Associations between VHL genotype and clinical phenotype in familial von Hippel-Lindau disease

BACKGROUND: Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary disorder associated with tumours and cysts in the central nervous system (CNS) and other visceral organs. Germline mutations in the VHL gene on chromosome 3p25-26 are considered the cause of this disease. MATERIALS AND METHODS: We studied six patients with VHL disease and their relatives. Loss of heterozygosity (LOH) was determined by five flanking microsatellite polymorphic markers in the VHL locus. Multiplex ligation-dependent probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qPCR) amplification were used to detect the genomic deletions. Single-strand conformation polymorphism (SSCP) analysis was applied to test for sequence variations. RESULTS: Three germline deletions in the VHL gene (142.9, 53.3 and 3.3 kb) were found by MLPA. These deletions were defined clearly by qPCR analyses. The142.9 kb germline deletion was significantly associated with patients with CNS haemangioblastomas (P < 0.01 by Fisher's exact test), and one missense mutation (Gln209Arg) was detected from a patient with a pancreatic cyst in the same family. LOH was also detected from a patient with bilateral renal cell carcinomas. CONCLUSION: Diverse genetic conditions are associated with the clinical manifestations of VHL disease. Genomic deletions that can be detected by MLPA or qPCR are major causes for this syndrome. Missense mutations and LOH accompanying the disease lead to complex clinical symptoms and genotypic determination can facilitate a clinical diagnosis because of their strong association.     

Characterization of microsatellite markers to diagnose ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) maps to chromosome 16p13.3 (PKD1) and to chromosome 4q21-23 (PKD2), with the likelihood of a third unmapped locus. The size and genomic complexity of the PKD1 gene make it impractical to detect mutations for prenatal diagnosis. Therefore, pedigree-based linkage analysis remains useful for diagnosis of ADPKD. Since, the complete genome sequences of chromosome 16p13.3 and 4q21-23 including PKD1 and PKD2, respectively, were reported very recently, in order to do more precise diagnosis of ADPKD, we tried to find microsatellite markers. We performed database searches of 2000 kb of genome sequence across the 16p13.3 and the 4q21-23. To determine the distribution of alleles and the degree of polymorphism of the microsatellites, genotyping experiments were performed on 48 Korean individuals. We found novel 14 microsatellite markers around ADPKD that are more polymorphic and closer to PKD1 or PKD2 than the known markers. The novel microsatellite markers were applied to diagnose ADPKD families. These novel microsatellite markers are not only useful for presymptomatic and prenatal diagnosis of ADPKD, but also applicable in the study of positional cloning, human evolution and tumor biology.     

Molecular studies of loss of heterozygosity in Chinese sporadic retinoblastoma patients

BACKGROUND: Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common and severe intra-ocular tumor affecting infants and young children. METHODS: Loss of heterozygosity (LOH) on chromosome 13 was investigated in 16 Chinese sporadic RB patients, using 14 microsatellite markers spanning the complete chromosome 13, to determine whether those alterations different from the alteration of RB1 gene on 13q14 may play a role in the development of RB. Loss of RB1 allele is commonly encountered in sporadic RB. Microdissected RB tissues and their matched blood DNAs were analyzed for PCR-based LOH by using fluorescence-based DNA sequencing technology. RESULTS: Of 16 RB cases, 13 showed LOH on chromosome 13. The frequency of LOH on 13q14 was about 75% (12/16), consistent with other reports. Investigation of parental origin of lost RB1 alleles showed that, in all these cases, the paternal alleles were preferentially lost. Aside from the RB1 locus, other regions with the frequency of LOH above 30% in these tumors were D13S265, D13S158, D13S170, D13S218, D13S285 and D13S159. In particular, the D13S265 locus at 13q31-32 showed the highest rate of allele loss (64%, 9/14 informative cases), suggesting the presence of 1 or several genes whose loss of function may contribute to the development of RB. Comparison of the genotypic characteristics of 3 sites of frequent LOH (D13S153, D13S263 and D13S265) with the clinicopathological phenotype, respectively, showed that LOH of each locus was preferentially associated with a significantly younger age at diagnosis of RB. CONCLUSIONS: LOH analysis at some specific loci on chromosome 13 may be of a value in RB patients as diagnostic markers.     

Usefulness of combined genetic data in Hungarian families affected by autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary diseases. Mutations of two known genetic loci (PKD1: 16p13.3 and PKD2: 4q21.2) can lead to bilateral renal cysts. The PKD1 locus is the more common (～85%), with a more severe phenotype. Because of the genetic complexity of ADPKD and the size and complexity of the PKD1 gene, pedigree-based linkage analysis is a useful tool for the genetic diagnosis in families with more than one subject affected. We tested linkage or non-linkage to the closely linked DNA markers flanking the PKD1 (D16S663 and D16S291) and one intragenic D16S3252 and PKD2 (D4S1563 and D4S2462) in 30 ADPKD-affected families, to determine the distributions of alleles and the degree of microsatellite polymorphisms (in 91 patients and 125 healthy subjects). To characterize the markers, used heterozygosity levels, polymorphism information content and LOD scores were calculated. The D16S663 marker included 12 kinds of alleles, while D16S291 had 10 alleles and D16S3252 had 8. D4S1563 had 12 alleles and D4S2462 had 11. In a search for a common ancestral relationship, we considered the patients’ alleles with the same repeat number. Only one haplotype was detected in more than one (2) unrelated families. The calculated two-point LOD scores indicated a linkage to PKD1 in 22 families (74%). In four families (13%) with a linkage to PKD2, the patients reached the end-stage renal disease after the age of 65 years. One family was linked to neither gene (3%), and in three families (10%) a linkage to both genes was possible. In the latter three families, the numbers of analyzed subjects were small (4–5), and/or some markers were only partially or non-informative. However, the elderly affected family members exhibited the clinical signs of the PKD1 form in these cases. The new Hungarian population genetic information was compared with available data on other populations.     

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.     

Gene mapping of familial autosomal dominant dilated cardiomyopathy to chromosome 10q21-23.

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial disorder, accounting for 60% of all cardiomyopathies. In 20-30% of cases, familial inheritance can be demonstrated; an autosomal dominant transmission is the usual type of inheritance pattern identified. Previously, genetic heterogeneity was demonstrated in familial autosomal dominant dilated cardiomyopathy (FDCM). Gene localization to chromosome 1 (1p1-1q1 and 1q32), chromosome 3 (3p25-3p22), and chromosome 9 (9q13-9q22) has recently been identified. We report one family with 26 members (12 affected) with familial autosomal dominant dilated cardiomyopathy in which linkage to chromosome 10 at the 10q21-q23 locus is identified. Using short tandem repeat polymorphism (STR) markers with heterozygosity > 70%, 169 markers (50% of the genome) were used before linkage was found to markers D10S605 and D10S201 with a pairwise LOD score = 3.91, theta = 0, penetrance = 100% for both markers. Linkage to 1p1-1q1, 1q32, 3p25-3p22, and 9q13-9q22 was excluded. We conclude that a new locus for pure autosomal dominant FDCM exists, and that this gene is localized to a 9 cM region of 10q21-10q23. The search for the disease causing gene and the responsible mutation(s) is ongoing.     

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide–dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.     

微卫星Mfd41和DCC的遗传不稳定性与慢性粒细胞白血病病程发展关系研究

目的：研究微卫星ＤＮＡｓ的遗传不稳定性与慢性粒细胞白血病（ＣＭＬ）加速、急变的关系。方法：采用基础ＰＣＲ银染方法对９例加速期和８例急变期ＣＭＬ患者的骨髓细胞与其慢性期标本位于染色体１７ｐ的Ｍｆｄ４１和１８ｑ的ＤＣＣ两个微卫星序列进行比较分析。结果：１７例ＣＭＬ加速期或急变期患者中有８例出现微卫星不稳定性（ＭＳＩ）或杂合性丢失（ＬＯＨ），占总病例数的４７．５％。６例（加速期２例，急变期４例）出现ＤＣＣ改变，占总病例数的３５．３％；２例（加速期和急变期各１例）出现Ｍｆｄ４１改变，占总病例数的１１．８％。结论：微卫星ＤＮＡｓ的遗传不稳定性可能参与ＣＭＬ加速或急变的演变     

Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and treatment

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder affecting 1 in 1,000 people in the general population and accounts for up to 10% of all patients on renal replacement therapy. Numerous fluid-filled epithelial cysts arise from different nephron segments as spherical dilatations or small out-pouchings, enlarge progressively and eventually become disconnected from the rest of the renal tubule. The development of cysts is accompanied by destruction of the renal parenchyma, interstitial fibrosis, cellular infiltration and loss of functional nephrons. ADPKD is not only a kidney disease but also a systemic disorder associated with intracranial arterial aneurysms, cardiac valvular defects, colonic diverticulosis and cyst formation in other organs such as the liver, spleen and pancreas. The identification of PKD1 and PKD2 together with the drive to elucidate the functions of their encoded proteins, polycystin-1 (PC1) and polycystin-2 (PC2), has led to an explosion of clinical and scientific interest in this common disorder. The aim of this review is to highlight recent advances in our understanding of ADPKD pathogenesis which are leading to exciting new treatment strategies.     

Paraovarian cyst: MR imaging features

BACKGROUND: Paraovarian cysts are common intrapelvic neoplasms, but the magnetic resonance (MR) findings of paraovarian cyst have never been reported. We investigated the spectrum of MR imaging features of paraovarian cyst. METHODS: MR images of 18 paraovarian cysts in 16 patients were reviewed retrospectively. MR images were evaluated for the size and location of paraovarian cysts, single or multicystic, signal intensity on T1- and T2-weighted images, and visualization of the normal ovary on the affected side. RESULTS: The normal ovary of the affected side was recognized in 13 lesions. Four of these 13 cysts were separated from the ipsilateral ovary. In seven cysts, the normal ovary was abutted by cysts but maintained its shape. In two cysts, the beak sign was recognized at the interface between the cyst and the ovary. Most other MR features were nonspecific. CONCLUSION: Most paraovarian cysts were homogeneous cystic masses near the ipsilateral round ligament and the uterus. Demonstration of a normal ipsilateral ovary close to, but separated from, the adnexal cyst may be an important MR finding for the diagnosis of paraovarian cysts.     

Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease.

A consistent phenotype observed in both human patients and several different mouse models of autosomal recessive polycystic kidney disease (ARPKD) is an increased activity of the epidermal growth factor receptor (EGFR) in the affected kidneys. To determine whether this increased activity of the EGFR is a functional event that is directly part of the disease pathway of renal cyst formation, we used a genetic approach to introduce a mutant EGFR with decreased tyrosine kinase activity into a murine model of ARPKD. We found that the modified form of the EGFR could block the increase in EGFR-specific tyrosine kinase activity that normally accompanies the development of renal cysts, and this correlated with an improvement in kidney function and a substantial decrease in cyst formation in the collecting ducts. These results suggest that changes in the expression of the EGFR contribute to the formation of cysts in the collecting ducts, and that drugs that target the tyrosine kinase activity of the EGFR may potentially be therapeutic in ARPKD.     

急性髓细胞白血病17号染色体短臂杂合性缺失的研究

目的探讨17号染色体短臂(17p13.3)的杂合性缺失(LOH)在急性髓细胞白血病(AML)中的作用。方法采用聚合酶链反应扩增、聚丙烯酰胺凝胶电泳和硝酸银染色技术,研究17p13.3上的D17S1866、D17S695、D17S849、D17S926、D17S643微卫星位点的LOH。结果 30例AML患者中发生LOH的共有17例(56.7%)25例次,D17S1866、D17S695、D17S849、D17S926、D17S643位点LOH的发生率依次为23.3%(7/30)、23.3%(7/30)、16.7%(5/30)、13.3%(4/30)和6.7%(2/30)。其中2例同时在D17S1866、D17S849位点发生LOH;2例同时在D17S1866、D17S695位点发生LOH;1例同时在D17S695、D17S849位点发生LOH;1例同时在D17S695、D17S926位点发生LOH;1例同时在D17S1866、D17S849、D17S643位点发生LOH。10例健康对照者均未发生LOH。结论 17p13.3上D17S1866、D17S695、D17S849、D17S926、D17S643位点可检测到较高频率的LOH,提示该区域的LOH可能在AML的发生中有一定作用。     

常染色体显性多囊肾病的预后评估及治疗

常染色体显性多囊肾病(autosomal dominant polycystic kidney disease, ADPKD)患病率为1‰~2‰, 属于罕见病, 临床主要表现为双侧肾囊肿且逐渐发展, 肾脏体积进行性增大, 肾功能逐步降低。PKD1基因突变约占81%, PKD2基因突变约占10.5%~22%。血管加压素(arginine vasopressin, AVP)和环磷酸腺苷(cyclic adenosine monophosphate, cAMP)信号通路在ADPKD囊肿发展过程中发挥重要作用。近年来发表的梅奥风险评估模型和PROPKD(predicting renal outcome in polycystic kidney disease)评分是ADPKD较好的预后评估模型, 已成为临床医生决策的重要依据。通过拮抗AVP受体, 抑制cAMP通路的托伐普坦已成为ADPKD首个特异治疗药物, 可有效抑制总肾脏体积的增长和保护肾功能。药物的长期安全性仍需进一步研究。     

Genetic linkage analysis, clinical features and prognosis of autosomal dominant polycystic kidney disease in Northern Ireland

Fifteen families with autosomal dominant poly-cystic kidneydisease were analysed for co-inheritance of the disease andDNA markers flanking the PKD1 locus. Eleven families demonstratedlinkage to PKD1 markers. Two families were unlinked to the PKD1locus (non-PKD1) and in two families the markers were uninformative.The clinical features and prognosis of 49 subjects with a PKD1genotype were compared with 17 non-PKD1 subjects. The age atdiagnosis in non-PKD1 subjects (37 ±11 years) was significantlylater than PKD1 subjects (25 ± 13 years, p< 0.001).Only two (12%) non-PKD1 subjects presented initially with clinicalfeatures of autosomal polycystic kidney disease compared to27 (55%) of PKD1 subjects (p< 0.002). Hypertension was morecommon in PKD1 compared to non-PKD1 subjects (29% vs. 12%),as was stage renal failure (25% vs. 6%). Seventy-five percentof non-PKD1 subjects had not developed end-stage renal failureby the age of 54 years compared to only 35% of PKD1 subjects. Most families with autosomal polycystic kidney disease in thispopulation have disease due to mutations at the PKD1 locus.However, the proportion of non-PKD1 families appears to be higherthan estimates for other populations. This study also confirmsinitial reports that subjects with a non-PKD1 genotype havea milder disease with a better prognosis than those with a PKD1genotype.     

The pathogenesis of autosomal dominant polycystic kidney disease

In individuals with autosomal dominant polycystic kidney disease (ADPKD), renal function deteriorates as the kidneys become replaced by multitudes of fluid-filled cysts. Although the PKD genes were identified a decade ago, the pathway(s) leading from mutation to disease remain the subject of intense investigation. As a result of this work, it has become apparent that the polycystins are multifunctional proteins that, in the broadest sense, appear to be involved in the transduction of a number of environmental cues into appropriate cellular responses. It is likely that the central pathogenetic pathway for cystogenesis stems from de-differentiation of tubular epithelial cells. Available evidence indicates that loss of polycystin activity leads to subtle derangements of cell calcium regulation through several possible pathways. Abnormal cell calcium homeostasis might then lead to altered differentiation in affected cells. The study of the polycystins has revealed some entirely novel insights into fundamental cell biology but these have not yet been satisfactorily integrated into a verified pathogenetic pathway for the development of ADPKD.     

The significance of the postmenopausal simple adnexal cyst

In order to help clarify the clinical problem of the postmenopausal woman with an adnexal cyst, 13 patients with sonographically detected relatively simple adnexal cysts were reviewed. Only one patient had a "borderline" malignancy; the remaining cysts were all benign. The 8 per cent malignancy rate in this small series suggests that the simple postmenopausal adnexal cyst may not necessarily be an ominous finding.     

Ciprofloxacin activity in cyst fluid from polycystic kidneys.

Renal cyst infection in patients with polycystic kidney disease (PKD) is often unresponsive to standard antimicrobial therapy, in part because of the failure of most antibiotics to adequately penetrate cyst fluid. Ciprofloxacin, a new quinolone antibiotic, possesses in vitro activity against most pathogens likely to be encountered in renal cyst infection. To study the potential usefulness of ciprofloxacin for the treatment of cyst infection, fluid from 70 cysts was obtained from seven patients with polycystic kidney disease who were receiving the drug. Cysts were categorized as nongradient or gradient by the sodium concentration in the fluid. The ciprofloxacin concentration within cysts was measured, and the cyst fluid bactericidal activity against likely cyst fluid pathogens was determined. The mean (+/- standard error) ciprofloxacin concentration was 12.7 +/- 2.9 micrograms/ml. Preferential accumulation of ciprofloxacin occurred in gradient cysts; these levels exceeded levels in serum by more than fourfold. Cyst fluid bactericidal activity titers were uniformly high against Escherichia coli and Proteus mirabilis, while less activity was observed against Streptococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.     

血浆DNA在肺癌诊断上的意义

目的研究血浆DNA 3P D3S1300和D3S1289位点杂合性缺失(LOH)作为肿瘤标志物在肺癌诊断中的意义.方法收集初诊69例肺癌患者全血及肿瘤组织,40例非肺癌病人全血,用PCR-银染法分析肿瘤患者血浆、肿瘤组织中及对照组血浆中DNA D3S1300和D3S1289 LOH情况,观察阳性率.结果 69例肺癌患者肿瘤组织DNA中D3S1300和D3S1289的LOH检出率分别为40.6%和31.9%,血浆检出率为29.0%和24.6%.对照组血浆仅有2例DNA检出D3S1300 LOH,3例检出D3S1289 LOH.与肺癌组相比P＜0.01.结论血浆DNA LOH可望作为一种新的肿瘤标志物.     

Genetic Heterogeneity in Saliva from Patients with Oral Squamous Carcinomas: Implications in Molecular Diagnosis and Screening

We performed microsatellite analysis at chromosomal regions frequently altered in head and neck squamous carcinoma on matched saliva and tumor samples from 37 patients who had oral squamous carcinoma. The results were correlated with the cytologic findings and traditional clinicopathologic factors to assess the diagnostic and biological potential of these markers. Our data showed that 18 (49%) of the saliva samples and 32 (86%) of the tumors had loss of heterozygosity (LOH) in at least one of the 25 markers studied. In saliva, the combination of markers D3S1234, D9S156, and D17S799 identified 13 (72.2%) of the 18 patients with LOH in saliva (P < 0.001). For tumors, markers D3S1234, D8S254, and D9S171 together identified 27 (84.3%) of the 32 tumors with LOH at any of the loci tested (P < 0.001). Eleven (55%) of the 20 saliva samples with cytologic atypia and seven (35%) of the 17 specimens without atypia had LOH. Significant correlation between LOH in tumor at certain markers and smoking and alcohol use was found. Our results indicate that: 1) epithelial cells in saliva from patients with head and neck squamous tumorigenesis provide suitable material for genetic analysis; 2) combined application of certain markers improves the detection of genetic alteration in these patients; 3) clonal heterogeneity between saliva and matching tumor supports genetic instability of the mucosal field in some of these patients; and 4) LOH at certain chromosomal loci appears to be associated with smoking and alcohol consumption.     

Carney complex, a familial multiple neoplasia and lentiginosis syndrome. Analysis of 11 kindreds and linkage to the short arm of chromosome 2.

Carney complex is an autosomal dominant syndrome characterized by multiple neoplasias, including myxomas at various sites and endocrine tumors, and lentiginosis. The genetic defect(s) responsible for the complex remain(s) unknown. We studied 101 subjects, including 51 affected members, from 11 North American kindreds with Carney complex. Blood samples were collected from patients and their family members. Hospital records, photographs, and tissue specimens of deceased individuals were reviewed. DNA was extracted from blood samples, patient-derived cell lines, and/or paraffin-embedded tissues. Linkage analysis was performed with highly polymorphic microsatellite markers, distributed over areas of the human genome harboring the most likely candidate genes. The most prevalent clinical manifestation in patients with Carney complex was spotty skin pigmentation, similar to that observed in Peutz-Jeghers and other lentiginosis syndromes. Skin and cardiac myxomas, Cushing syndrome, and acromegaly were present in 62, 30, 31 and 8 percent of the patients, respectively. Linkage was obtained for three markers on the short arm of chromosome 2 (2p16), with a maximum two-point lod score of 5.97 at theta = 0.03 for the marker CA-2 (odds in favor of linkage 10(6):1. The flanking markers CA7 and D2S378 defined a region of approximately 6.4 cM that is likely to contain the gene(s) associated with Carney complex. Candidate genes in the proximity, including the propiomelanocortin and the DNA-mismatch repair hMSH2 genes, were excluded. We conclude that the genetic defect(s) responsible for Carney complex map(s) to the short arm of chromosome 2 (2p16). This region has exhibited cytogenetic aberrations in atrial myxomas associated with the complex, and has been characterized by microsatellite instability in human neoplasias.