Diagnostic dilemmas in Fabry disease: a case series study on GLA mutations of unknown clinical significance.

Fabry disease' (FD) phenotype is heterogeneous: alpha-galactosidase A gene mutations (GLA) can lead to classical or non-classical FD, or no FD. The aim of this study is to describe pitfalls in diagnosing non-classical FD and assess the diagnostic value of plasma globotriaosylsphingosine. This is a case series study. Family 1 (p.A143T) presented with hypertrophic cardiomyopathy (HCM), absent classical FD signs, high residual alpha-galactosidase A activity (AGAL-A) and normal plasma globotriaosylsphingosine. Co-segregating sarcomeric mutations were found. Cardiac biopsy excluded FD. In family 2 (p.P60L), FD was suspected after kidney biopsy in a female with chloroquine use. Males had residual AGAL-A, no classical FD signs and minimally increased plasma globotriaosylsphingosine, indicating that p.P60L is most likely non-pathogenic. Non-specific complications and histology can be explained by chloroquine and alternative causes. Males of two unrelated families (p.R112H) show AGAL-A <5%, but slightly elevated plasma globotriaosylsphingosine (1.2-2.0 classical males >50 nmol/l). Histological evidence suggests a variable penetrance of this mutation. Patients with GLA mutations and non-specific findings such as HCM may have non-classical FD or no FD. Other (genetic) causes of FD-like findings should be excluded, including medication inducing FD-like storage. Plasma globotriaosylsphingosine may serve as a diagnostic tool, but histology of an affected organ is often mandatory.

Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance.

BACKGROUND: Screening in subjects with left ventricular hypertrophy (LVH) reveals a high prevalence of Fabry disease (FD). Often, a diagnosis is uncertain because characteristic clinical features are absent and genetic variants of unknown significance (GVUS) in the α-galactosidase A (GLA) gene are identified. This carries a risk of misdiagnosis, inappropriate counselling and extremely expensive treatment. We developed a diagnostic algorithm for adults with LVH (maximal wall thickness (MWT) of >12 mm), GLA GVUS and an uncertain diagnosis of FD. METHODS: A Delphi method was used to reach a consensus between FD experts. We performed a systematic review selecting criteria on electrocardiogram, MRI and echocardiography to confirm or exclude FD. Criteria for a definite or uncertain diagnosis and a gold standard were defined. RESULTS: A definite diagnosis of FD was defined as follows: a GLA mutation with ≤ 5% GLA activity (leucocytes, mean of reference value, males only) with ≥ 1 characteristic FD symptom or sign (neuropathic pain, cornea verticillata, angiokeratoma) or increased plasma (lyso)Gb3 (classical male range) or family members with definite FD. Subjects with LVH failing these criteria have a GVUS and an uncertain diagnosis. The gold standard was defined as characteristic storage in an endomyocardial biopsy on electron microscopy. Abnormally low voltages on ECG and severe LVH (MWT>15 mm) <20 years exclude FD. Other criteria were rejected due to insufficient evidence. CONCLUSIONS: In adults with unexplained LVH and a GLA GVUS, severe LVH at young age and low voltages on ECG exclude FD. If absent, an endomyocardial biopsy with electron microscopy should be performed.

A novel lamin A/C mutation in a Dutch family with premature atherosclerosis.

OBJECTIVE: We report a novel lamin A/C (LMNA) mutation, p.Glu223Lys, in a family with extensive atherosclerosis, diabetes mellitus and steatosis hepatis. METHODS: Sequence analysis of LMNA (using Alamut version 2.2), co-segregation analysis, electron microscopy, extensive phenotypic evaluation of the mutation carriers and literature comparison were used to determine the loss of function of this mutation. RESULTS: The father of three siblings died at the age of 45 years. The three siblings and the brother and sister of the father were referred to the cardiovascular genetics department, because of the premature atherosclerosis and dysmorphic characteristics observed in the father at autopsy. The novel LMNA mutation, p.Glu223Lys, was identified in the proband and his two sons. Clinical evaluation revealed atherosclerosis, insulin resistance and hypertension in the proband and dyslipidemia and hepatic steatosis in all the patients with the mutation. CONCLUSION: Based on the facts that in silico analysis predicts a possibly pathogenic mutation, the mutation co-segregates with the disease, only fibroblasts from mutation carriers show nuclear blebbing and a similar phenotype was reported to be due to missense mutations in LMNA we conclude that we deal with a pathogenic mutation. We conclude that the phenotype is similar to Dunnigan-type familial partial lipodystrophy.

Familial LCAT deficiency: from renal replacement to enzyme replacement.

Familial LCAT deficiency (FLD) is a recessive lipid disorder ultimately leading to end-stage renal disease (ESRD). We present two brothers with considerable variation in the age at which they developed ESRD. Kidney biopsies revealed both tubular and glomerular pathology. To date, no causal therapy is available, yet enzyme replacement therapy is in development.

Analysis of placental tissue in Fabry disease with and without enzyme replacement therapy.

There are only a few reports on the histology of placental tissue of pregnancies from mothers with Fabry disease. Fabry disease is a lysosomal disorder caused by alpha-galactosidase A deficiency. Extensive glycosphingolipid (GSL) accumulation in fetal and maternal placenta tissue obtained from a Fabry mother and her affected male newborn has previously been reported. Here we report the evaluation of placenta tissue of two pregnancies in Fabry mothers, one of an unaffected male newborn (placenta A) and one of an affected female newborn (placenta B). The mother of the female affected offspring was treated with recombinant alpha-galactosidase A (enzyme replacement therapy, ERT) during the pregnancy (placenta B). Storage material was only detected in smooth muscle cells of the umbilical cord of placenta B. No accumulation was seen in both placentae. Combing these results with the outcome in two earlier described placentae, a heterogeneous picture emerges. This may be due to differences in disease severity in the mothers or severity of disease in their offspring. In addition, a possible effect of ERT on placental GSL accumulation could also explain lack of GSL storage in placenta B.

Pulsatile perfusion preservation of warm ischaemia-damaged experimental kidney grafts.

BACKGROUND: Cold storage using histidine-tryptophan-ketoglutarate (HTK) solution is used widely in clinical practice for the preservation of warm ischaemia-damaged kidney grafts. This study assessed the efficacy of pulsatile machine perfusion in combination with Polysol for the preservation of warm ischaemia-damaged kidney grafts. METHODS: After induction of warm ischaemia by clamping of the left renal pedicle for 30 min, pigs were subjected to left nephrectomy. Thereafter, grafts were preserved for 20 h by cold storage with HTK (CS-HTK) or Polysol (CS-PS), or machine preservation with Polysol (MP-PS). Subsequently, contralateral kidneys were removed and preserved kidneys were transplanted. Control pigs underwent unilateral nephrectomy. Renal function was assessed daily for 1 week. Kidney biopsies were analysed for morphology and proliferative response. RESULTS: Renal function of warm ischaemia-damaged grafts preserved using MP-PS was comparable to that of non-ischaemic controls. MP-PS and CS-PS groups showed improved renal function compared with the CS-HTK group, with more favourable results for MP-PS than for CS-PS. The proliferative response of tubular cells in the CS-HTK group was higher than in all other groups. CONCLUSION: This study demonstrated that the function of warm ischaemia-damaged kidney grafts after pulsatile perfusion preservation was comparable to that of non-ischaemic controls.

[Neonatal respiratory distress caused by primary ciliary dyskinesia]. / Neonatale respiratoire problemen door primaire ciliaire dyskinesie.

Two newborns, both boys, presented with unexplained respiratory distress. One developed recurrent pneumonias in the first neonatal week and was diagnosed with primary ciliary dyskinesia at the age of 2.5 years. The other had respiratory problems besides a situs inversus totalis and was diagnosed with primary ciliary dyskinesia in the neonatal period. Although 65-90% of children with primary ciliary dyskinesia present with neonatal respiratory distress, the disease is often diagnosed after a considerable delay. Primary ciliary dyskinesia should be considered in newborns with unexplained respiratory problems and in children with recurrent respiratory problems. The disease is diagnosed by taking a nasal brush biopsy of the cilia and examining it using electron microscopy or using phase contrast microscopy. Early diagnosis and adequate treatment may prevent further lung damage.

The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females.

BACKGROUND/AIMS: The mouse mdr2 gene encodes a P-glycoprotein expressed in the canalicular membrane of the hepatocyte. Mice in which this gene has been inactivated (mdr2 -/-) show a defect in biliary phospholipid and cholesterol secretion and develop non-suppurative cholangitis. We hypothesized that secretion of bile salts without lipids initiates this liver disease. METHODS: To delineate the pathologic process, mdr2 (-/-) mice were fed different bile salt-supplemented diets for 22 weeks after weaning. Aspects of liver pathology including eosinophilic bodies, portal inflammation, ductular proliferation, mitotic activity and fibrosis were semi-quantitatively scored. RESULTS: It was observed that liver pathology was more severe in female than in male mice when fed a purified control diet. This correlated with a more hydrophobic bile salt composition of female vs. male bile. When increasing amounts of cholate were added to the diet (0.01% and 0.1%), the secretion of taurocholate increased and this was accompanied by a more severe liver pathology. At the high dose of cholate (0.1%), the bile salt compositions of male and female mice became similar, as did the severity of the histological score. Addition of cholate to the diet did not induce liver pathology in (+/+) mice. Addition of ursodeoxycholate to the diet (0.5%) led to a near complete replacement of biliary bile salts by tauroursodeoxycholate and this reduced pathology and dissipated the difference between males and females. CONCLUSIONS: These observations support our hypothesis that liver pathology in the mdr2 (-/-) mouse is caused by bile salts and depends on the hydrophobicity c.q. cytotoxicity of biliary bile salts.

Effects of Ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene.

BACKGROUND & AIMS: The mouse mdr2 gene encodes a P-glycoprotein expressed in the hepatocanalicular membrane. Inactivation of this gene causes lack of biliary phospholipid and cholesterol secretion and non-suppurative cholangitis. The aim of this study was to investigate the role of bile salt hydrophobicity in induction of liver pathology in mdr2 (-/-) mice. METHODS: Mice (+/+) wild type or (-/-) knockout for the mdr2 gene were fed with either purified control diet or this diet supplemented with cholate (0.1%) or ursodeoxycholate (0.5%) for 3, 6, or 22 weeks after weaning. Liver histology was semiquantitatively scored. RESULTS: Each mouse fed bile acid became the major constituent of the bile salt pool. The cholate diet during 22 weeks induced only very mild liver pathology in (+/+) mice. By contrast, lever histology had already deteriorated after 3 weeks in the (-/-) mice and caused pronounced inflammatory nonsuppurative cholangitis and fibrosis in the 75% of mice that survived. Dietary ursodeoxycholate had no effect on histology in (+/+) mice but improved liver pathology significantly in (-/-) mice compared with purified control diet; the decrease of ductular proliferation and portal inflammation was most prominent after 22 weeks. CONCLUSIONS: The cholangiolitis and its sequelae in the mdr2 knockout mice depend on bile salt hydrophobicity.

Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.

The mouse mdr2 gene (and its human homologue MDR3, also called MDR2) encodes a P-glycoprotein that is present in high concentration in the bile canalicular membrane of hepatocytes. The 129/OlaHsd mice with a homozygous disruption of the mdr2 gene (-/- mice) lack this P-glycoprotein in the canalicular membrane. These mice are unable to secrete phospholipids into bile, showing an essential role for the mdr2 P-glycoprotein in the transport of phosphatidylcholine across the canalicular membrane. The complete absence of phospholipids from bile leads to a hepatic disease, which becomes manifest shortly after birth and shows progression to an end stage in the course of 3 months. The liver pathology is that of a nonsuppurative inflammatory cholangitis with portal inflammation and ductular proliferation, consistent with toxic injury of the biliary system from bile salts unaccompanied by phospholipids. Thus, the mdr2 (-/-) mice can serve as an animal model for studying mechanisms and potential interventions in nonsuppurative inflammatory cholangitis (in a generic sense) in human disease, be it congenital or acquired. When the mice are 4 to 6 months of age, preneoplastic lesions develop in the liver, progressing to metastatic liver cancer in the terminal phase. The mdr2 (-/-) mice therefore also provide a tumor progression model of value for the study of hepatic carcinogenesis. Interestingly, also in this regard, the model mimicks human disease, because chronic inflammation of the biliary system in humans may similarly carry increased cancer risk.

Septata intestinalis frequently isolated from stool of AIDS patients with a new cultivation method.

Two species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.

Developmental stages in experimental liver metastases: relation to invasiveness.

We have previously reported that an invasive morphotype can be evoked in a rat colon carcinoma by transplanting it into pre-induced subcutaneous granulation tissue. We have now studied the interaction of the same tumor with liver tissue, which is extremely poor in connective tissue in comparison with the subcutaneous site. Tumor cells were injected into the portal system and the resulting experimental liver metastases were examined by electron microscopy and immunohistochemistry. Early metastases consisted of well-differentiated acini, fully surrounded by connective tissue that was derived from the periportal stroma. In a later stage, this connective tissue was overgrown by tumor cells and, almost immediately, acinar differentiation was lost. Most metastases eventually reached the liver capsule, which reacted by forming a layer of granulation tissue. Only in this layer, we observed invasion by thin tumor cell strands, which were often intimately associated with fibroblasts or with blood capillaries. The tumor cells remained smooth and rounded during this process. After fully penetrating the granulation tissue, the tumor cell strands reached the liver surface, where they formed poorly structured papillary masses that were nearly devoid of stroma. Our observations indicate that, even in a relatively homogeneous organ like the liver, the tumor-host interaction is highly complex and dynamic. They also confirm the notion that granulation tissue stimulates tumor invasiveness. Finally, they show that tumor cells can actively invade host tissues without exhibiting a "fibroblastic" morphology.

Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique.

AIMS: To assess the value of a new rapid fluorescence method for the diagnosis of microsporidiosis in HIV seropositive patients. METHODS: Microsporidian spores in stools were demonstrated by using the fluorochrome stain Uvitex 2B. The new technique was evaluated in three groups of HIV seropositive patients with diarrhoea. Group 1: 19 patients with biopsy confirmed E bieneusi infection (186 stool samples); group 2: 143 consecutive patients from whom faeces were submitted for routine investigation of diarrhoea (318 samples); group 3: 16 patients with small intestinal biopsy specimens negative for microsporidia (55 samples). The new method was used to monitor spore shedding during experimental treatment with paromomycin and albendazole in four patients. RESULTS: Brightly fluorescent spores were detected in all stool samples of patients in group 1. In group 2 16 (11%) patients had spores in their stool samples. E bieneusi was found in 11 patients; in the other five another genus of microsporidia, Encephalitozoon, was recognised. Encephalitozoon spores were also found in the urine of three of these patients and in the maxillary sinus aspirate of two of them, suggesting disseminated infection. The results were confirmed by electron microscopic examination. In group 3 negative biopsy specimens were confirmed by negative stool samples in all cases. Treatment with albendazole and paromomycin did not affect the spore shedding in three patients with E bieneusi infection. By contrast, in a patient with Encephalitozoon sp infection albendazole treatment resulted in clinical improvement together with complete cessation of spore excretion in the stool. CONCLUSION: The Uvitex 2B fluorescence method combines speed, sensitivity, and specificity for the diagnosis and treatment evaluation of intestinal and disseminated microsporidiosis.

Electron microscopy as an essential technique for the identification of parasites in aids patients.

We use EM with increasing frequency for the identification of opportunistic parasitic infections in HIV-infected individuals. Apart from Pneumocytis carinii, Toxoplasma, Cryptosporidium, and Leishmania, we studied several aspects of microsporidiosis. Infection with the intestinal microsporidian Enterocytozoon was found in as much as 27% of AIDS patients with chronic diarrhoea without other pathogens. EM diagnosis of microsporidiosis is commonly performed on intestinal biopsies, but we have recently demonstrated spores of microsporidium with a non-invasive technique, viz. in faeces (1). However, EM of biopsy material remains the reference technique to distinguish the various species. Combining faeces and biopsy examination, we identified another group of microsporidians, Encephalitozoon sp., in the small intestine of AIDS patients with chronic diarrhoea (Fig. 1). Encephalitozoon sp. with identical ultrastructure was found in urine and sinus discharge, suggesting dissemination of the infection. In the maxillary sinus of one patient, we demonstrated E. bieneusi, a parasite which had previously been found only in small intestine and bile duct epithelium (2) (Fig. 2). After albendazole treatment, Encephalitozoon sp. disappeared from faeces, urine and nasal discharge. Although ultrastructural damage was noted in the developmental cycle of E. bieneusi in biopsies after treatment with albendazole, spores continued to be present in the faeces. These results demonstrate the great value of EM in the diagnosis of several parasitic diseases, especially microsporidiosis.

Rapid contrasting of extracellular elements in thin sections.

Standard methods for contrasting ultrathin sections generally have their greatest effect on cells and cellular components, whereas extracellular elements remain relatively electron-lucent. Occasionally, some extracellular elements even fail completely to react with the staining solutions. We describe a method for rendering a uniformly high contrast to extracellular tissue components. This consists of a brief prestaining of grids with diluted tannic acid in distilled water. Simple, rapid, and versatile, this procedure can be routinely applied to all tissue samples examined by electron microscopy. As an additional advantage, the method greatly enhances the electron density of intracellular glycogen. Higher concentrations of tannic acid give increased electron density, especially to elastin, and can therefore be used as an elastin stain.

Heterogeneity of lung cancer: the problem of sample error in diagnostic electron microscopy.

We studied the ultrastructure of superficial and deep samples of 40 resected primary lung carcinomas. Tumour cell differentiation was semiquantitatively assessed and differences between samples of a same tumour were evaluated. In two instances were major differences in ultrastructural diagnosis found between the samples of the same tumour. A further 9 cases showed one predominant differentiation in one sample, but two equally predominant differentiations in the second sample. The other 29 tumours did show occasional minor differences between the samples, but these differences did not result in differences in ultrastructural diagnosis.

Rapid deparaffinization for electron microscopy.

The authors present a method by which the number of steps required for the ultrastructural study of material originally embedded in paraffin can be greatly reduced. The process reduces deparaffinization, post-fixation, and re-embedding to four steps that take little more than 30 min to complete.