Novel mutations in the duplicated region of the polycystic kidney disease 1 (PKD1) gene provides supporting evidence for gene conversion

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human single-gene disorders, and is the most common inherited form of cystic kidney disease. It is estimated that approximately 85% of ADPKD is due to mutations in the PKD1 gene, which is located on chromosome 16p13.3. Mutation analysis in this gene is difficult, because more than two-thirds of reiterated several times at 16p13.1. In this study, mutation screening in 90 ADPKD patients was carried out on exons in the duplicated region of the PKD1 gene (23-34), using genomic long-range PCR followed by nested PCR and single-strand conformation polymorphism (SSCP), and finally cycle sequencing. Two nonconservative missense mutations were detected in exons 25 and 31, and two conservative mutations were found in exons 24 and 29. A novel splicing mutation, which is expected to cause skipping of exon 30, was detected in one case. Moreover, six intronic variants, three silent variants, and one polymorphic variant were detected in this study. Comparison between some of these changes and published sequences from the homologous genes on 16p13.1, revealed supporting evidence for the gene conversion theory as a mechanism responsible for some of the mutations in the PKD1 gene. Factors likely to facilitate gene conversion in this region of the PKD1 gene are discussed.     

Mutational analysis within the 3' region of the PKD1 gene in Japanese families.

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease that causes renal failure. One of the genes that is responsible for this disease, PKD1, has been identified and characterized. Many mutations of the PKD1 gene have been identified in the Caucasian population. We investigated the occurrence of mutations in this gene in the Japanese population. We analyzed each exon in the 3' single copy region of the gene between exons 35 and 46 in genomic DNA obtained from 69 patients, using a PCR-based direct sequencing method. Four missense mutations (T3509M, G3559R, R3718Q, R3752W), one deletion mutation (11307del61bp) and one polymorphism (L3753L) were identified, and their presence confirmed by allele-specific oligonucleotide (ASO) hybridization. These were novel mutations, except for R3752W, and three of them were identified in more than two families. Mutation analysis of the PKD1 gene in the Japanese population is being reported for the first time.     

Genomic structure and identification of novel mutations in usherin, the gene responsible for Usher syndrome type IIa

Usher syndrome type IIa (USHIIa) is an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa. This disorder maps to human chromosome 1q41. Recently, mutations in USHIIa patients were identified in a novel gene isolated from this chromosomal region. The USH2A gene encodes a protein with a predicted molecular weight of 171.5 kD and possesses laminin epidermal growth factor as well as fibronectin type III domains. These domains are observed in other protein components of the basal lamina and extracellular matrixes; they may also be observed in cell-adhesion molecules. The intron/exon organization of the gene whose protein we name "Usherin" was determined by direct sequencing of PCR products and cloned genomic DNA with cDNA-specific primers. The gene is encoded by 21 exons and spans a minimum of 105 kb. A mutation search of 57 independent USHIIa probands was performed with a combination of direct sequencing and heteroduplex analysis of PCR-amplified exons. Fifteen new mutations were found. Of 114 independent USH2A alleles, 58 harbored probable pathologic mutations. Ten cases of USHIIa were true homozygotes and 10 were compound heterozygotes; 18 heterozygotes with only one identifiable mutation were observed. Sixty-five percent (38/58) of cases had at least one mutation, and 51% (58/114) of the total number of possible mutations were identified. The allele 2299delG (previously reported as 2314delG) was the most frequent mutant allele observed (16%; 31/192). Three new missense mutations (C319Y, N346H, and C419F) were discovered; all were restricted to the previously unreported laminin domain VI region of Usherin. The possible significance of this domain, known to be necessary for laminin network assembly, is discussed in the context of domain VI mutations from other proteins.     

The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.     

Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease

DNA sequence analysis of the human Tg737 gene was performed in 36 patients with the autosomal recessive form of polycystic kidney disease (ARPKD). Coding exons and their adjacent splice sites were screened for mutations. Pathogenic exon or splice region mutations were not identified although one exonic and two intronic polymorphic sites were discovered. These results are in agreement with another study that has recently reported linkage to Chromosome (Chr) 6p21-cen in a set of 16 ARPKD families. STS mapping has localized the gene to a YAC contig that includes D13S175 on chromosome 13q12.1. The polymorphisms found in the hTg737 gene will permit its future evaluation as a candidate gene for other recessive cystic renal diseases and as a modifier gene in human PKD.     

Mutations in NPC1 Highlight a Conserved NPC1-Specific Cysteine-Rich Domain

Niemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3' portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.     

Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent inherited disorders. The majority of cases are due to mutation of the PKD1 gene, on 16p13.3, while in most of the remainder the disease maps to the PKD2 locus, at chromosome 4q21-q23. Recently, the PKD2 gene has been positionally cloned and three nonsense mutations within the coding sequence of the gene identified. Here we report a systematic mutation screening of all 15 exons of the PKD2 gene in chromosome 4-linked ADPKD families, using heteroduplex and SSCP analyses. We have identified and characterized seven novel mutations, with a detection rate of approximately 90% in the population studied. All of the mutations result in the premature stop of translation: four nonsense changes and three deletions. The deletions are all frameshifting, of four T nucleotides in one case and one G nucleotide in the other two. All mutations are unique and are distributed throughout the gene without evidence of clustering. Comparison of specific mutations with the clinical profile in ADPKD2 families shows no clear correlation. Received: 5 April 1997 / Accepted: 31 July 1997     

Identification of two novel polycystic kidney disease-1-like genes in human and mouse genomes

Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.     

Mutation detection in the repeated part of the PKD1 gene.

The principle cause of one of the most prevalent genetic disorders, autosomal dominant polycystic kidney disease, involves mutations in the PKD1 gene. However, since its identification in 1994, only 27 mutations have been published. Detection of mutations has been complicated because the greater part of the gene lies within a genomic region that is reiterated several times at another locus on chromosome 16. Amplification of DNA fragments in the repeated part of the PKD1 gene will lead to coamplification of highly homologous fragments derived from this other locus. These additional fragments severely hamper point-mutation detection. None of the point mutations published to date are located in the repeated part of the PKD1 gene. However, we have reduced the problems posed by the strong homology, by using the protein-truncation test, and we have identified eight novel mutations, seven of which are located in the repeated part of the PKD1 gene.     

Mutation detection of PKD1 identifies a novel mutation common to three families with aneurysms and/or very-early-onset disease

It is known that several of the most severe complications of autosomal-dominant polycystic kidney disease, such as intracranial aneurysms, cluster in families. There have been no studies reported to date, however, that have attempted to correlate severely affected pedigrees with a particular genotype. Until recently, in fact, mutation detection for most of the PKD1 gene was virtually impossible because of the presence of several highly homologous loci also located on chromosome 16. In this report we describe a cluster of 4 bp in exon 15 that are unique to PKD1. Forward and reverse PKD1-specific primers were designed in this location to amplify regions of the gene from exons 11-21 by use of long-range PCR. The two templates described were used to analyze 35 pedigrees selected for study because they included individuals with either intracranial aneurysms and/or very-early-onset disease. We identified eight novel truncating mutations, two missense mutations not found in a panel of controls, and several informative polymorphisms. Many of the polymorphisms were also present in the homologous loci, supporting the idea that they may serve as a reservoir for genetic variability in the PKD1 gene. Surprisingly, we found that three independently ascertained pedigrees had an identical 2-bp deletion in exon 15. This raises the possibility that particular genotypes may be associated with more-severe disease.     

The sequence, expression, and chromosomal localization of a novel polycystic kidney disease 1-like gene, PKD1L1, in human

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Since the first two polycystins were cloned, three new members, polycystin-L, -2L2, and -REJ, have been identified. In this study, we describe a sixth member of the family, polycystin-1L1, encoded by PKD1L1 in human. The full-length cDNA sequence of PKD1L1, determined from human testis cDNA, encodes a 2849-amino-acid protein and 58 exons in a 187-kb genomic region. The deduced amino acid sequence of polycystin-1L1 has significant homology with all known polycystins, but the longest stretches of homology were found with polycystin-1 and -REJ over the 1453- and 932-amino-acid residues, respectively. Polycystin-1L1 is predicted to have two Ig-like PKD, a REJ, a GPS, a LH2/PLAT, a coiled-coil, and 11 putative transmembrane domains. Several rhodopsin-like G-protein-coupled receptor (GPCR) signatures are also found in polycystin-1L1. Dot-blot analysis and RT-PCR revealed that human PKD1L1 is expressed in testis and in fetal and adult heart. In situ hybridization analysis showed that the most abundant and specific expression of Pkd1l1 was found in Leydig cells, a known source of testosterone production, in mouse testis. We have assigned PKD1L1 to the short arm of human chromosome 7 in bands p12--p13 and Pkd1l1 to mouse chromosome 11 in band A2 by fluorescence in situ hybridization. We hypothesize a role for polycystin-1L1 in the heart and in the male reproductive system.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.     

Remarkable sequence conservation of the last intron in the PKD1 gene

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.     

Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20–27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD. Received: 13 July 1996 / Revised: 6 August 1996