A complete mutation screen of PKHD1 in autosomal-recessive polycystic kidney disease (ARPKD) pedigrees

BACKGROUND: Autosomal-recessive polycystic kidney disease (ARPKD) is an important neonatal nephropathy characterized by fusiform dilation of collecting ducts, congenital hepatic fibrosis, and in some cases Caroli's disease. The ARPKD gene, PKHD1, has recently been identified. Herein we describe an effective method for PKHD1 mutation screening and the results from analysis of a novel ARPKD cohort. METHODS: The coding region of PKHD1 was amplified as 79 fragments and analyzed for base pair changes by denaturing high-performance liquid chromatography (DHPLC). Forty-seven ARPKD and 14 pedigrees with congenital hepatic fibrosis and/or Caroli's disease, were screened for PKHD1 mutations. RESULTS: Thirty-three different mutations were detected on 57 alleles (51.1% ARPKD, 32.1% congenital hepatic fibrosis/Caroli's disease). In the 22 pedigrees where both mutations were identified, two were homozygous for 9689delA and the remainder were compound heterozygotes; a combination of truncating, missense and splicing changes. Patients with two truncating mutations all died in the perinatal period. Two frequent truncating mutations were identified: 9689delA (9 alleles) and 5896insA (8 alleles) plus some more common missense changes; haplotype analysis indicated most were ancestral mutations. CONCLUSION: DHPLC has been established as a rapid mutation screening method for ARPKD. The mutation detection rate was high in severely affected patients (85%), lower in those with moderate ARPKD (41.9%), and low, but significant, in adults with congenital hepatic fibrosis/Caroli's disease (32.1%). The prospects for gene-based diagnostics are complicated by the large gene size, marked allelic heterogeneity, and clinical diversity of the ARPKD phenotype. Identification of some common mutations, especially in specific populations, will aid mutation screening.     

A comprehensive screen of genes implicated in craniosynostosis

Recent advances in human molecular genetics have identified mutations in the TWIST, FGFR-1, FGFR-2 and FGFR-3 genes to be important causes of craniosynostosis. Despite this, however, mutations cannot be identified in the majority of patients. This study reports the first comprehensive screen of mutations in TWIST, FGFR-1, FGFR-2 and FGFR-3 genes in a cohort of patients with craniosynostosis. This has led to the identification of Saethre-Chotzen syndrome to be a new microdeletion disorder and reports the first example of a gene-environment interaction leading to craniosynostosis. In addition, investigation of the expression patterns of the Fgfr and Twist genes in the normal developing mouse coronal suture has identified the TWIST protein to be important in cranial suture initiation and biogenesis. These findings have significant clinical implications and will form the basis of future attempts to develop novel therapies aimed at inhibiting cranial suture fusion.     

A novel nonsense mutation in the androgen receptor gene causes the complete androgen insensitivity syndrome

We identified an unusual novel nonsense mutation in exon 3 of the androgen receptor (AR) gene in a patient with complete androgen insensitivity that was persistence of Wolffian derivatives. Sequence analysis revealed a substitution (C→T) at position 2211 and a deletion of G at position 2213 in exon 3 of the AR gene, resulting in the conversion of arginine(CGG) to a stop codon (TGA) of the AR. Western blotting demonstrated a truncated AR with around 70 kd was expressed. Histology of patient's testes showed that seminiferous tubules were totally filled with Sertoli cells without germ cells. Immunohistochemistry revealed positive AR localization in the nuclei of Sertoli cells and epithelia of efferent ductule and vas deferens. AR immunoexpression was stronger in the epithelia of efferent ductule and vas deferens than in Sertoli cells. The study extends the spectrum of exon 3 mutations in the AR gene.     

利用凝集素芯片检测多囊肾病特异iPS细胞表面糖谱的研究

目的:建立一套凝集素芯片检测活体iPS细胞表面糖谱的方法。方法:以ADPKD-iPS为对象,运用不同活细胞染料,尝试不同的染色时间,找到最佳的iPS细胞与凝集素芯片结合的实验方案。结果:利用SYBR green,染色30min可以获得最佳的信号强度和信噪比。结论:已成功建立了一套利用凝集素芯片对活体iPS细胞进行表面糖谱检测的方法。     

Treatment of autosomal dominant polycystic kidney disease (ADPKD): the new horizon for children with ADPKD

Polycystic kidney disease (PKD) is the most common inherited renal disorder. Patients with PKD remain clinically asymptomatic for decades, while significant anatomic and physiologic systemic changes take place. Sequencing of the responsible genes and identification of their protein products have significantly expanded our understanding of the pathophysiology of PKD. The molecular basis for cystogenesis is being unraveled, leading to new targets for therapy and giving hope to millions of people suffering from PKD. This has direct implications for children with PKD with regard to screening for the disease and identification of high-risk individuals. In this article we provide a review of the clinical manifestations in children with autosomal dominant polycystic kidney disease (ADPKD), the genetic and molecular basis for the disease, and a concise review of potential therapies being evaluated.     

Sirolimus Therapy to Halt the Progression of ADPKD

Activation of mammalian target of rapamycin (mTOR) pathways may contribute to uncontrolled cell proliferation and secondary cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD). To assess the effects of mTOR inhibition on disease progression, we performed a randomized, crossover study (The SIRENA Study) comparing a 6-month treatment with sirolimus or conventional therapy alone on the growth of kidney volume and its compartments in 21 patients with ADPKD and GFR ≥40 ml/min per 1.73 m². In 10 of the 15 patients who completed the study, aphthous stomatitis complicated sirolimus treatment but was effectively controlled by topical therapy. Compared with pretreatment, posttreatment mean total kidney volume increased less on sirolimus (46 ± 81 ml; P = 0.047) than on conventional therapy (70 ± 72 ml; P = 0.002), but we did not detect a difference between the two treatments (P = 0.45). Cyst volume was stable on sirolimus and increased by 55 ± 75 ml (P = 0.013) on conventional therapy, whereas parenchymal volume increased by 26 ± 30 ml (P = 0.005) on sirolimus and was stable on conventional therapy. Percentage changes in cyst and parenchyma volumes were significantly different between the two treatment periods. Sirolimus had no appreciable effects on intermediate volume and GFR. Albuminuria and proteinuria marginally but significantly increased during sirolimus treatment. In summary, sirolimus halted cyst growth and increased parenchymal volume in patients with ADPKD. Whether these effects translate into improved long-term outcomes requires further investigation.Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disorder with major renal manifestations, which occurs in 1 of 400 to 1000 individuals.¹ ADPKD is genetically heterogeneous. Mutations of the two genes PKD1 (85% of the cases) and PKD 2 (15% of cases), encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, are implicated in the disease development.² The functions of PC1 and PC2 have not been defined with certainty; however, PC1 is thought to interact with and regulate PC2, which is a member of a subfamily of transient receptor potential channels³ and may act as a cation channel allowing Ca²⁺ entry from the extracellular environment. Consistent with the PC1/PC2 complex having a role in Ca²⁺ regulation, PKD epithelial cells display altered intracellular Ca²⁺ homeostasis,⁴ which alters the response to increased levels of intracellular cAMP.^5–7Another change consistently found in PKD cells is activation of the Ser/Thr kinase mammalian target of rapamycin (mTOR), an enzyme that coordinates cell growth, cell-cycle progression, and proliferation.⁸ mTOR is made up of two distinct complexes: mTORC1 and TORC2. The direct downstream targets of mTORC1, the eukaryotic initiation factor 4E-binding protein and ribosomal protein S6 kinase (p70S6K1),^9,10 tightly regulate the translational initiation machinery to control cell growth and proliferation.⁸ In vitro studies demonstrated that the N-terminal cytoplasmic domain of PC1 co-localizes and interacts with tuberin.¹¹ Activated phospho-mTOR and p70S6K are induced in cyst-lining epithelial cells in cysts from human and mouse kidneys.¹¹ Moreover, p70S6K is increased in Han:SPRD rat kidneys with PKD.¹² These observations led to the hypothesis that defects in PC1 in ADPKD promote disruption of the tuberin-mTOR complex, leading to aberrant mTOR activation and signaling.¹¹ There is also evidence that IGF-1 by binding to its receptor is a major regulator of the mTOR pathway via signaling to phosphatidylinositol-3 kinase, protein kinase B (Akt), and mTOR.⁸ Increase in IGF-1 mRNA levels in the kidneys of the pcy mouse model of PKD¹³ and in IGF-1 protein in Han:SPRD rats¹⁴ has been reported. In addition, the amount of phospho-Akt in cystic Pkd1^−/− mouse kidneys was more than that in wild-type kidneys.¹⁵ Thus, if mTOR is such a converging point in PKD cells, it would be worthwhile as a possible drug target for treatment of renal cystic disorders.Sirolimus (originally referred to as rapamycin) is a macrocyclic lactone that is derived from Streptomyces hygroscopicus and exerts antiproliferative and growth-inhibiting effects as well as antifibrotic effect by inhibition of the mTOR enzyme.^16,17 The drug has been used in kidney transplant recipients as part of maintenance immunosuppressive therapy¹⁸ and more recently as an antitumor agent^19,20 and in drug-eluting stents to prevent coronary artery stenosis.²¹ Short-term treatment with sirolimus markedly reduced kidney size and lowered renal total cyst volume (TCV) density in PKD animal models.^11,12,22 In addition, in renal transplant recipients who had progressed to ESRD because of ADPKD, the size of native kidney and liver cysts decreased while on mTOR inhibitor therapy but did not change appreciably during treatment with other immunosuppressants.^11,23Thus, to assess formally the risk/benefit profile of mTOR inhibitor therapy in PKD, we designed the Sirolimus Treatment in Patients with Autosomal Dominant Polycystic Kidney Disease: Renal Efficacy and Safety (SIRENA; http://clinicaltrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00491517","term_id":"NCT00491517"}}NCT00491517), a proof-of-concept, randomized clinical trial aimed to compare the changes in total kidney volume (TKV) and in the kidney''s various compartments. This was assessed by serial computed tomography (CT) scan evaluations during 6 months of treatment with sirolimus or conventional therapy alone in 21 patients with ADPKD and normal or moderately decreased kidney function. The study secondarily evaluated whether and to which extent treatment-induced changes in kidney volume and structure translated into concomitant changes in GFR as assessed by standard techniques. The results of these analyses formed the basis of this report.     

A case of complete testicular feminization: Laparoscopic orchiectomy and analysis of androgen receptor gene mutation

BACKGROUND: Analysis of an androgen receptor gene mutation and a bilateral laparoscopic orchiectomy were performed on a 19-year-old patient diagnosed as a case of complete testicular feminization. METHODS: DNA sequencing of an androgen receptor gene mutation and laparoscopic orchiectomy were performed. RESULTS: A novel point mutation substituting a proline residue (CCG) for a leucine residue (CTG) was observed in codon 892 of exon 8 in the hormone-binding domain of the androgen receptor gene. Bilateral intra-abdominal testes were resected uneventfully by means of laparoscopic orchiectomy. CONCLUSION: We conclude that genetic analysis of androgen receptor gene mutation is essential for diagnosis of teon and laparoscopic orchiectomy is a useful therapeutic alteration as a minimally invasive treatment.     

A genome-wide DNA microsatellite association screen to identify chromosomal regions harboring candidate genes in diabetic nephropathy

In an effort to accelerate the identification of susceptibility genes in diabetic nephropathy, the first genome-wide fluorescence-based DNA microsatellite (n=6000) association screen was performed, using pools of genomic DNA derived from Irish patients with (cases; n=200) and without (controls; n=200) type 1 diabetic nephropathy. Allele image profiles were generated for 5353 (89.2%) microsatellite markers for both case and control pools. Allele counts (estimated from allele image profiles) were compared in case versus control groups, and empirical P values were generated. Markers then were ranked on the basis of their empirical P values (lowest to highest). Repeat PCR amplification and electrophoresis of pooled samples were performed systematically on ranked markers until the 50 most associated markers with consistent results were identified. DNA samples that composed the pools then were genotyped individually for these markers. Two markers on chromosome 10, D10S558 (Pcorrected=0.005) and D10S1435 (Pcorrected=0.016), revealed statistically significant associations with diabetic nephropathy. An additional four markers (D6S281, D4S2937, D2S291, and D17S515) also are worthy of further investigation. Relevant functional candidate genes have been identified in the vicinity of these markers, demonstrating the feasibility of low-resolution genome-wide microsatellite association screening to identify possible candidate genes for diabetic nephropathy.     

Molecular pathogenesis of ADPKD: the polycystin complex gets complex

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases with an incidence of 1:400 to 1:1000. It is characterized by the progressive development and enlargement of focal cysts in both kidneys, typically resulting in end-stage renal disease (ESRD) by the fifth decade. The cystogenic process is highly complex with a cellular phenotype consistent with "dedifferentiation" (i.e., a high proliferative rate, increased apoptosis, altered protein sorting, changed secretory characteristics, and disorganization of the extracellular matrix). Although cystic renal disease is the major cause of morbidity, the occurrence of nonrenal cysts, most notably in the liver (occasionally resulting in clinically significant polycystic liver disease) and the increased prevalence of other abnormalities including intracranial aneurysms, indicate that ADPKD is a systemic disorder. Following the identification of the first ADPKD gene, PKD1, 10 years ago and PKD2 2 years later, considerable progress has been made in defining the etiology and understanding the pathogenesis of this disorder, knowledge that is now leading to the development of several promising new therapies. The purpose of this review is to summarize our current state of knowledge as to the structure and function of the PKD1 and PKD2 proteins, polycystin-1 and -2, respectively, and explore how mutation at these loci results in the spectrum of changes seen in ADPKD.     

Analysis of xenoantigenicity of HLA class I molecules by a complete series of human-mouse hybrid genes

To investigate the xenoantigenicity of HLA class I antigens, a complete series of human/mouse chimeric major histocompatibility complex class I genes was constructed by using the human HLA-B7 and mouse H-2Kb genes. All of these hybrid antigens were stably expressed on L cells at high levels. Nine anti-HLA class I monoclonal antibodies, including 5 monomorphic, 3 broad polymorphic, and 2 specific polymorphic ones, were tested by binding to these cell lines. Our data suggest that the alpha 2 domain of HLA-B7 plays an important role in the formation of the antigenic determinants recognized by all of the anti-HLA class I mAbs tested. However, the antigenic determinants of the monomorphic mAbs were complex. In some cases, all of the three extracellular domains were required for expression of the epitope. The location of xenoimmunogenic site of HLA-B7 antigen was also examined by immunizing (B6xC3H)F1 and C3H mice with the parental and hybrid antigens. Our data suggest that the alpha 1 and alpha 2 domains of HLA-B7 must be expressed together for production of antibodies against monomorphic determinants of HLA class I antigens, while the alpha 3 alone can induce xenoantibodies in (B6xC3H)F1 mice but not in C3H mice.     

A novel androgen receptor mutation resulting in complete androgen insensitivity syndrome and bilateral Leydig cell hyperplasia

Androgens drive male secondary sexual differentiation and maturation. Mutations in the androgen receptor (AR) gene cause a broad spectrum of abnormal phenotypes in humans, ranging from mild through partial to complete androgen insensitivity. We have analyzed the AR gene by using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing and have studied gonads histologically in a familial case of complete androgen insensitivity syndrome. Sequence analysis of the AR gene showed a novel C2578T missense mutation, resulting in the replacement of a highly conserved leucine residue with phenylalanine (L859F) in ligand-binding domain of the receptor. The residue L859, located in helix 10 of the androgen receptor, plays a significant role in overall architecture of ligand-binding pocket. The mutation was absent from the father, normal brother of the patients, and 100 normal males recruited in this study as controls. The inheritance of the mutation in the family clearly shows that C2578T is the underlying mutation for the eventual phenotype in the patients. Histology of patient's gonads showed Leydig cell hyperplasia, with a few or no spermatogonium. It is thought that AR gene mutations result in hormonal imbalance, resulting in the high levels of luteinizing hormone (LH) and ultimately Leydig cell hyperplasia or tumor formation. In the present study, we have reported a rare familial case of Leydig cell hyperplasia despite consistently normal LH levels. The finding will help in giving counseling to this family and prevent the transmission of the mutated X chromosome to the coming generations.     

A chemical mutagenesis screen to identify modifier genes that interact with growth hormone and TGF-beta signaling pathways

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as >3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p<0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.     

Surveillance on mutation carriers of DNA mismatch repair genes

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited cancer susceptibility syndrome signifying a very high risk of colorectal and endometrial cancer at young age. It also entails an increased risk of a variety of other tumours, such as ovarian, gastric, uroepithelial and biliary tract cancer. The underlying pathogenic mutation lies in one of the five known DNA mismatch repair genes (MSH2, MLH1, PMS1, PMS2, and MSH2). The majority of HNPCC patients and families can at present be identified and the underlying mutation detected by genetic diagnostics. This provides the opportunity for predictive genetic testing to exclude or identify the mutation carrier status of the family members at risk. Mutation-negative individuals can then be relieved from any extra cancer threat. For mutation-positive individuals a preventive surveillance programme offers substantial benefits in reducing the cancer incidence, almost precluding death of colorectal cancer and reducing overall mortality.     

A novel E153X point mutation in the androgen receptor gene in a patient with complete androgen insensitivity syndrome

Aim: To study a 46, XY newborn patient with a phenotype suggestive of an androgen insensitivity syndrome toconfirn an anomaly in the AR gene. Methods: Genomic DNA from leukocytes was isolated in order to analyze SRYgene by PCR and sequencing of the eight exons of AR gene. Isolation of human Leydig cell mesenchymal precursorsfrom the testis was performed in order to study testosterone production and response to hCG stimulation in culture.Results: Surgical exploration disclosed two testes, no Wolffian structures and important Mullerian derivatives. TheSRY gene was present in peripheral blood leukocytes. Sequencing of the AR gene evidenced a previously unreported Gto T transversion in exon 1 that changed the normal glutamine 153 codon to a stop codon. Interstitial cell culturesproduced sizable amounts of testosterone and were responsive to hCG stimulation. Conclusion: This E153X nonsensepoint mutation has not been described previously in cases of AIS, and could lead to the synthesis of a short truncated(15     

肝癌微环境中影响转移相关通路的基因富集分析

目的 探讨肝癌微环境对肝癌发生门静脉转移起重要作用的基因簇或通路,为肝癌的治疗和研究提供理论依据.方法 利用基因组富集（GSEA）的方法,对美国国家生物技术信息中心（NCBI）的综合性的基因表达与杂交排序数据库（GEO数据库）中的11例肝癌无门静脉癌栓（PVTT）和9例肝癌合并PVTT的癌旁相对正常组织基因表达谱芯片进行分析,分析其在肿瘤相关通路中的富集特征与核心富集基因.结果 找到15条相关的信号通路,这些通路与癌旁微环境中的免疫功能状态有关,对于肝癌发生PVTT转移可能起重要作用.结论 肝癌发生门静脉转移除了与肿瘤细胞本身的恶性增殖和侵袭有关以外,还与周围微环境中的免疫状态和炎症反应有重要联系.     

Family History of Renal Disease Severity Predicts the Mutated Gene in ADPKD

Mutations of PKD1 and PKD2 account for 85 and 15% of cases of autosomal dominant polycystic kidney disease (ADPKD), respectively. Clinically, PKD1 is more severe than PKD2, with a median age at ESRD of 53.4 versus 72.7 yr. In this study, we explored whether a family history of renal disease severity predicts the mutated gene in ADPKD. We examined the renal function (estimated GFR and age at ESRD) of 484 affected members from 90 families who had ADPKD and whose underlying genotype was known. We found that the presence of at least one affected family member who developed ESRD at age ≤55 was highly predictive of a PKD1 mutation (positive predictive value 100%; sensitivity 72%). In contrast, the presence of at least one affected family member who continued to have sufficient renal function or developed ESRD at age >70 was highly predictive of a PKD2 mutation (positive predictive value 100%; sensitivity 74%). These data suggest that close attention to the family history of renal disease severity in ADPKD may provide a simple means of predicting the mutated gene, which has prognostic implications.Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder, with a prevalence of one in 500 to 1000 in the general population. It is the third most common single cause of ESRD in the United States, accounting for 5% of people with ESRD.^1–5 ADPKD is genetically heterogeneous, with two disease genes (PKD1 on chromosome 16 and PKD2 on chromosome 4) accounting for most of the cases. Mutations of PKD1 and PKD2 are thought to account for 85 and 15% of cases, respectively, in linkage-characterized European populations.^6,7 Although the clinical manifestations overlap completely between two gene types, there is a strong locus effect on renal disease severity. Patients with PKD1 have significantly more severe renal disease than patients with PKD2, with larger kidneys and earlier onset at ESRD (median age 53.4 versus 72.7 yr, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may exist for type 1¹⁰ but not type 2 ADPKD.¹¹ In addition, significant intrafamilial renal disease variability is evident, which is thought to be due to genetic and environmental modifiers.^11,12PKD1 is a large, complex gene containing 46 exons spanning 50 kb, with 33 of these exons at the 5′ end being duplicated elsewhere on chromosome 16. PKD2 is a single-copy gene, consisting of 15 exons spanning a 68-kb genomic region. There is marked allelic heterogeneity for both gene types, with 314 truncating mutations having been described in PKD1 and 91 truncating mutations in PKD2.^1,2 PKD1 encodes polycystin 1 (PC1), a large receptor-like protein, and PKD2 encodes polycystin 2 (PC2), a nonselective cation channel that transports calcium. Both PC1 and PC2 physically interact to form a complex that regulates intracellular levels of calcium and are located in the primary cilia of renal tubular cells. Recent studies suggested that the polycystin complex in the primary cilia of renal tubular cells serves as a mechanosensor for urine flow and that dysfunction of this mechanosensor may lead to cellular proliferation and cystogenesis.^1,2Recent advances in our understanding of the molecular pathobiology of ADPKD have led to the discovery of a number of drugs (e.g., tolvaptan, somatostatin, mammalian target of rapamycin inhibitors) that may target cyst growth and delay renal disease progression.^1,2 Several of these promising drugs are being or will be tested in clinical trials, and disease-modifying treatment may become a reality in the not-too-distant future.¹ In this context, the knowledge of ADPKD gene type may allow for the optimization of the design of such clinical trials, and identification of those affected individuals who are most likely to benefit from these novel therapies should they become available; however, the gene type is seldom known for most families in the clinical setting. Although molecular genetic testing, either by linkage or direct mutation analysis, can elucidate the gene type, such testing has its limitations.¹³ Linkage studies require DNA samples from several affected family members and are of limited utility in small families or de novo cases. Mutation-based screening for ADPKD is expensive and yields a definitive pathogenic mutation in only 42 to 63% of cases because the large and complex structure of PKD1 results in many unclassified missense variants whose pathogenicity often cannot be predicted with complete certainty.^14,15In this study, we explored whether renal disease severity can be used as clinical predictors of underlying gene type in families with ADPKD. To predict PKD1, we explored various cutoffs of early age at ESRD as indicative of severe renal disease. To predict PKD2, we explored various cutoffs of late age with renal sufficiency or at ESRD as indicative of milder renal disease. We then evaluated the performance characteristics of these cutoffs to define the optimal criteria for clinical prediction of ADPKD gene type.     

A novel E153X point mutation in the androgen receptor gene in a patient with complete androgen insensitivity syndrome

AIM: To study a 46, XY newborn patient with a phenotype suggestive of an androgen insensitivity syndrome to confirm an anomaly in the AR gene. METHODS: Genomic DNA from leukocytes was isolated in order to analyze SRY gene by PCR and sequencing of the eight exons of AR gene. Isolation of human Leydig cell mesenchymal precursors from the testis was performed in order to study testosterone production and response to hCG stimulation in culture. RESULTS: Surgical exploration disclosed two testes, no Wolffian structures and important Müllerian derivatives. The SRY gene was present in peripheral blood leukocytes. Sequencing of the AR gene evidenced a previously unreported G to T transversion in exon 1 that changed the normal glutamine 153 codon to a stop codon. Interstitial cell cultures produced sizable amounts of testosterone and were responsive to hCG stimulation. CONCLUSION: This E153X nonsense point mutation has not been described previously in cases of AIS, and could lead to the synthesis of a short truncated (153 vs 919 residues) non functional AR probably responsible for the phenotype of complete androgen insensitivity syndrome (CAIS).     

静脉动脉化再植全拇指旋转撕脱性完全离断成功一例报告

目的报道全拇指旋转撕脱性完全离断一例应用静脉动脉化方法再植获得成功.方法2000年11月7日,在指动脉吻合四次后无法恢复断指血供的情况下,将拇指尺侧指动脉与指腹桡侧皮下静脉吻合作为供血通路,吻合三条指背静脉作回血通路.结果拇指色泽红润、指腹饱满、张力适中、指温正常及弹性好,两周后复查,患指成活.结论静脉动脉化是旋转撕脱性完全离断全拇指再植的一种可行新方法.     

Use of in vitro DNA amplification to screen family members for an insulin gene mutation

The DNA polymerase chain reaction can be a powerful tool for amplifying selected segments of genomic DNA for investigation of point mutations that are inaccessible via classic restriction-fragment-length polymorphism analysis. We have applied this method to an analysis of the incidence of heterozygosity for the mutant insulin allele insulin Wakayama (A3 Val----Leu) in two unrelated Japanese families having the hyperinsulinemic mutant insulin syndrome. The results indicate that this method is simple, sensitive, and accurate and should be useful for screening larger (diabetic) populations to detect single-base substitutions in the insulin gene that lead to either altered (pro)insulin structure and/or insulin production.