral pathologies and malignant pathologies of the thyroid. TT has numerous advantages over less radical approaches, such as the resolution of the thyroid pathology, avoidance of recurrences, and improved response to life-long substitutive organotherapy. TT has a negligible rate of recurrence. Near Total Thyroidectomy (NTT) is associated with a low rate of recurrence. Subtotal Thyroidectomy (ST), in which a portion of the thyroid gland is deliberately left in the thyroid lodge, has a considerably higher rate of recurrence. The incidence of complications with TT is similar to that with other techniques of thyroid exeresis. However, despite the radical intent of surgeons, a real TT is not always carried out. The complete removal of all the thyroid tissue employing TT is not the norm and micro/macroscopic remnants almost always remain. The literature on these tissue remnants is often based on techniques that are not very accurate in terms of determining the diameters of the tissue remaining. In our study, conducted by colour echo-doppler of the thyroid lodge in 102 patients who had undergone TT for benign thyroid pathologies, we demonstrated significant thyroid tissue remnants after TT in 34 cases of 102 (33,3%). Therefore, out of a total of 102 so-called "total thyroidectomies", only 68 (66,7%) were really total, whereas 12 patients (11,76%) had near total thyroidectomy, leaving tissue remnants < 1 cm, and 22 patients (21,57%) had subtotal thyroidectomy, with tissue remnants > or = 1 cm.

Contribution of BRCA1 and BRCA2 germline mutations to the incidence of early-onset breast cancer in Cyprus.

In Cyprus, the prevalence of breast cancer associated with BRCA1 and BRCA2 mutations in young women is unknown. In this study, we present the results of mutational analysis of the BRCA1 and BRCA2 genes in 26 Cypriot women diagnosed with breast cancer by the age of 40. The entire coding regions, including splice sites, of the BRCA1 and BRCA2 genes were sequenced using cycle sequencing. We identified four pathogenic mutations: two in BRCA1 [c.1840A>T (K614X), c.5310delG (5429delG)] and two in BRCA2 [c.3531-3534delCAGC (3758del4), c.8755delG (8984delG)] in six of 26 unrelated patients. The BRCA2 mutation c.3531-3534delCAGC (3758del4) is novel and the BRCA1 mutation c.1840A>T (K614X) is reported for the first time in Cypriot patients. The BRCA2 Cypriot founder mutation c.8755delG (8984delG) was detected in three unrelated patients. Additionally, we identified one novel BRCA1 missense mutation, two novel polymorphisms and three novel intronic variants of which BRCA1 c.4185+3A>G (IVS12+3A>G) may be pathogenic. Of the six BRCA1/2 mutation carriers, only four had a family history. These results show that the prevalence of BRCA1 and BRCA2 mutations in Cypriot women diagnosed with early-onset breast cancer is high. We conclude that Cypriot women with early-onset breast cancer should be offered BRCA1/2 testing irrespective of their family history.

Novel germline mutations in the APC gene of Cypriot patients with familial and sporadic adenomatous polyposis.

Familial adenomatous polyposis (FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer. FAP is caused by mutations in the adenomatous polyposis coli (APC) tumour suppressor gene that has a high penetrance. The disease is characterized by the occurrence of hundreds to thousands of colorectal polyps, which if left untreated give rise to colorectal cancer. In Cyprus, there are no molecular data available as yet on families with FAP. This work presents the results of APC analysis in our population for the first time. The APC gene was analyzed in 33 DNA samples from 20 individuals belonging to four FAP families and 13 patients with sporadic polyposis. We identified three truncating mutations, four missense mutations and 11 polymorphisms. It is of interest that two of the three truncating mutations, 2307delA and Q1242X, are novel, which supports the existence of a unique genetic pool in the Cypriot population. This ethnic molecular study in addition to highlighting population heterogeneity also contributes to phenotype-genotype associations that are essential for the clinical management of FAP families in Cyprus.

Mitochondrial encephalomyopathies: a review of routine morphological diagnostic methods with emphasis on the role of electron microscopy.

Mitochondrial encephalomyopathies (MEs) are a group of clinically and genetically heterogeneous diseases. They can be caused by defects in both mitochondrial or nuclear coded genes. Their phenotypic expression is governed by unique biological phenomena such as the dual genetic control, mitotic segregation, heteroplasmy and threshold effects. Currently, the correct diagnosis of ME relies on a multidisciplinary approach which includes clinical information as well as laboratory data from muscle morphology, biochemistry and molecular genetics. Among the morphological methods, histology, histochemistry and electron microscopy were historically instrumental in the diagnosis of MEs. However, with the development of molecular genetics, the diagnostic value of morphology and of electron microscopy in particular have been questioned. The aim of the present review is to present a comparative assessment of the diagnostic contribution of histology, histochemistry and electron microscopy in a group of 48 patients with a diagnosis of ME.

Morphological methods in the diagnosis of mitochondrial encephalomyopathies: the role of electron microscopy.

Mitochondrial encephalomyopathies (MEs) encompass a heterogeneous group of disorders that frequently present a diagnostic challenge to clinicians. Historically, MEs were diagnosed by finding ragged red fibers in the muscle biopsy and confirmatory evidence was provided by the presence of numerical and/or ultrastructural abnormalities in mitochondria. In most centers diagnosis involves clinical evaluation and the morphological, histochemical, and biochemical investigation of a skeletal muscle biopsy. However, with the availability of mitochondrial DNA analysis, the necessity and role of morphological methods and, in particular, electron microscopy has been questioned. The aim of this study was to delineate the role of electron microscopy in the diagnosis of MEs.

A simplified method for measuring the thickness of glomerular basement membranes.

Measurement of the thickness of glomerular basement membranes is required for the diagnosis of thin membrane nephropathy. Over the years various morphometric methods have been used but some are laborious so there is a need for establishing a simplified method for measuring thickness. In the present study 20 renal biopsies were used to carry out a comparative morphometric analysis between 2 methods. The first method was based on measuring thickness at the maximum number of available points, whereas for the second method, thickness was measured at only 5 points per loop. Since both methods gave mean values that are not statistically different in each patient, the authors recommend that the simplified method be used routinely for diagnosis.

A comparative morphological study in 33 cases of respiratory chain encephalomyopathies.

Mitochondrial encephalomyopathies (ME) are clinically and genetically heterogeneous syndromes ranging from pure myopathies to complex multisystem disorders. This phenotypic and genotypic variability, coupled with the lack of a laboratory gold standard marker for the diseases, makes diagnosis a challenging process. Mitochondrial DNA analysis and biochemical assay of muscle homogenates are quite specific diagnostically but have low sensitivity in unselected cases suspected of ME. We decided to evaluate four routine morphological methods in 33 cases of definite or probable ME in an effort to assess the reliability of each of these techniques in diagnosing ME.

Correlation between morphology, immunohistochemistry and molecular pathology in hereditary and sporadic breast cancer cases.

Breast cancer still represents a serious health problem and is currently the most frequent malignancy in the female population in developed countries. In Cyprus, there are 300 new cases annually. In the present study, histology, electron microscopy, immunohistochemistry, and Western blot analysis were used to investigate 100 cases of invasive breast carcinoma. In addition, mutation analysis for the BRCA1 gene was carried out in patient DNA from 26 families with multiple cases of breast/ovarian cancers. Of note are the results of molecular biology which show that there are no germline truncating mutations in the BRCA1 gene in these 26 Cypriot breast cancer families. Furthermore, Western blot analysis revealed the presence of multiple BRCA1 bands in homogenates of tumor and normal tissues, and immunoelectron microscopy showed the presence of nuclear staining for BRCA1 antibodies.

Amyloid myopathy: evidence for mechanical injury to the sarcolemma.

Myopathy is a rare clinical manifestation in primary systemic amyloidosis. The clinical phenotype and muscle histology are well described but the pathophysiological mechanisms remain poorly understood. We report a 40-year-old man who presented with hypertrophic cardiomyopathy and a limb girdle syndrome associated with deposition of amyloid and free lambda light chains in skeletal muscle. Electron microscopy showed amyloid fibrils, physically disrupting the plasma membrane and basal lamina, while laminin immunocytochemistry revealed a reduction of laminin beta1 and upregulation of laminin alpha1. We believe that one of the possible pathophysiological mechanisms in amyloid myopathy is mechanical disruption of the sarcolemma by the abutting amyloid fibrils.

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction.

The spectrum of the beta-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 beta-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (-A) and IVS-I-129 (A-->C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found to coexist in one beta-globin gene: Sri Lankan patients homozygous for the beta0 codon 16 (-C) frameshift were also homozygous for the beta+ codon 10 (C-->A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C-->A) mutation is just a rare polymorphism on an ancestral allele, on which the beta0 codon 16 (-C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the beta-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

BRCA1 germline mutations in Cypriot breast cancer patients from 26 families with family history.

Germline mutations in the BRCA1 gene are causative for a variable number of hereditary breast/ovarian cancers. The data presented in this study are based on genetic analysis of the BRCA1 gene in 49 DNA samples from breast cancer patients with a positive family history. A combination of manual direct DNA sequencing and SSCP analysis was used to screen the entire coding region of BRCA1. Overall 13 variants were detected which included 5 missense mutations, 3 polymorphisms and 5 intronic changes. Further genetic analysis of the 13 variants was carried out using 50 control DNA samples. Our results showed that 12 out of the 13 variants detected in the DNA of the patients group, were also present in the control group. It appears that the Greek Cypriot families studied so far have an unexpectebly low frequency of deleterious mutations in the BRCA1 gene. This is the first report on BRCA1 mutation analysis in Cyprus.