The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.     

Identification of a locus which shows no genetic recombination with the autosomal dominant polycystic kidney disease gene on chromosome 16.

The major site for mutations leading to autosomal dominant polycystic kidney disease (ADPKD) is at the PKD1 locus, previously mapped to 16p13. Three additional probes have now been mapped within an existing array of genetic markers flanking this locus. One of these, CMM65b (D16S84), shows no recombination with PKD1 in 201 informative meioses. The others, Fr3-42 (D16S21) and EKMDA2 (D16S83), are shown to be the closest telomeric flanking markers. Somatic cell hybrids containing derivative chromosome 16s were used to construct a physical map of the region. Cosmid overlap cloning of the D16S84 region allowed a t(16;1) translocation breakpoint to be mapped at the molecular level, orientating the extended D16S84 locus with respect to the chromosome. The new markers and physical map described here provide an improved framework for attempts to clone the PKD1 region and to identify polycystic kidney disease mutations.     

Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.     

Linkage Disequilibrium in the Region of the Autosomal Dominant Polycystic Kidney Disease Gene (PKDI)

The gene for autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p, between the flanking markers D16S84 and D16S125 (26.6prox). This region is 750 kb long and has been cloned. We have looked at the association of 10 polymorphic markers from the region, with the disease and with each other. This was done in a set of Scottish families that had previously shown association with D16S94, a marker proximal to the PKD1 region. We report significant association between two CA repeat markers and the disease but have not found evidence for a single founder haplotype in these families, indicating the presence of several mutations in this population. Our results favor a location of the PKD1 gene in the proximal part of the candidate region.     

A long-range restriction map between the alpha-globin complex and a marker closely linked to the polycystic kidney disease 1 (PKD1) locus

Two polymorphic loci and two additional probes that map close to CMM65, which is tightly linked to the polycystic kidney disease 1 (PKD1) locus in chromosome band 16p13.3, are described. These new probes were isolated from a library that was enriched by preparative pulsed-field gel electrophoresis (PFGE) for sequences from a 320-kb NotI fragment that includes CMM65. Through the use of a panel of somatic cell hybrids and PFGE, the new polymorphic loci, PNL56S and NKISP1, were localized within 60 kb and approximately 250 kb distal to CMM65, respectively. A long-range restriction map linking these new probes and the distal markers EKMDA2, CMM103, and alpha-globin was constructed. These latter probes have been localized to regions approximately 900 kb, 1.2 Mb, and 1.9 Mb distal to CMM65, respectively. The entire region was found to be unusually rich in CpG dinucleotides. The new polymorphic probes and the long-range map will aid both the search for the PKD1 locus and the detailed characterization of this distal region of 16p.     

Fine-mapping of the spinal muscular atrophy locus to a region flanked by MAP1B and D5S6.

The microtubule-associated protein 1B (MAP1B) locus has been mapped in close proximity to spinal muscular atrophy (SMA) on chromosome 5q13. We have identified a second microsatellite within a MAP1B intron, which increases the heterozygosity of this locus to 94%. Two unambiguous recombination events establish MAP1B as a closely linked, distal flanking marker for the disease locus, while a third recombinant establishes D5S6 as the proximal flanking marker. The combination of key recombinants and linkage analysis place the SMA gene in an approximately 2-cM interval between loci D5S6 and MAP1B. Physical mapping and cloning locate MAP1B within 250 kb of locus D5S112. The identification and characterization of a highly polymorphic gene locus tightly linked to SMA will facilitate isolation of the disease gene, evaluation of heterogeneity, and development of a prenatal test for SMA.     

Chromosome jumping from flanking markers defines the minimal region for alf/hsdr-1 within the albino-deletion complex.

The locus alf/hsdr-1, defined by the albino-deletion complex on mouse chromosome 7, is essential for neonatal survival. Animals homozygous for a subset of the deletions die shortly after birth due to impaired gene expression in liver parenchymal cells and kidney proximal tubular cells. Here, we describe a detailed analysis of the region containing alf/hsdr-1 by means of chromosome jumping from flanking markers. Three chromosome jumping libraries based on the restriction enzymes XmaI and SalI were constructed. Isolation of eight jumping clones distributed over 450 kb allowed more than 240 kb to be cloned in genomic lambda and cosmid libraries. Five of the probes map within the minimal genetic interval for alf/hsdr-1, which is defined by the proximal borders of the deletions c10R75M and c11DSD. The breakpoints of these deletions were precisely mapped, which allowed alf/hsdr-1 to be localized to a 310-kb interval.     

A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.     

Gene Conversion Alone Accounts for More than 90% of Recombination Events at the Am Locus of Neurospora Crassa

We have used closely flanking molecular markers located ~4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am(1) am(6). Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (~0.07) than estimated previously using more distant flanking markers. This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences. Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (~0.35), our observation may have general significance.     

Saturating the region of the polycystic kidney disease gene with NotI linking clones.

A NotI-linking library was constructed from a radiation hybrid containing fragments of human chromosome 16. The clones were mapped on a panel of somatic cell hybrids, and 10 different NotI site-containing clones were localized close to and between genetic markers flanking the PKD1 locus. With pulsed-field gel analysis the clones were shown to be distributed over four adjacent ClaI fragments covering 1,200 kb.     

Limits of resolution of genetic linkage studies: implications for the positional cloning of human disease genes.

Positional cloning studies to identify disease genes are being carried out for many human genetic diseases. Such studies often include a genome-scan linkage analysis to identify the rough chromosomal location of a disease gene, fine structure genetic mapping to define and narrow the chromosomal interval in which the disease gene may be located, and physical mapping and gene identification in the genetically defined interval to clone the disease gene. During the planning of a positional cloning study, it is important to know that, if linkage is found, the genetic interval identified is likely to be sufficiently narrow to be dissected efficiently by methods of physical mapping and gene identification. Thus, we wish to know the limits of resolution of a genetic linkage study. In this paper, I determine for Mendelian diseases the distributions and moments of three measures of linkage resolution: (1) in a set of N chromosomes, the distance between the nearest crossovers that flank a disease locus, (2) the distance between the nearest genetic markers that flank the pair of flanking crossovers after a genome scan, and (3) the distance between the nearest flanking markers after additional randomly placed markers are generated and typed in an identified interval. These results provide explicit sample-size guidelines for future positional cloning studies of Mendelian diseases and make possible a more objective evaluation of whether a proposed positional cloning study is likely to be successful. I also briefly discuss the more difficult problem of linkage resolution for complex genetic diseases.     

Reciprocal crossovers and a positional preference for strand exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2

Smith-Magenis syndrome (SMS) is caused by an approximately 4-Mb heterozygous interstitial deletion on chromosome 17p11.2 in approximately 80%-90% of affected patients. Three large ( approximately 200 kb), complex, and highly homologous ( approximately 98%) low-copy repeats (LCRs) are located inside or flanking the SMS common deletion. These repeats, also known as "SMS-REPs," are termed "distal," "middle," and "proximal." The directly oriented distal and proximal copies act as substrates for nonallelic homologous recombination resulting in both the deletion associated with SMS and the reciprocal duplication: dup(17)(p11.2p11.2). Using restriction enzyme cis-morphism analyses and direct sequencing, we mapped the regions of strand exchange in 16 somatic-cell hybrids that harbor only the recombinant SMS-REP. Our studies showed that the sites of crossovers were distributed throughout the region of homology between the distal and proximal SMS-REPs. However, despite approximately 170 kb of high homology, 50% of the recombinant junctions occurred in a 12.0-kb region within the KER gene clusters. DNA sequencing of this hotspot (positional preference for strand exchange) in seven recombinant SMS-REPs narrowed the crossovers to an approximately 8-kb interval. Four of them occurred in a 1,655-bp region rich in polymorphic nucleotides that could potentially reflect frequent gene conversion. For further evaluation of the strand exchange frequency in patients with SMS, novel junction fragments from the recombinant SMS-REPs were identified. As predicted by the reciprocal-recombination model, junction fragments were also identified from this hotspot region in patients with dup(17)(p11.2p11.2), documenting reciprocity of the positional preference for strand exchange. Several potential cis-acting recombination-promoting sequences were identified within the hotspot. It is interesting that we found 2.1-kb AT-rich inverted repeats flanking the proximal and middle KER gene clusters but not the distal one. The role of any or all of these in stimulating double-strand breaks around this positional recombination hotspot remains to be explored.     

Genetic heterogeneity in adult dominant polycystic kidney disease in Cypriot families

Polycystic kidney disease is an inherited heterogeneous disorder that affects approximately 11000 Europeans. It is characterized mainly by the formation of cysts in the kidney that lead to end-stage renal failure with late age of onset. Three loci have been identified, PKD1 on the short arm of chromosome 16, which has recently been isolated and characterized, PKD2 on the long arm of chromosome 4, and a third locus of unknown location, that is apparently much rarer. In families that transmit the PKD2 gene there is a significantly later age of onset of symptoms, compared with families that transmit the PKD1 gene, and in general they present with milder progression of symptomatology. For the first time we attempted molecular genetic analysis in seven Cypriot families using highly polymorphic markers around the PKD1 and PKD2 genes. Our data showed that there is genetic and phenotypic heterogeneity among these families. For four of the families we obtained strong evidence for linkage to the PKD1 locus. In two of these families linkage to PKD1 was strengthened by excluding linkage to PKD2 with the use of marker D4S423. In three other families we showed linkage to the PKD2 locus. In the largest of these families one recombinant placed marker D4S1534 distal to D4S231, thereby rendering it the closest proximal marker known to us to date. The application of molecular methods allowed us to make presymptomatic diagnosis for a number of at-risk individuals.     

Probe,VK5B,is located in the same interval as the autosomal dominant adult polycystic kidney disease locus,PKD1

Summary The polymorphic DNA probe VK5B (D16S94) was mapped by genetic linkage in families from the Centre d'Etude de Polymorphisme Humain (CEPH) as being in the same interval as the autosomal dominant adult polycystic kidney disease locus (PKD1). The maximum likelihood estimate of the genetic location of VK5B using multipoint linkage analysis was 9.6cM proximal to {ie286-01} (D16S85) and 5.4cM distal to CRI-0327 (D16S63), in males. The VK5B probe may be useful in PKD1 families for prenatal and presymptomatic diagnosis of the disease. Additional typing of PKD1 families is required to determine whether the location of VK5B is distal or proximal to (PKD1).     

Hereditary neuralgic amyotrophy: evidence for genetic homogeneity and mapping to chromosome 17q25

Hereditary neuralgic amyotrophy (HNA) is a rare autosomal dominant disorder on chromosome 17q, associated with recurrent, episodic, painful brachial plexus neuropathy. Dysmorphic features, including hypotelorism, long nasal bridge and facial asymmetry, are frequently associated with HNA. To assess genetic homogeneity, determine the cytogenetic location, and identify flanking markers for the HNA locus, six pedigrees were studied with multiple DNA markers from distal chromosome 17q. The results in all pedigrees supported linkage of the HNA locus to chromosome 17. A maximum combined lod score (Ζ = 10.94, ￡ = 0.05) was obtained with marker D17S939 and the maximum multipoint lod score was 22.768 in the interval defined by D17S802– D17S939. An analysis of crossovers placed the HNA locus within an approximate 4.0-cM interval flanked by D17S1603 and D17S802. Analysis of DNA from a human/mouse somatic cell hybrid with linked markers suggests that band 17q25 harbors the HNA locus. These results support genetic homogeneity within HNA and define a specific interval and a precise cytogenetic location in chromosome 17q25 for this disorder. Received: 24 June 1997 / Accepted: 21 August 1997     

Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population.

Forty-one Spanish families with polycystic kidney disease 1 (PKD1) were studied for evidence of linkage disequilibrium between the disease locus and six closely linked markers. Four of these loci--three highly polymorphic microsatellites (SM6, CW3, and CW2) and an RFLP marker (BLu24)--are described for the first time in this report. Overall the results reveal many different haplotypes on the disease-carrying chromosome, suggesting a variety of independent PKD1 mutations. However, linkage disequilibrium was found between BLu24 and PKD1, and this was corroborated by haplotype analysis including the microsatellite polymorphisms. From this analysis a group of closely related haplotypes, consisting of four markers, was found on 40% of PKD1 chromosomes, although markers flanking this homogeneous region showed greater variability. This study has highlighted an interesting subpopulation of Spanish PKD1 chromosomes, many of which have a common origin, that may be useful for localizing the PKD1 locus more precisely.     

Evidence for negative interference: clustering of crossovers close to the am locus in Neurospora crassa among am recombinants.

In response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, Fincham proposed that recombination events at the Neurospora am locus rarely have an associated crossover. Fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am in these recombinants. In this article we reevaluate the data from three mapping studies of the am locus and report molecular evidence that shows crossovers to be clustered immediately proximal to am in am recombinants.     

A human Y-chromosome specific repeated DNA family (DYZ1) consists of a tandem array of pentanucleotides.

We have determined the complete nucleotide sequence of a 3564 bp EcoRI fragment which represents a major component of the human Y-chromosome specific repeated DNA family (DYZ1). Sequencing result showed a tandem array of pentanucleotides after five nucleotides were inserted or deleted at four positions. 229 out of the 713 pentanucleotides were TTCCA, and 297 were its single-base substituents. Southern hybridization analyses of male genomic DNAs showed that several endonuclease cleavage sites were located at intervals of 3.56kb in the DYZ1 locus. This indicates that the DYZ1 repeated DNA family evolved and expanded by unequal crossovers which occurred at distances of 3.56kb. As there is a uniformly distributed array of pentanucleotides on this locus, it is not a sequence homology that determines the distance of unequal crossovers. A higher order of chromatin structure may be involved in the determination of distance in unequal crossovers.     

The Localization of a Gene Causing X-Linked Cleft Palate and Ankyloglossia (CPX) in an Icelandic Kindred Is between DXS326 and DXYS1X

The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. We have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Z_max = 4.16 at θ = 0.08 (DXS326) and Z_max = 5.80 at θ = 0.06 (DXYS1X).