Unveiling the genetic etiology of primary ciliary dyskinesia: When standard genetic approach is not enough

PurposePrimary ciliary dyskinesia (PCD) is a ciliopathy caused by dysfunction of motile cilia. As there is still no standard PCD diagnostics, the final diagnosis requires a combination of several tests. The genetic screening is a hallmark for the final diagnosis and requires high-throughput techniques, such as whole-exome sequencing (WES). Nevertheless, WES has limitations that may prevent a definitive genetic diagnosis. Here we present a case that demonstrates how the PCD genetic diagnosis may not be trivial.Materials/methodsA child with PCD and situs inversus totalis (designated as Kartagener syndrome (KS)) was subjected to clinical assessments, ultrastructural analysis of motile cilia, extensive genetic evaluation by WES and chromosomal array analysis, bioinformatic analysis, gene expression analysis and immunofluorescence to identify the genetic etiology. His parents and sister, as well as healthy controls were also evaluated.ResultsHere we show that a disease-causing variant in the USP11 gene and copy number variations in CRHR1 and KRT34 genes may be involved in the patient PCD phenotype. None of these genes were previously reported in PCD patients and here we firstly show its presence and immunolocalization in respiratory cells.ConclusionsThis work highlights how the genetic diagnosis can turn to be rather complex and that combining several approaches may be needed. Overall, our results contribute to increase the understanding of the genetic factors involved in the pathophysiology of PCD/KS, which is of paramount importance to assist the current diagnosis and future development of newer therapies.     

Quick and reliable galactomannan detection in crude minced lung specimens from haematological patients with suspected invasive fungal infection: results from a case series

Invasive aspergillosis (IA) is the leading direct or contributory cause of death in patients with haematological malignancies. Early diagnosis remains difficult and often elusive due the heterogeneity of clinical presentations and the low sensitivity of both histological examination and cultures of specimens obtained from patients at risk. We report two cases of IA, both of which lacked both histological and cultural evidence of IA from pulmonary specimens. In both patients, detection of galactomannan (GM) by enzyme immunoassay (EIA) on pulmonary tissue homogenates led to the diagnosis of IA, which was confirmed by Aspergillus DNA (real time PCR). In conclusion, we provide preliminary evidence that lung homogenates may be prepared for GM EIA assays, which may contribute to quick diagnosis of IA on otherwise negative samples. We feel that our results open up the opportunity of a prospective and comparative evaluation of this diagnostic technique.     

Differential diagnosis of pheochromocytomas and paragangliomas

Paragangliomas are of two types, sympathetic and parasympathetic, depending on the type of paraganglion in which they arise. The term pheochromocytoma is reserved for tumors arising in the adrenal medulla. These tumors are usually fairly easy to diagnose. However, several areas are the subject of debate, including the identification of malignant potential, the diagnosis of medullary hyperplasia, and the recognition of composite tumors. Some histologic features can cause problems in differential diagnosis. Paragangliomas may have spindle cell morphology or contain pigment, requiring distinction from mesenchymal tumors and melanoma, respectively. Extensive degenerative change in phenochromocytomas may mimic adrenal cortical tumors. This short review addresses the diagnosis of pheochromocytomas and paragangliomas and discusses useful approaches in the aforementioned problem areas.     

Gas-liquid chromatography in microbiological diagnosis

With gas-liquid chromatography, it is possible to detect chemical compounds which are specific of micro-organisms: either metabolic products, or structural components. This technique permits to simplify the diagnosis: it is the case for anaerobic bacteria, where the analysis of the fermentation acids produced in a 48 hours liquid culture enables to rapidly establish the diagnosis, at least as far as the genus is concerned. It may also increase the accuracy of the diagnosis by searching such or such metabolite or structural component which are specific of a species or a sub-species. Some authors have already determined automatic systems of bacterial identification; these systems are based on a computer analysis of the chromatographic profile of cellular fatty acids. Finally it is important to emphasize that liquid-gas chromatography may markedly shorten the delay of identification by allowing, in some cases, a direct diagnosis in pathological samples.     

A qualitative exploration of mothers' and fathers' experiences of having a child with Klinefelter syndrome and the process of reaching this diagnosis

Klinefelter syndrome (KS) is a common genetic condition that is currently under-diagnosed. The phenotype is broad, with physical, medical and psychosocial features ranging from mild to severe. When a child is diagnosed with KS, the parents may spend months to years searching for a diagnosis. This study used a qualitative methods approach to explore parents'' experiences of having a child with KS and receiving a diagnosis. Fifteen semistructured one-to-one in-depth interviews were conducted to explore their experiences and views. The interviews were then transcribed, coded and thematically analysed. The interviews revealed that parents had diverse experiences related to: the timing of the diagnosis of their child and reasons why their child was investigated for KS; the information that was provided at the time of diagnosis; the supports that were available and the concerns that parents held for the future of their child. The conclusions from this study were that parents'' experiences of having a child with KS and receiving a diagnosis were complex and multifaceted. This experience was shaped by the timing of when the diagnosis was received, who provided the diagnosis, what information was provided from health-care professionals and that which parents may have encountered on the internet. The long-term experiences for parents were also impacted by the level of support they received. These findings have implications for the process by which KS is recognised by the health-care community and supports available for families.     

Diagnostic des maladies à prions humaines : quel rôle pour le biologiste ?

Human prion diseases are rare neurodegenerative diseases, due to proteinaceous infectious particles, named prions. The most frequent of these rare diseases is Creutzfeldt-Jakob disease, which can be sporadic, inherited or acquired (iatrogenic or variant). The diagnosis is based on the post-mortem examination of the brain. During the life of the patient, neuronal markers may be detected in cerebro-spinal-fluid, the prion protein gene PRNP may be screened for pathogenic mutations linked to inherited prion disease forms, and the pathogenic prion protein may be evidenced in the tonsils of patients affected with the variant form of the disease. Research are in progress in order to improve the differential diagnosis of these diseases.     

Impotence in diabetes: An overview

Diabetes mellitus is the leading cause of organic impotence; upwards of 40% of diabetic men suffer from impotence. The author describes the various factors and mechanisms involved in diabetic impotence and discusses the secondary psychological reactions that may develop. Guidelines are given for diagnosis (including a discussion of the special laboratory studies that may be required) and treatment (which is dictated by the partic ular diagnosis).     

Development of touch down-multiplex PCR for the diagnosis of toxoplasmosis

Purpose: The diagnosis of toxoplasmosis is challenging since conventional methods like culture and immunofluorescence are not universally available. Serology, which is used regularly might be negative during early phase of infection and in immunosuppressed patients or may remain positive for a long time. Several molecular tests have been used for the diagnosis of toxoplasmosis, but none of them have an internal control which would inform us regarding the presence of polymerase chain reaction (PCR) inhibitors thus, undermining the confidence of a laboratory physician. Materials and Methods: We designed a multiplex PCR containing primers targeting human beta globin gene which would act as internal control and two primers against the B1 gene and 5s gene which aid in sensitive detection of T. gondii. Results: Multiplex PCR had a sensitivity of 83.3% and specificity of 100%. Conclusion: Multiplex PCR may provide a sensitive and specific tool for diagnosis of human toxoplasmosis.     

Gene technology from the viewpoint of genetic counseling

Gentechnology detects genetic defects at the DNA level. Direct analysis, which may be performed without family investigations, is the most reliable and therefore the most desirable means of detection. Indirect analysis on the other hand-using restriction fragment length polymorphisms (RFLP) - requires family investigations and the pedigrees are not always informative; furthermore, meiotic recombination may occur leading to erroneous conclusions. Pre-symptomatic diagnosis of a severe disease may cause serious psychical and ethical problems. Prenatal diagnosis by gentechnology may be made after amniocentesis or chorionic villi sampling.     

Metastatic meningioma detected in pleural fluid

Meningiomas with both malignant cytologic features and clinical behavior are rare. A 39-yr-old man with recurrent meningioma developed a pleural effusion which, on cytologic examination, contained metastatic meningioma. The diagnosis was subsequently confirmed histologically and ultrastructurally. In conclusion, metastatic meningiomas can retain meningotheliomatous cytologic features which may allow a diagnosis to be made of clinically unanticipated tumor spread. Diagn. Cytopathol. 1998;18:453–457. © 1998 Wiley-Liss, Inc.     

Hemangioma of the right atrium: imaging and pathology

BackgroundCardiac hemangiomas are benign neoplasms which have been reported to appear as well-circumscribed, homogenous, enhancing masses at imaging.Methods and resultsWe report a 49-year-old woman with a cardiac hemangioma detected by echocardiography, computed tomography, and magnetic resonance imaging. The multiple imaging modalities showed features which have been reported in cardiac hemangiomas. The tumor was surgically excised and the diagnosis of cardiac hemangioma was made.ConclusionsCardiac hemangiomas are rare tumors with a variety of imaging features which may suggest the diagnosis.     

Endobronchial tuberculosis: an overview

Endobronchial tuberculosis (EBTB), of which the incidence has been increasing in recent years, is a special type of pulmonary tuberculosis. The endobronchial tuberculose focuses often injure the tracheobronchial wall and lead to tracheobronchial stenosis. The tracheobronchial stenosis may cause intractable tuberculosis and make patients become chronic infection sources of tuberculosis, or may even cause pulmonary complications and result in death. The etiological confirmation of Mycobacterium tuberculosis is most substantial for diagnosis. However, because the positive rate of acid-fast bacillus staining for sputum smears is low and the clinical and radiological findings are usually nondistinctive, the diagnosis of EBTB is often mistaken and delayed. For early diagnosis, a high index of awareness of this disease is required and the bronchoscopy should be performed as soon as possible in suspected patients. The eradication of Mycobacterium tuberculosis and the prevention of tracheobronchial stenosis are two most substantial treatment goals. To get treatment goals, the diagnosis must be established early and aggressive treatments must be performed before the disease progresses too far.     

Sarcoidosis: histopathological definition and clinical diagnosis.

Sarcoidosis is best defined in histopathological terms as 'a disease characterised by the presence in all of several affected organs and tissues of non-caseating epithelioid-cell granulomas, proceeding either to resolution or to conversion into hyaline connective tissue'. Although the defining characteristics are thus histopathological, diagnosis during life depends largely upon clinical, radiological, and immunological findings. The amount of support required from histology varies greatly from case to case. Though histology from one site cannot in itself establish the diagnosis of sarcoidosis, a generalised disease, detailed histological study of biopsy tissue makes an important and often essential contribution. In many instances, complete lack of necrosis, an intact reticulin pattern, and failure to demonstrate infective agents permit an unequivocal statement of compatibility with this diagnosis; however, a compatible clinical picture and absence of evidence of known causes of local granulomatous reactions or of other generalised granulomatous diseases are required for definitive diagnosis. In some, the histological pattern deviates in some particular from the accepted 'typical' pattern; there may be a little necrosis, the follicular pattern of the granuloma may be less than perfect, and exclusion of known infective agents can never be absolute. In such instances, subsequent surveillance, including possible response to treatment, may show a clinical course justifying a diagnosis of sarcoidosis, and necropsy may establish it; but it must be recognised that in a few cases, particularly those in which the clinical evidence of disease is confined to one organ, diagnosis is likely to remain in doubt for long periods. Reports on the histology of the Kveim test should be made without knowledge of clinical findings and in terms of the presence and quality of granulomatous response. A granulomatous reaction to a validated test suspension makes a contribution to diagnosis similar to the finding of granulomas in an additional organ or tissue.     

Sarcoma and Look-Alikes: The Important Role of Ultrastructural Evaluation

Due to the variable light microscopic appearance of sarcomas, a large number of neoplasms may need to be considered in the differential diagnosis in some situations. In difficult cases, the surgical pathologist must approach the differential diagnosis using ancillary diagnostic techniques and should do so in an orderly fashion, recognizing that arriving at the correct diagnosis is without a doubt the most important goal. It is also imperative to take into account the expense and time that may be involved in arriving to the definitive diagnosis, which could influence how the workup is conducted. Therefore, the most reasonable route to address the differential diagnosis in order to make a final solid diagnostic assessment should be taken. Whether immunohistochemistry, electron microscopy, cytogenetics, and/or molecular diagnostics should be employed in a given case becomes the dilemma. In some instances a combination of the above-mentioned techniques is important not only to obtain an unequivocal diagnosis, but also to provide important additional information regarding prognosis and to facilitate the patient's management.     

Diagnosis of Huntington disease: A model for the stages of psychological response based on experience of a predictive testing program

Persons diagnosed as affected with Huntington's disease (HD) may have similar stages of psychological response to the clinical presentation of the illness. Here we describe a model of these stages of response based on our experience during a predictive testing program for HD. During the warning Stage, asymptomatic persons are aware of their risk status for HD and develop defenses which favor adaptation to their genetic risk. In response to the initial signs and symptoms of HD (the Incipient Stage) unconscious working through of this realization occurs while it is still kept out of conscious awareness. When symptoms become obvious such that recognition of disease onset is inevitable (Break-through Stage) the possibility of the diagnosis of HD is assimilated. After the delivery of the diagnosis during the Adjustment Stage, short-and long-term adaptive responses to living with HD occur. Recognition of the stage of psychological response of a patient who presents with HD is important prior to delivering a clinical diagnosis. In a significant minority of cases, the psychological readiness lags behind the clinical symptomatology and premature presentation of diagnosis may result in significant untoward adverse events. Understanding of the stages of response may provide a frame-work for evaluating the psychological state of the person with HD and determining their readiness to receiving the diagnosis. This model may have relevance to the psychological responses of patients to the diagnosis of other late onset autosomal dominant disorders. © 1993 Wiley-Liss, Inc.     

Ubiquitin as a marker of cell injury in nonalcoholic steatohepatitis

Ubiquitin (UB), an intracellular protein that binds to other proteins to target them for proteolysis, is associated with Mallory hyalin (MH), which supports a biopsy diagnosis of nonalcoholic steatohepatitis (NASH). We analyzed 54 liver biopsy specimens from 49 patients with a clinical diagnosis of NASH for immunoreactive UB and multiple features of necroinflammation, fibrosis, and Prussian blue-positive iron to determine whether the presence of immunoreactive UB increases detection of MH or correlates with other features of cell injury or mutations of the HFE gene. MH and UB were graded. Analysis for HFE gene mutations was performed in 48 patients. Biopsy diagnoses were distributed as follows: NASH, 42; steatosis, 10; and nonspecific changes, 2. UB was present in 20 specimens and MH in 23. Of 31 specimens with 0 MH, 6 had UB; of 14 with 1 + (questionable) MH, 7 had 1+ or 2+ UB. UB correlated positively and significantly with the diagnosis and grade of NASH, presence of MH, cell swelling, lobular inflammation, and fibrosis. Immunostaining for UB may enhance detection of MH in questionable cases, support the diagnosis of NASH, and indicate which patients may be at risk for progression of disease.     

Intestinal anisakidosis: Histopathological findings and differential diagnosis

Anisakidosis is a human zoonotic disease caused by the ingestion of raw or undercooked or not adequately salted, pickled, or smoked fish containing larval nematodes of the Anisakis species. The incidence of this infection is higher in geographical zones with traditional consumption of raw fish. However, in the last years, prevalence raised in low risk zones due to the increase popularity of Asian cuisine. According to where the larvae settle in the gastrointestinal tract, anisakidosis may have different clinical symptoms. In particular, intestinal anisakidosis may mimic several surgical conditions, including appendicitis, ileitis, diverticulitis or inflammatory bowel disease. For this reason, diagnosis is often histopathological. In the present paper, we describe the clinico-pathological findings of six novel cases of intestinal anisakidosis occurred in southern Italy, and provide clues for the differential diagnosis toward Crohn's disease and eosinophilic enteritis, which have similar histopathological characteristics. Awareness of the existence of intestinal anisakidosis may facilitate its recognition and correct diagnosis, which is of fundamental importance for appropriate therapeutic approach.     

Number of levels needed for diagnosis of cervical biopsies

CONTEXT: Three levels of histologic sections are routinely prepared for small biopsies in many surgical pathology laboratories. The first level is superficial and may not be representative of the entire biopsy, and may therefore represent wasted resources and time for technologists and pathologists alike. OBJECTIVE: To determine if disposing of the first of 3 standard levels materially affects the diagnosis of cervical biopsies. DESIGN: We retrospectively reviewed levels 2 and 3 of 241 cervical biopsies and compared the review diagnosis with the original diagnosis, using 6 diagnostic categories: I, benign lesions; II, human papillomavirus-associated changes or low-grade dysplasia; III, high-grade dysplasia/carcinoma in situ; IV, invasive carcinoma; V, insufficient tissue for diagnosis; and VI, further workup needed. If there was a discrepancy between the original and review diagnostic categories, then we examined level 1 to determine if this would resolve the disagreement. SETTING: The surgical pathology laboratory. PATIENTS: Women undergoing cervical biopsy. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The frequency with which level 1 information changed the diagnostic category determined with levels 2 and 3 only. RESULTS: After review of levels 2 and 3, the diagnosis in 42 (17%) of 241 cases was in disagreement with the original diagnosis. Upon review of level 1, the review category was changed to that of the original diagnosis in only one case. CONCLUSIONS: The first of 3 levels contributed little to reaching a diagnosis in these cervical biopsies. Control of interobserver variation would seem to be superior to preparation of additional levels as a strategy for reducing diagnostic error.     

Gene expression profiling of oral squamous cell carcinoma using laser microdissection and cDNA microarray

Cancer diagnosis and therapy are performed on the basis of clinical stage and clinicopathological findings; however, sensitivity to therapy and prognosis may not always be the same even when considering similar cancers because it is difficult to recognize adequate biological characteristics of a cancer when determining cancer therapy. To enable personalized medicine for cancer diagnosis and therapy, which may solve this problem, we used laser microdissection and cDNA microarrays to study the gene expression profile of oral squamous cell carcinoma. Moreover, to establish an objective evaluation with this system, we examined which type of gene expression profile corresponded to the biological characteristics of this cancer. We identified several genes that were up- or downregulated in the majority of cases and clarified genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases. It was suspected that the genes that were commonly up- or downregulated in the majority of cases were important for histogenesis and acquisition of invasion and proliferation capability and that the genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases played a large role in cancer metastasis. Using the expression profile of these genes, it may be possible to evaluate cellular state and metastatic potential and use them as tumor markers. Alternately, we showed averaged gene expression profiles in cases with or without metastasis; this may reveal a profile that could evaluate metastatic potential, which is an important element in the biological characteristics of cancer. In conclusion, our system using laser microdissection and cDNA microarray may contribute to cancer diagnosis and therapy and improvement in the quality of life of cancer patients.