An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons

In this report, we show that the Caenorhabditis elegans gene osm-5 is homologous to the Chlamydomonas gene IFT88 and the mouse autosomal recessive polycystic kidney disease (ARPKD) gene, Tg737. The function of this ARPKD gene may be evolutionarily conserved: mutations result in defective ciliogenesis in worms [1], algae [2], and mice [2, 3]. Intraflagellar transport (IFT) is essential for the development and maintenance of motile and sensory cilia [4]. The biochemically isolated IFT particle from Chlamydomonas flagella is composed of 16 polypeptides in one of two Complexes (A and B) [5, 6] whose movement is powered by kinesin II (anterograde) and cytoplasmic dynein (retrograde) [7-9]. We demonstrate that OSM-5 (a Complex B polypeptide), DAF-10 and CHE-11 (two Complex A polypeptides), and CHE-2 [10], a previously uncategorized IFT polypeptide, all move at the same rate in C. elegans sensory cilia. In the absence of osm-5, the C. elegans autosomal dominant PKD (ADPKD) gene products [11] accumulate in stunted cilia, suggesting that abnormal or lack of cilia or defects in IFT may result in diseases such as polycystic kidney disease (PKD).     

Exon skipping in IVD RNA processing in isovaleric acidemia caused by point mutations in the coding region of the IVD gene

Isovaleric acidemia (IVA) is a recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). We have reported elsewhere nine point mutations in the IVD gene in fibroblasts of patients with IVA, which lead to abnormalities in IVD protein processing and activity. In this report, we describe eight IVD gene mutations identified in seven IVA patients that result in abnormal splicing of IVD RNA. Four mutations in the coding region lead to aberrantly spliced mRNA species in patient fibroblasts. Three of these are amino acid altering point mutations, whereas one is a single-base insertion that leads to a shift in the reading frame of the mRNA. Two of the coding mutations strengthen pre-existing cryptic splice acceptors adjacent to the natural splice junctions and apparently interfere with exon recognition, resulting in exon skipping. This mechanism for missplicing has not been reported elsewhere. Four other mutations alter either the conserved gt or ag dinucleotide splice sites in the IVD gene. Exon skipping and cryptic splicing were confirmed by transfection of these mutations into a Cos-7 cell line model splicing system. Several of the mutations were predicted by individual information analysis to inactivate or significantly weaken adjacent donor or acceptor sites. The high frequency of splicing mutations identified in these patients is unusual, as is the finding of missplicing associated with missense mutations in exons. These results may lead to a better understanding of the phenotypic complexity of IVA, as well as provide insight into those factors important in defining intron/exon boundaries in vivo.     

Autosomal recessive polycystic kidney disease does not map to the second gene locus for autosomal dominant polycystic kidney disease on chromosome 4

Linkage analysis in 19 families with autosomal recessive polycystic kidney disease (ARPKD) has shown that ARPKD is not linked to the recently assigned second gene locus for autosomal dominant polycystic kidney disease (ADPKD) on chromosome 4q (PKD2). Thus, there is strong evidence that ADPKD and ARPKD have different gene loci.     

Germline PKHD1 mutations are protective against colorectal cancer

The autosomal recessive polycystic kidney disease (ARPKD) gene, PKHD1, has been implicated in the genesis or growth of colorectal adenocarcinoma, as a high level of somatic mutations was found in colorectal tumor tissue. To determine whether carriers of a single PKHD1 mutation are at increased risk of colorectal carcinoma, we assessed the prevalence of the commonest European mutation, T36M. First, we assayed a European cohort of ARPKD patients and found T36M was responsible for 13.1% of mutations. We then investigated two European cohorts with colorectal adenocarcinoma versus two control cohorts of similar age and gender. Screening for the most common PKHD1 mutation, T36M, we detected 15:3,603 (0.42%) controls versus 1:3,767 (0.027%) colorectal cancer individuals, indicating that heterozygous PKHD1 mutations are not a risk factor and are protective (p=0.0002). We also show that the carriage rate for PKHD1 mutations in the European population is higher than previous accepted at 3.2% (1:31 genomes).     

Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) belongs to a group of congenital hepatorenal fibrocystic syndromes characterized by dual renal and hepatic involvement of variable severity. Despite the wide clinical spectrum of ARPKD (MIM 263200), genetic linkage studies indicate that mutations at a single locus, PKHD1 (polycystic kidney and hepatic disease 1), located on human chromosome region 6p21.1–p12, are responsible for all phenotypes of ARPKD. Identification of cystic disease genes and their encoded proteins has provided investigators with critical tools to begin to unravel the molecular and cellular mechanisms of PKD. PKD cystic epithelia share common phenotypic abnormalities despite the different genetic mutations that underlie the disease. Recent studies have shown that many cyst-causing proteins are expressed in multimeric complexes at distinct subcellular locations within epithelia. This co-expression of cystoproteins suggests that cyst formation, regardless of the underlying disease gene, results from perturbations in convergent and/or integrated signal transduction pathways. To date, no specific therapies are in clinical use for ameliorating cyst growth in ARPKD. However, studies noted in this review suggest that therapeutic targeting of the cAMP and epidermal growth factor receptor (EGFR)-axis abnormalities in cystic epithelia may translate into effective therapies for ARPKD and, by analogy, autosomal dominant polycystic kidney disease (ADPKD). A particularly promising approach appears to be the targeting of downstream intermediates of both the cAMP and EGFR axis. This review focuses on ARPKD and presents a concise summary of the current understanding of the molecular genetics and cellular pathophysiology of this disease. It also highlights phenotypic and mechanistic similarities between ARPKD and ADPKD.The authors are supported by the National Institutes of Health (grant no. 1-P50-DK57306), the PKD Foundation (grant no. 76a2r), and the Children’s Research Institute, Children’s Hospital of Wisconsin.     

The Severe Perinatal Form of Autosomal Recessive Polycystic Kidney Disease Maps to Chromosome 6p21.1-p12: Implications for Genetic Counseling

Autosomal recessive polycystic kidney disease (ARPKD) is a one of the most common hereditary renal cystic diseases in children. Its clinical spectrum is widely variable with most cases presenting in infancy. Most affected neonates die within the first few hours of life. At present, prenatal diagnosis relies on fetal sonography, which is often imprecise in detecting even the severe form of the disease. Recently, in a cohort of families with mostly milder ARPKD phenotypes, an ARPKD locus was mapped to a 13-cM region of chromosome 6p21-cen. To determine whether severe perinatal ARPKD also maps to chromosome 6p, we have analyzed the segregation of seven microsatellite markers from the ARPKD interval in 22 families with the severe phenotype. In the majority of the affected infants, ARPKD was documented by histopathology. Our data confirm linkage and refine the ARPKD region to a 3.8-cM interval, delimited by the markers D6S465/D6S427/D6S436/D6S272 and D6S466. Taken together, these results suggest that, despite the wide variability in clinical phenotypes, there is a single ARPKD gene. These linkage data and the absence of genetic heterogeneity in all families tested to date have important implications for DNA-based prenatal diagnoses as well as for the isolation of the ARPKD gene.     

Autosomal recessive and dominant forms of polycystic kidney disease are not allelic

Linkage analysis has been carried out in 11 kindreds with autosomal recessive polycystic kidney disease (ARPKD) using the genetic marker 3'HVR, closely linked (theta = 0.05) to the gene of the autosomal dominant type. Close linkage (theta less than or equal to 0.20) between the locus of the marker and that of ARPKD can be excluded. These data strongly suggest that the loci for the autosomal recessive and dominant forms of polycystic kidney disease are not allelic.     

A novel mutation identified in PKHD1 by targeted exome sequencing: Guiding prenatal diagnosis for an ARPKD family

Autosomal recessive polycystic kidney disease (ARPKD) is a rare hereditary renal cystic disease involving multiple organs, mainly the kidney and liver. Parents who had an affected child with ARPKD are in strong demand for an early and reliable prenatal diagnosis to guide the future pregnancies. Here we provide an example of prenatal diagnosis of an ARPKD family where traditional antenatal ultrasound examinations failed to produce conclusive results till 26th week of gestation. Compound heterozygous mutations c.274C>T (p.Arg92Trp) and c.9059T>C (p.Leu3020Pro) were identified using targeted exome sequencing in the patient and confirmed by Sanger sequencing. Further, the mother and father were revealed to be carriers of heterozygous c.274C>T and c.9059T>C mutations, respectively. Molecular prenatal diagnosis was performed for the current pregnancy by direct sequencing plus linkage analysis. Two mutations identified in the patient were both found in the fetus. In conclusion, compound heterozygous PKHD1 mutations were elucidated to be the molecular basis of the patient with ARPKD. The newly identified c.9059T>C mutation in the patient expands mutation spectrum in PKHD1 gene. For those ultrasound failed to provide clear diagnosis, we propose the new prenatal diagnosis procedure: first, screening underlying mutations in PKHD1 gene in the proband by targeted exome sequencing; then detecting causative mutations by direct sequencing in the fetal DNA and confirming results by linkage analysis.     

Molecular analysis of the fibrinogen gene cluster in 16 patients with congenital afibrinogenemia: novel truncating mutations in the FGA and FGG genes

Congenital afibrinogenemia is an autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease in a non-consanguineous Swiss family. These were homozygous deletions of approximately 11 kb of the fibrinogen alpha chain gene (FGA). Our subsequent study revealed that the majority of cases were attributable to truncating mutations in FGA, with the most common mutation affecting the donor splice site in FGA intron 4 (IVS4+1 G-->T). Here, we report 13 further unrelated patients with mutations in FGA, confirming the relative importance of this gene compared with FGG and FGB in the molecular aetiology of afibrinogenemia. Three other patients were homozygous for mutations in FGG. Eight novel mutations were identified: five in FGA and three in FGG. Sufficient mutation data is now available to permit an effective strategy for the genetic diagnosis of congenital afibrinogenemia.     

Only one splice variant of the human TAZ gene encodes a functional protein with a role in cardiolipin metabolism

Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the TAZ gene and is characterized by cardiomyopathy, short stature, neutropenia, and 3-methylglutaconic aciduria. Recently it was found that BTHS patients exhibit a profound cardiolipin deficiency although the biosynthetic capacity to synthesize this lipid from its precursor phosphatidylglycerol is entirely normal. Like BTHS patients, a Saccharomyces cerevisiae strain, in which the yeast orthologue of the human TAZ gene has been disrupted, exhibits an abnormal cardiolipin profile as determined by tandem mass spectrometry. Additionally, this yeast strain grows poorly on non-fermentable carbon sources. We have used both properties of this yeast disruptant as a read-out system to test the physiological functionality of each of 12 different splice variants that have been reported for the human TAZ gene. Our results demonstrate that only the splice variant lacking exon 5 was able to complement the retarded growth of the yeast disruptant on selective plates and restore the cardiolipin profile to the wild type pattern. We conclude that this splice variant most likely represents the only physiologically important mRNA, at least with regard to cardiolipin metabolism.     

The search for mutations in the gene for the beta subunit of the cGMP phosphodiesterase (PDEB) in patients with autosomal recessive retinitis pigmentosa.

The finding of a mutation in the beta subunit of the cyclic GMP (cGMP) phosphodiesterase gene causing retinal degeneration in mice (the Pdeb gene) prompted a search for disease-causing mutations in the human phosphodiesterase gene (PDEB gene) in patients with retinitis pigmentosa. All 22 exons including 196 bp of the 5' region of the PDEB gene have been assessed for mutations by using single-strand conformational polymorphism analysis in 14 patients from 13 unrelated families with autosomal recessive retinitis pigmentosa (ARRP). No disease-causing mutations were found in this group of affected individuals of seven different ancestries. However, a frequent intronic and two exonic polymorphisms (Leu489----Gln and Gly842----Gly) were identified. Segregation analysis using these polymorphic sites excludes linkage of ARRP to the PDEB gene in a family with two affected children.     

Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase. In order to characterize mutations, RNA was isolated from cultured fibroblasts from 13 unrelated patients with neonatal citrullinemia. Ten mutations were identified by sequencing of amplified cDNA. Seven single base missense mutations were identified: Gly14----Ser, Ser180----Asn, Arg157----His, Arg304----Trp, Gly324----Ser, Arg363----Trp, and Gly390----Arg. Six of these missense mutations involved conversion of a CpG dinucleotide in the sense strand to TpG or CpA, and six of the seven mutations alter a restriction enzyme site in the cDNA. Two mutations were observed in which the sequences encoded by a single exon (exon 7 or 13) were absent from the cDNA. One mutation is a G----C substitution in the last position of intron 15 resulting in splicing to a cryptic splice site within exon 16. There is extreme heterogeneity of mutations causing citrulinemia. This heterogeneity may prove typical for less common autosomal recessive human genetic diseases.     

Atmin modulates Pkhd1 expression and may mediate Autosomal Recessive Polycystic Kidney Disease (ARPKD) through altered non-canonical Wnt/Planar Cell Polarity (PCP) signalling

Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a genetic disorder with an incidence of ~1:20,000 that manifests in a wide range of renal and liver disease severity in human patients and can lead to perinatal mortality. ARPKD is caused by mutations in PKHD1, which encodes the large membrane protein, Fibrocystin, required for normal branching morphogenesis of the ureteric bud during embryonic renal development. The variation in ARPKD phenotype suggests that in addition to PKHD1 mutations, other genes may play a role, acting as modifiers of disease severity. One such pathway involves non-canonical Wnt/Planar Cell Polarity (PCP) signalling that has been associated with other cystic kidney diseases, but has not been investigated in ARPKD. Analysis of the Atmin^Gpg6 mouse showed kidney, liver and lung abnormalities, suggesting it as a novel mouse tool for the study of ARPKD. Further, modulation of Atmin affected Pkhd1 mRNA levels, altered non-canonical Wnt/PCP signalling and impacted cellular proliferation and adhesion, although Atmin does not bind directly to the C-terminus of Fibrocystin. Differences in ATMIN and VANGL2 expression were observed between normal human paediatric kidneys and age-matched ARPKD kidneys. Significant increases in ATMIN, WNT5A, VANGL2 and SCRIBBLE were seen in human ARPKD versus normal kidneys; no substantial differences were seen in DAAM2 or NPHP2. A striking increase in E-cadherin was also detected in ARPKD kidneys. This work indicates a novel role for non-canonical Wnt/PCP signalling in ARPKD and suggests ATMIN as a modulator of PKHD1.     

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.     

Mouse splice mutant generation from ENU-treated ES cells--a gene-driven approach

Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.