Abstract 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.

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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. Introduction The gene responsible for the most severe form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has several notable features. It has a compact genomic structure with 46 exons clustered within a 50 kb genomic segment. It encodes a relatively long mRNA of 14 kb that is predicted to produce a membrane glycoprotein of 4302 amino acids that participates in cell-cell or cell-matrix interactions. Approximately 70% of the gene's length is present in at least three faithful copies at 16p13.1 (,).

The duplicated region extends from exon 1 to intron 34 and includes all intervening sequences. The PKD1 copies are transcribed but their respective mRNA molecules can be distinguished from authentic PKD1 transcripts on the basis of size. It is not known whether the homologous transcripts are translated into proteins.

Finally, bisecting intron 21 of PKD1 is an unusual polypyrimidine tract of ∼2.5 kb (,). This element is also present in the PKD1 homologs (unpublished data). Although mutations in PKD1 account for ∼85% of all forms of ADPKD, relatively few mutations have been reported since the gene's identification in 1994 and most have been clustered in the unique portion of the gene (,). The major obstacle to mutation detection in the 5′ region of the gene has been the presence of the homologs described above whose sequence is nearly identical to that of PKD1. Recently, our group has devised a strategy for mutation detection in the duplicated region of PKD1. Our method uses one gene-specific primer, PKD1, as an anchor in combination with a primer from the duplicated portion to amplify PKD1-specific templates (3′LR, ) that are ∼10 kb in length and include exons 23–34 or exons 23–38.

We have demonstrated that 3′LR is PKD1 specific once it has been diluted sufficiently (1:10 4 or 1:10 5) to remove genomic contamination. The diluted long-range PCR product can therefore be used for nested PCR of any exon contained within it.

These products can then be analyzed for PKD1 mutations with conventional methods such as heteroduplex or single strand conformation polymorphism (SSCP). Using this technique, we previously have identified an unusual cluster of nearly identical base pair transitions involving exon 23 in two unrelated individuals.

These changes were predicted to result in multiple non-conservative amino acid substitutions in a short stretch of the protein. Both the unusual pattern of these mutations and their apparent independent origin prompted us to test whether they might have arisen as a result of a gene conversion event between PKD1 and its homologs.

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Results summarizes the base pair substitutions found in patients JHU273 and JHU086. These mutations were detected in a screen of ∼80 affected individuals. The restriction sites that are created or destroyed by each sequence change are noted in the table and the corresponding restriction maps for the 5′ portion of exon 23 are shown. Each change was confirmed by restriction analysis and the data for JHU086 are presented in A and B. Although JHU273 and JHU086 were found to have similar base pair changes, they were not identical. Each patient contained the same sequence substitutions at base pairs 8446, 8490 and 8502 of the published cDNA sequence (accession no.

L33243), but JHU086 was found to have an additional C→G transversion at base pair 8498. This substitution alters amino acid number 2763 (accession no. A38971), converting a leucine to a valine. It was possible to confirm that these patients differed at base pair 8498 by restriction digest since the first portion of exon 23 in JHU086 but not JHU273 contained an additional Tsp45I site (data not shown).

JHU273 initially was reported to have an additional sequence substitution not present in either normal subjects or JHU086. Although this change (C8688T) could not be assayed directly by restriction analysis, we had concluded it was a true change because it was found in clones derived from two affected family members. We were unable to confirm this change, however, in either the 3′LR or the blood of JHU273 using a PCR-based strategy that creates a StyI site in the presence of the mutant sequence. The structure of the PKD1 gene is shown including the location of the 46 exons that encode its protein product, polycystin.

The duplicated portion of PKD1 is represented by an unfilled box extending from exon 1 to intron 34. The striped box in intron 21 identifies the position of the 2.5 kb polypyrimidine tract. The horizontal boxes below the genomic structure represent PCR products used in this study (3′LR, exon 23). The 3′LR is a PKD1-specific long-range PCR product that is amplified using either KG8R25 or KG8R5 in combination with TWF1. Mutation analysis of exon 23 was performed on nested products amplified from 3′LR using primers TWF1 and 23R1. The vertical lines intersecting the exon 23 PCR product indicate the approximate position of the base pair substitutions detected in JHU086 and JHU273 that are listed in.

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The line marked by an asterisk indicates the position of the nucleotide substitution that is present only in JHU086. The structure of the PKD1 gene is shown including the location of the 46 exons that encode its protein product, polycystin. The duplicated portion of PKD1 is represented by an unfilled box extending from exon 1 to intron 34. The striped box in intron 21 identifies the position of the 2.5 kb polypyrimidine tract. The horizontal boxes below the genomic structure represent PCR products used in this study (3′LR, exon 23).

The 3′LR is a PKD1-specific long-range PCR product that is amplified using either KG8R25 or KG8R5 in combination with TWF1. Mutation analysis of exon 23 was performed on nested products amplified from 3′LR using primers TWF1 and 23R1. The vertical lines intersecting the exon 23 PCR product indicate the approximate position of the base pair substitutions detected in JHU086 and JHU273 that are listed in. The line marked by an asterisk indicates the position of the nucleotide substitution that is present only in JHU086. The base pair substitutions identified in JHU086 and JHU273 are also found in the cell line N23HA that contains the PKD1 homologs. The 5′ portion of exon 23 was amplified as described in Materials and Methods from N23HA (lane 4), 145.19 (lane 3), a mutant allele cloned from JHU086 (lane 1) and 3′LR from JHU086 (lane 2).

The products were digested with each of the four restriction enzymes indicated in ( A) and ( B). Complete digestion with Tsp45l required prolonged incubation which resulted in a high degree of non-specific activity and smearing of the 172 bp band seen in the N23HA digest. The base pair substitutions identified in JHU086 and JHU273 are also found in the cell line N23HA that contains the PKD1 homologs. The 5′ portion of exon 23 was amplified as described in Materials and Methods from N23HA (lane 4), 145.19 (lane 3), a mutant allele cloned from JHU086 (lane 1) and 3′LR from JHU086 (lane 2).

The products were digested with each of the four restriction enzymes indicated in ( A) and ( B). Complete digestion with Tsp45l required prolonged incubation which resulted in a high degree of non-specific activity and smearing of the 172 bp band seen in the N23HA digest. Summary of sequence variants detected in JHU086 and JHU273 In order to determine whether these mutations were the result of a gene conversion event, we took advantage of two cell lines. The first is a rodent-human somatic cell hybrid, N23HA, that contains most of chromosome 16 including the homologs but lacks the PKD1 region at 16p13.3 (,). The second is a radiation hybrid, 145.19, that contains a small (∼3 Mb) segment of chromosome 16p13.3 which includes PKD1 but not the homologous loci (,). The 5′ portion of exon 23, containing the cluster of base pair substitutions, was amplified from N23HA, 145.19 and from the appropriately diluted 3′LR of JHU086. As a positive control, the PCR product was also amplified from a clone containing the mutant sequence.

The PCR products were digested by each of the four restriction enzymes that either gained or lost a site in the mutant sequence. The results are presented in A and B. The cell line 145.19 (lane 3) shows the normal restriction pattern for each enzyme as predicted by the published PKD1 sequence.

In contrast, a combination of restriction patterns is observed for N23HA (lane 4). For each enzyme, the normal pattern is found in addition to that present in the cloned mutant allele (lane 1), specifically gain of BstUI and Tsp45I sites and loss of FspI and MslI sites. Alignment of the exon 23 sequence of PKD1 (N), two homologous loci (H1, H2), JHU086 and JHU273. The numbers identify the position of the segment with respect to the PKD1 cDNA sequence in accession no. The dotted lines represent a region of complete homology shared by all sequences.

Nucleotides that differ from the published PKD1 sequence are boxed. These findings suggest that not all of the homologous loci harbor identical sequence changes. Further evidence in support of this conclusion is provided in. A survey of the GenBank database identified the sequence of a bacterial artificial chromosome (BAC) that maps to 16p13.11 and contains two homologous copies (H1, H2) of PKD1 (accession no. An alignment of the exon 23-like sequences of H1 and H2 reveals significant sequence differences. H1 more closely resembles PKD1 in the region of interest whereas the sequence of H2 exactly matches that of JHU086. The latter observation strongly suggests that an H2-like locus served as template for the JHU086 gene conversion event.

It should be noted that the sequence of JHU273 differs from both H1 and H2, implying the existence of at least one other class of homologous sequence. We next sought to determine whether the base pair substitutions detected in the cell line N23HA and present in the 16p13.11 BAC were common to homologs in a broader spectrum of individuals. The 5′ portion of exon 23 was therefore amplified from both total genomic DNA and 3′LR of two affected (lanes 1 and 2) and two unaffected subjects (lanes 3 and 4). None of these four individuals previously had exhibited heteroduplex formation in PKD1 exon 23. Restriction patterns for total genomic DNA and 3′LR were compared for each of the four enzymes ( BstU I, Tsp45I, FspI and MslI). The representative result for one affected and one unaffected individual is shown in.

When the product amplified from total genomic DNA (containing both PKD1 and its homologs) was subjected to restriction digest, a pattern similar to that observed for N23HA was detected (lanes 2 and 4). The base pair substitutions detected in the cell line N23HA are common to homologs in a broader spectrum of individuals.

The 5′ portion of exon 23 was amplified from total genomic DNA (lanes 2 and 4) and 3′LR (lanes 1 and 3) of two affected (lanes 1 and 2) and two unaffected subjects (lanes 3 and 4) and then digested with the enzymes as indicated. A small amount of full-length product is left in lanes 1 and 3 even after prolonged incubation with MslI and FspI because these enzymes cut with lower efficiency. Data for Tsp 45I are not shown. The base pair substitutions detected in the cell line N23HA are common to homologs in a broader spectrum of individuals. The 5′ portion of exon 23 was amplified from total genomic DNA (lanes 2 and 4) and 3′LR (lanes 1 and 3) of two affected (lanes 1 and 2) and two unaffected subjects (lanes 3 and 4) and then digested with the enzymes as indicated. A small amount of full-length product is left in lanes 1 and 3 even after prolonged incubation with MslI and FspI because these enzymes cut with lower efficiency. Data for Tsp 45I are not shown.

Discussion We have used PKD1-specific reagents to identify a novel type of mutation in two patients consisting of a similar but non-identical cluster of base pair substitutions in the 5′ portion of exon 23. These changes would be predicted to alter several amino acids within a short region of polycystin.

Although we previously had reported that this unusual grouping of base pair substitutions was consistent with the pattern of mutation associated with triple helix formation and faulty transcription-coupled repair, the data presented here suggest a different mutational mechanism. We have demonstrated using the cell line N23HA and total blood DNA from several individuals that these substitutions appear to reflect the sequence present in at least a subset of PKD1 homologs. Sequence analysis of a subset of homologous loci supports this conclusion.

It therefore seems more likely that the mutations in JHU273 and JHU086 arose as the result of two independent gene conversion events between PKD1 and a homologous copy located more proximally on chromosome 16. The subtle differences in sequence noted between JHU086 and JHU273 is probably due to conversion between different homologous loci. It is difficult to establish the exact boundaries of the conversion tract in either case because the homologous sequences have not yet been defined completely.

Nonetheless, the minimum size for the tract in both JHU086 and JHU273 must be 56 bp. If one assumes that H2 is the likely template for JHU086, one can estimate a maximum tract size of 127 bp since JHU086 and H2 differ at positions 8418 and 8545 of the PKD1 sequence.

Gene conversion is a well recognized phenomenon that was first studied extensively in yeast and has been defined as the non-reciprocal exchange of genetic information. It has been postulated that gene conversion and recombination may be related processes that involve pairing of homologous sequences except that in gene conversion genetic information is transferred from the donor gene to the recipient without the donor being modified in the process Gene conversion has been identified as a mechanism of mutation for a number of human genes and their related pseudogenes. An interesting example is 21-hydroxylase deficiency, the most common form of congenital adrenal hyperplasia. In this disorder, gene conversion accounts for ∼75% of mutant alleles (,).

The functional 21-hydroxylase gene ( CYP21) and a non-functional pseudogene ( CYP21P) on chromosome 6 are located on tandemly repeated 30 kb segments that share ∼98% sequence identity. The pseudogene contains small deletions, insertions and point mutations that prevent synthesis of a functional enzyme and are deleterious when they become part of the CYP21 gene.

There are a number of factors that may play a role in the high rate of gene conversion noted for 21-hydroxylase and perhaps PKD1. These include the presence of loci with high degrees of sequence identity over an extended region as well as the number and proximity of these homologous loci. Both CYP21 and PKD1 share 95% sequence identity over many thousands of base pairs with their respective pseudogenes (,). This could promote homologous pairing and therefore recombination. In support of this, it has been found that in some eukaryotic systems, as shared homology decreases in length, recombination rate (and gene conversion) decreases concomitantly.

Although CYP21 and PKD1 resemble each other in some respects, they differ in the number and proximity of their homologous loci. The PKD1 gene is replicated in at least three copies that are located megabases away while CYP21 has only one tandemly repeated unit. How these differences might affect the rate of gene conversion is not clear.

One might predict that in the case of PKD1, the existence of multiple highly homologous pseudogenes might increase the frequency of genetic exchange. However, at least one study looking at interchromosomal gene conversion events using a binary transgenic mouse system suggested the opposite. The transgene copy number was not found to be related to conversion frequency although the homologous sequences used in the model were relatively short (∼2.5 kb). The importance of proximity in the frequency of gene conversion also seems unclear since interchromosomal gene conversion events have been documented both in the transgenic murine system described above and for at least one other human gene. Multiple adjacent nucleotide substitutions have been described in the von Willebrand factor gene on chromosome 12 that mimic the sequence of its pseudogene located on chromosome 22.

Therefore, there may be other factors such as genome context or particular sequence elements that may be more significant than physical distance alone in promoting non-reciprocal recombination. As noted previously, both PKD1 and its homologs contain unusual polypyrimidine tracts that are situated in adjacent introns (unpublished data). These elements have been postulated to form triple helices under appropriate conditions that could conceivably contribute to mutagenesis by more than one mechanism. In one example using a bacterial system, triplex formation by a poly(dG)-poly(dC) tract separating two homologous sequences was shown to promote recombination when transcription of the downstream gene was activated (,).

Since recombination and gene conversion are closely related, factors that increase recombination might also be expected to increase the frequency of gene conversion. Ultimately the role of these polypyrimidine elements in generating mutations via gene conversion or other mechanisms will need to be confirmed using cell culture systems. In summary, we believe that gene conversion is a likely cause of mutation in PKD1.

We have screened a relatively small number of individuals and have identified two with apparent mutations generated via gene conversion events in the same region of exon 23. It remains to be determined whether gene conversion occurs in other locations within the duplicated portion of PKD1 or whether this process also plays a role in generating somatic mutations, a step thought to be rate limiting in cyst formation. This study highlights the importance of reagents proven to be locus specific in identifying PKD1 mutations that may reflect sequence differences also present in the homologous loci.

Materials and Methods Patient recruitment Affected individuals were recruited from dialysis centers and nephrology clinics. The diagnosis of ADPKD was established using standard criteria (,).

Family members of probands were recruited to participate after receiving permission from the donors. Blood samples were obtained after receiving informed consent and in accordance with institutional guidelines. Long-range PCR Genomic DNA was isolated from whole blood or the cell lines, 145.19 and N23HA using the Puregene kit (Gentra) and the manufacturer's protocol. 3′LR was amplified using 300 ng of genomic DNA as template and the primers TWF1 (5′-CTGCACTGACCTCACGCATGT-3′, genomic position 37 678–37 698) and KG8R25 (5′-GTTGCAGCCAAGCCCATGTTA-3′, genomic position 47 320–47 340), or KG8R5 (5′-GCGCTTTGCAGACGGTAGGCG-3′, genomic position 48 579–48 599).

PCR was performed using conditions previously described with the rTth DNA polymerase, XL (Perkin Elmer-Cetus). PCR The long-range template 3′LR was diluted serially and its specificity was confirmed as previously described. Two microliters of the diluted (10 4 or 10 5) 3′LR or 200 ng of genomic DNA was then used as template for amplification of the first portion of exon 23 using the primers TWF1 and 23R1 (5′-GCCAAAGGGAAAGGGATTGGA-3′). PCR was performed as follows: denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 s, 67°C for 30 s, 72°C for 30 s and a final extension of 72°C for 10 min. The total PCR volume was 30 µl using 2 U of Taq DNA polymerase (Boehringer Mannheim), and a final MgCl 2 concentration of 1.5 mM. Restriction digests Restriction digests were performed in a total volume of 30 µl by incubating 5–10 µl of the PCR product with the appropriate enzyme according to the manufacturer's protocol.

The entire digest was then run on a 4% Nusieve gel and products visualized with ethidium bromide. Acknowledgements We are grateful to all ADPKD family members for their invaluable participation. We thank Ms Sidney McGaughey for her assistance in the preparation of the manuscript.

This work was supported by grants from the NIH (G.G.G., DK48006), Maryland NKF (T.W.) and JHU Institutional Solo Cup Award (T.W.). Is the Irving Blum Scholar of The Johns Hopkins University School of Medicine.