Gene conversion is a likely cause of mutation in PKD1

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.      

Thirteen novel mutations of the replicated region of PKD1 in an Asian population

BACKGROUND: Mutations of PKD1 are thought to account for approximately 85% of all mutations in autosomal dominant polycystic kidney disease (ADPKD). The search for PKD1 mutations has been hindered by both its large size and complicated genomic structure. To date, few mutations that affect the replicated segment of PKD1 have been described, and virtually all have been reported in Caucasian patients. METHODS: In the present study, we have used a long-range polymerase chain reaction (PCR)-based strategy previously developed by our laboratory to analyze exons in the replicated region of PKD1 in a population of 41 unrelated Thai and 6 unrelated Korean families with ADPKD. We have amplified approximately 3.5 and approximately 5 kb PKD1 gene-specific fragments (5'MR and 5'LR) containing exons 13 to 15 and 15 to 21 and performed single-stand conformation analysis (SSCA) on nested PCR products. RESULTS: Nine novel pathogenic mutations were detected, including six nonsense and three frameshift mutations. One of the deletions was shown to be a de novo mutation. Four potentially pathogenic variants, including one 3 bp insertion and three missense mutations, were also discovered. Two of the nonconservative amino acid substitutions were predicted to disrupt the three-dimensional structure of the PKD repeats. In addition, six polymorphisms, including two missense and four silent nucleotide substitutions, were identified. Approximately 25% of both the pathogenic and normal variants were found to be present in at least one of the homologous loci. CONCLUSION: To our knowledge, this is the first report of mutation analysis of the replicated region of PKD1 in a non-Caucasian population. The methods used in this study are widely applicable and can be used to characterize PKD1 in a number of ethnic groups using DNA samples prepared using standard techniques. Our data suggest that gene conversion may play a significant role in producing variability of the PKD1 sequence in this population. The identification of additional mutations will help guide the study of polycystin-1 and better help us to understand the pathophysiology of this common disease.     

Shwachman syndrome: Exocrine pancreatic dysfunction and variable phenotypic expression

BACKGROUND & AIMS: Shwachman syndrome is an inherited condition with multisystemic abnormalities, including exocrine pancreatic dysfunction. The aim of this study was to evaluate the occurrence and progression of features in a large cohort of patients. METHODS: Clinical records of 25 patients with Shwachman syndrome were reviewed. RESULTS: Mean birth weight (2.92 +/- 0.51 kg) was at the 25th percentile. However, by 6 months of age, mean heights and weights were less than the 5th percentile. After 6 months of age, growth velocity was normal. Severe fat maldigestion due to pancreatic insufficiency was present in early life (fecal fat, 26% +/- 17% of fat intake; age, < 2 years). Serial assessment of exocrine pancreatic function showed persistent deficits of enzyme secretion, but 45% of patients showed moderate age-related improvements leading to pancreatic sufficiency. Neutropenia was the most common hematologic abnormality (88%), but leukopenia, thrombocytopenia, and anemia were also frequently encountered. Patients with hypoplasia of all three bone marrow cellular lines (n = 11) had the worst prognosis; 5 patients died, 2 of sepsis and 3 of acute myelogenous leukemia. Other findings included hepatomegaly and/or abnormal liver function test results and skeletal abnormalities. CONCLUSIONS: A wide and varied spectrum of phenotypic abnormalities among patients with Shwachman syndrome is described. Pancreatic acinar dysfunction is an invariable abnormality. Patients with severe bone marrow involvement may have a guarded prognosis. (Gastroenterology 1996 Dec;111(6):1593-602)     

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.     

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid cell-specific monoclonal antibodies

Second or third chemotherapy-induced remissions in acute myelogenous leukemia (AML) are limited by early relapse of the leukemia. We developed monoclonal antibodies (MoAbs) that are cytotoxic to myeloid leukemia cells to treat bone marrow from these patients ex vivo for autologous transplantation. In this pilot study, bone marrow was harvested from ten patients with AML in remission, treated with one or two complement-fixing MoAbs, PM-81 and AML-2-23, which react with myeloid differentiation antigens, incubated with rabbit complement, and cryopreserved. These MoAbs were chosen because they have broad reactivity with AML cells but not with pluripotent progenitor cells. At the time of transplant, 6 patients were in second complete remission, 1 each was in third complete or partial remission, and 2 were in early first relapse. The patients were treated with cyclophosphamide (60 mg/kg a day for 2 days) and total body irradiation (200 cGy twice a day for 3 days) and given infusions of MoAb-treated bone marrow. Full bone marrow reconstitution was observed in eight patients; two patients did not recover platelets. Seven of the ten patients are surviving and disease-free at 21.0, 15.0, 13.0, 10.0, 6.0, 3.0, and 2.0 months posttransplant. Treating bone marrow with MoAbs to myeloid differentiation antigens does not interfere with pluripotential stem cell engraftment. Longer follow-up and a controlled study are necessary to prove that the apparent efficacy of this therapeutic approach in some patients is attributable to MoAb-mediated killing of leukemia cells.     

The Future of Polycystic Kidney Disease Research—As Seen By the 12 Kaplan Awardees

Polycystic kidney disease (PKD) is one of the most common life-threatening genetic diseases. Jared J. Grantham, M.D., has done more than any other individual to promote PKD research around the world. However, despite decades of investigation there is still no approved therapy for PKD in the United States. In May 2014, the University of Kansas Medical Center hosted a symposium in Kansas City honoring the occasion of Dr. Grantham''s retirement and invited all the awardees of the Lillian Jean Kaplan International Prize for Advancement in the Understanding of Polycystic Kidney Disease to participate in a forward-thinking and interactive forum focused on future directions and innovations in PKD research. This article summarizes the contributions of the 12 Kaplan awardees and their vision for the future of PKD research.