Molecular investigation and long-term clinical progress in Greek Cypriot families with recessive distal renal tubular acidosis and sensorineural deafness due to mutations in the ATP6V1B1 gene.

The spectrum of distal renal tubular acidosis (dRTA) includes a genetically heterogeneous group of inherited conditions of both autosomal-dominant and recessive mode of inheritance. The basic defect is linked to the renal part of acid-base homeostasis, which is partly achieved by the regulated luminal secretion of H+ at the apical surface of the alpha-intercalated cells of renal collecting ducts. This is coupled to bicarbonate reabsorption with chloride counter transport across the basolateral membranes. Here, we describe the molecular findings of the first two Greek Cypriot families with recessive dRTA and the long-term clinical findings in four of five affected members. DNA linkage analysis with four polymorphic markers flanking the ATP6V1B1 gene on chromosome 2 gave evidence for positive linkage; direct DNA analysis by automated DNA sequencing revealed that patients in one family were homozygous for mutation 229+1G>T (IVS7+1G>T) and that patients in the second family were compound heterozygous for 229+1G>T and R157C. The mutations were found on four different haplotypes. Both the mutations were previously reported in patients of Turkish origin. Three known polymorphic variants were also identified. The five patients demonstrated the whole clinical spectrum of the disease including death in infancy, failure to thrive, rickets, nephrocalcinosis, nephrolithiasis, and episodes of hypokalemic paralysis. Some of the family members are now in their mid 30s and late 20s, and nephrolithiasis with recurrent renal colics is their main problem. Renal function has remained normal. In conclusion, early diagnosis in infancy and prompt treatment with alkali and potassium supplements is of great benefit to the patient with dRTA and ensures normal growth. The identification of responsible mutations facilitates antenatal or postnatal diagnosis in concerned families and improves the prognosis.

Non-isotopic RNase cleavage assay for mutation detection in MEFV, the gene responsible for familial Mediterranean fever, in a cohort of Greek patients.

BACKGROUND: The MEFV gene is responsible for familial Mediterranean fever (FMF). Several disease associated mutations have been identified. The range of genetic variation in MEFV in Greek patients has not been determined. OBJECTIVE: To describe a method that facilitates the routine screening of the entire coding sequence of MEFV (excluding exon 1). METHODS: The non-isotopic RNase cleavage assay (NIRCA) was optimised and used as a first step screening method to screen exons 2 to 10 of MEFV. Exons 2 and 10 were analysed separately at DNA level, while exons 3 to 9 were analysed together at cDNA level. The sample group consisted of 26 FMF patients diagnosed using established clinical criteria, six asymptomatic relatives, 12 patients with atypical clinical manifestations, nine patients suffering from various inflammatory diseases, and three normal individuals. All were analysed by NIRCA for mutations in the MEFV gene and direct sequencing was applied subsequently to confirm the results. RESULTS: MEFV mutations were identified in 25 of 26 typical FMF patients and in two of 12 patients with atypical manifestations. NIRCA results were in concordance with sequencing findings in all sequences analysed, suggesting that the method is highly reliable in this disease. Sixteen alterations of MEFV were identified (eight missense mutations and eight single nucleotide polymorphisms). CONCLUSIONS: NIRCA can be used for rapid screening of the coding sequence of the MEFV gene in patients suspected of suffering from FMF.

Novel PKD1 deletions and missense variants in a cohort of Hellenic polycystic kidney disease families.

The autosomal dominant form of polycystic kidney disease is a very frequent genetically heterogeneous inherited condition affecting approximately 1 : 1000 individuals of the Caucasian population. The main symptom is the formation of fluid-filled cysts in the kidneys, which grow progressively in size and number with age, and leading to end-stage renal failure in approximately 50% of patients by age 60. About 85% of cases are caused by mutations in the PKD1 gene on chromosome 16p13.3, which encodes for polycystin-1, a membranous glycoprotein with 4302 amino acids and multiple domains. Mutation detection is still a challenge owing to various sequence characteristics that prevent easy PCR amplification and sequencing. Here we attempted a systematic screening of part of the duplicated region of the gene in a large cohort of 53 Hellenic families with the use of single-strand conformation polymorphism analysis of exons 16-34. Our analysis revealed eight most probably disease causing mutations, five deletions and three single amino acid substitutions, in the REJ domain of the protein. In one family, a 3-bp and an 8-bp deletion in exons 20 and 21 respectively, were co-inherited on the same PKD1 chromosome, causing disease in the mother and three sons. Interestingly we did not find any termination codon defects, so common in the unique part of the PKD1 gene. In the same cohort we identified 11 polymorphic sequence variants, four of which resulted in amino acid variations. This supports the notion that the PKD1 gene may be prone to mutagenesis, justifying the relatively high prevalence of polycystic kidney disease.

Mutations of the human polycystic kidney disease 2 (PKD2) gene.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited nephropathy, usually of late onset (onset between third to seventh decade), primarily characterized by the formation of fluid-filled cysts in the kidneys. It is one of the most frequent inherited conditions affecting approximately 1:1,000 Caucasians. Two major genes have been identified and characterized in detail: PKD1 and PKD2, mapping on chromosomes 16p13.3 and 4q21-23, respectively. A third gene, PKD3, has been implicated in selected families. Polycystic kidney disease of types 1 or 2 follows a very similar course of symptoms, both being multisystem pleiotropic disorders of indistinguishable picture on clinical grounds. The only difference is that patients with PKD2 mutations run a milder course compared to PKD1 carriers, with an average 10-20 years later age of onset and lower probability to reach end-stage-renal failure. The proteins polycystin-1 and -2 are trans-membranous glycoproteins hypothesized to participate in a common signaling pathway, interacting with each other and with other proteins, and coordinately expressed in normal and cystic tissue. Renal cysts most probably arise after a second somatic event, which inactivates the inherited healthy allele of the same gene, or perhaps one of the alleles of the other gene counterpart, generating a trans-heterozygous state. This article reviews the reported mutations in PKD2. Mutations of all kinds have been reported over the entire sequence of the PKD2 gene, with no apparent significant clustering and with some evidence of genotype/phenotype correlation. Most families harbor their own private mutations but a few recurrent events have been reported in unrelated families.

Novel NPR1 polymorphic variants and its exclusion as a candidate gene for medullary cystic kidney disease (ADMCKD) type 1.

Autosomal dominant medullary cystic kidney disease (ADMCKD) is an adult-onset heterogeneous genetic nephropathy characterized by salt wasting and end-stage renal failure. The gene responsible for ADMCKD-1 was mapped on chromosome 1q21 and it is flanked proximally by marker D1S498 and distally by D1S2125, encompassing a region of approximately 8 cm. Within this region there are a large number of transcribed genes including NPR1 that encodes the atrial natriuretic peptide receptor 1. This receptor plays a crucial role in regulation of blood pressure by facilitating salt excretion. Based on its function we hypothesized this gene as a reasonable candidate for the MCKD1 locus. DNA mutation screening was performed on the entire NPR1 gene-coding sequence and some of the 5' prime-UTR and 3'-UTR sequences. The samples investigated belonged to patients of five large ADMCKD-1 Cypriot families. The screening revealed two novel polymorphisms, one intragenic at amino acid position 939, which was occupied by either arginine or glutamine, and a second one located in the 3' prime-UTR, 29 nucleotides downstream of the NPR1 stop codon. The latter was a single nucleotide C insertion/deletion in a stretch of three or four Cs. No relationship was present between any allele of the two polymorphisms and the disease, as both alleles were observed in both affected and healthy subjects. In addition, no association was observed between the disease and another rare 8-bp deletion polymorphism at the 5' prime-UTR of NPR1 and the disease. Based on these findings it is unlikely that NPR1 is the same as the MCKD1 gene, although it is presently unknown whether it plays a disease modifying role.

Autosomal dominant polycystic kidney disease: molecular genetics and molecular pathogenesis.

Mutations in three different genes, PKD1, PKD2 and PKD3, can cause a very similar clinical picture of the autosomal dominant form of polycystic kidney disease (ADPKD). Apparently, mutations in the PKD3 gene, which is still unmapped, are very rare, whereas PKD1 defects account for about 85% of cases. Although ADPKD is a frequent monogenic disorder affecting approximately 1:1000 individuals in the Caucasian population, progress in understanding its pathology was somewhat slow until relatively recently when the PKD1 and PKD2 genes were mapped and cloned. They are both large, being approximately 52 kb and 68 kb in length respectively, and in addition, PKD1 is fairly complex, thus complicating mutation detection. The gene products, polycystin-1 and polycystin-2, are trans-membranous glycoproteins and are considered to be involved in signalling pathways, in cooperation with additional partners. Immunostaining studies in both humans and mice have revealed information regarding the localization of polycystins and their role in the development and maintenance of nephrons. Recent experimentation from various laboratories has shown that loss of heterozygosity and acquired somatic second hits may account, at least partly, for the inter- and intrafamilial phenotypic heterogeneity of the disease, while at the same time, the existence of other modifying loci is also hypothesized. The two-hit hypothesis is admittedly a very attractive one in that it can explain many of the features of the disease, whereas recent data regarding a trains-heterozygous model for cystogenesis adds to the complexity of the molecular mechanisms that can lead to pathogenesis.

Screening of the PKD1 duplicated region reveals multiple single nucleotide polymorphisms and a de novo mutation in Hellenic polycystic kidney disease families.

Mutations in the PKD1 gene account for approximately 85% of cases with autosomal dominant polycystic kidney disease (ADPKD1; MIM# 601313), which is considered one of the most frequent monogenic disorders, with a frequency of approximately 1:1000. The main symptom is the formation of fluid-filled cysts in the kidneys and less often in other organs, such as the liver and pancreas. Since the cloning of the gene many mutations have been identified, although the screening is hampered by several unique features of this gene, the most significant one being that approximately 70% of the sequence at the 5'-end, is reiterated elsewhere on chromosome 16 with homology approaching 95%. Here, we used an oligonucleotide primer anchored in the unique part in exon 34, paired with a forward primer in exon 23 for specifically amplifying PKD1 sequences. We screened for mutations in samples from 32 Hellenic ADPKD families. We detected seven sequence variants, five of which most probably are single nucleotide polymorphisms (SNPs), especially useful for linkage analysis and disease association studies. One is a missense change, segregating with ADPKD in one family. The last one is a missense non-conservative change, H2921P, which appeared de novo in the proband, concurrently with the disease phenotype, and was passed on to another two generations. Two siblings who inherited the same haplotype as the proband, but not the de novo mutation, were not affected. This is only the fourth case of a molecularly documented de novo mutation in ADPKD. Somatic mosaicism in peripheral blood leukocytes of the proband was tested and excluded. Hum Mutat 16:176, 2000.

Genetic evidence for a trans-heterozygous model for cystogenesis in autosomal dominant polycystic kidney disease.

Polycystic kidney disease (ADPKD) is a condition with an autosomal dominant mode of inheritance and adult onset. Two forms of the disease, ADPKD1 and ADPKD2, caused by mutations in PKD1 and PKD2, respectively, are very similar, except that ADPKD1 patients run a more severe course. At the cellular level, ADPKD1 was first shown to be recessive, since somatic second hits are perhaps necessary for cyst formation. The near identical phenotype had suggested that ADPKD1 and ADPKD2 might have a similar pathogenesis and that the two gene products, poly- cystins 1 and 2, are part of a common developmental pathway. Work in transgenic mice showed that somatic loss of Pkd2 expression is necessary for renal cyst formation, and recently we showed that somatic mutations inactivating the inherited healthy allele were present in 9 of 23 cysts from a human ADPKD2 kidney, supporting a two-hit loss-of-function model for ADPKD2 cystogenesis. Here, we provide the first direct genetic evidence that polycystins 1 and 2 do interact, perhaps as part of a larger complex. In cystic DNA from a kidney of an ADPKD1 patient, we showed somatic mutations not only in the PKD1 gene of certain cysts, but also in the PKD2 gene of others, generating a trans -heterozygous state with mutations in both genes. One mutation in PKD1 is of germinal nature and the mutation in the PKD2 gene is of somatic nature. The implications of such a situation are enormous, not only for ADPKD, but also for many other conditions with phenotypic heterogeneity and age-dependent penetrance.

Autosomal dominant polycystic kidney disease-type 2. Ultrasound, genetic and clinical correlations.

BACKGROUND: Ultrasound, genetic and clinical correlations are available for ADPKD-1, but lacking for ADPKD-2. The present study was carried out to address: (i) the age-related diagnostic usefulness of ultrasound compared with genetic linkage studies; (ii) the age-related incidence and prevalence of relevant symptoms and complications; and (iii) the age and causes of death in patients with ADPKD-2. METHODS: Two hundred and eleven alive subjects, from three ADPKD-2 families at 50% risk, were evaluated by physical examination, consultation of hospital records, biochemical parameters, ultrasound and with genetic linkage and DNA mutation analyses. Nineteen deceased and affected family members were also included in the study. RESULTS: Of the 211 alive members, DNA linkage studies and direct mutation analyses showed that 106 were affected and 105 were not. Ultrasound indicated 94 affected, 108 not affected and nine equivocal results in nine children under the age of 15. For all ages, the false-positive diagnostic rate for ultrasound was 7.5% and the false-negative rate was 12.9%. The difference between ultrasound and DNA findings was most evident in children aged 5-14 years where the ultrasound was correct in only 50% and wrong or inconclusive in the remaining 50%. The mean age of the 106 alive, ADPKD-2 genetically affected patients was 37.9 years (range: 6-66 years). Among them, 23.5% had experienced episodes of renal pain, 22.6% were treated for hypertension, 22.6% had experienced at least one urinary tract infection, 19.8% had nephrolithiasis, 11.3% had at least one episode of haematuria, 9.4% had asymptomatic liver cysts, 7.5% had developed chronic renal failure and 0.9% had reached end-stage renal failure. Of the 19 deceased members, nine died before reaching end-stage renal failure at a mean age of 58.7 years (range: 40-68 years), mainly due to vascular complications, while the remaining 10 died on haemodialysis at a mean age of 71.4 years (range: 66-82 years). CONCLUSIONS: DNA analysis is the gold standard for the diagnosis of ADPKD-2, especially in young people. Ultrasound diagnosis is highly dependent on age. Under the age of 14, ultrasound is not recommended as a routine diagnostic procedure, but ultrasound becomes 100% reliable in excluding ADPKD-2 in family members at 50% risk, over the age of 30. ADPKD-2 represents a mild variant of polycystic kidney disease with a low prevalence of symptoms and a late onset of end-stage renal failure.

Autosomal dominant medullary cystic kidney disease: evidence of gene locus heterogeneity.

Autosomal dominant medullary cystic kidney disease (ADMCKD; synonym: medullary cystic disease, MCD) is an autosomal dominant kidney disorder, sharing morphological and clinical features with recessive juvenile nephronophthisis (NPH), such as reduced urinary concentration ability and multiple renal cysts at the corticomedullary junction. While in NPH end-stage renal disease (ESRD) occurs in adolescence, ADMCKD leads to ESRD in adulthood. Recently a gene locus for ADMCKD has been localized to chromosome 1q21 in two large Cypriot families. This prompted us to examine linkage in three ADMCKD-families, using the same set of polymorphic microsatellite markers spanning the critical region on chromosome 1q21. Haplotype analysis revealed that none of the three families showed linkage to this locus, thus demonstrating evidence for genetic locus heterogeneity. Additional linkage analysis studies need to be performed in order to identify further gene loci cosegregating with this autosomal dominant kidney disorder.

Medullary cystic kidney disease with hyperuricemia and gout in a large Cypriot family: no allelism with nephronophthisis type 1.

We describe a large Cypriot family with an interstitial type of nephropathy, inherited as an autosomal dominant trait that led to end stage renal failure between 51 to 78 years of age (mean 62.2 years). Twenty-three people are known to be affected, but several younger relatives with normal renal function may remain undiagnosed because of the absence of precise clinical and laboratory diagnostic criteria. This nephropathy is associated with medullary renal cysts, hypertension, hyperuricemia, and gout. Several relatives have typical medullary cystic disease (MCD), while in the others the findings are compatible with this diagnosis. Due to the similarity of clinical and pathologic findings, earlier reports had suggested that MCD may be allelic to autosomal recessive familial juvenile nephronophthisis, which was mapped recently to chromosome band 2q13. Linkage analysis of the present family with a closely linked marker excluded linkage to the above locus. Linkage was also excluded to the PKD1 locus of adult polycystic kidney disease type 1, and up to 5 cM on either side, on chromosome 16. We suggest that because of the element of hyperuricemia and gout found in this family, although with reduced penetrance, it may represent a variant of autosomal dominant MCD of the adult type. This variability may be the result of allelic or locus heterogeneity. Molecular genetic approaches including linkage analysis on appropriate families will certainly assist in classifying such related genetically heterogeneous disorders.

Loss of heterozygosity in polycystic kidney disease with a missense mutation in the repeated region of PKD1.

Loss of heterozygosity (LOH) is a molecular phenomenon that denotes the loss of one of the two alleles at a specific locus. It is frequently associated with tumour suppressor genes in various cancers and also with hyperproliferative disorders, although not exclusively. Interestingly, in conditions where there is an inherited germline mutation, the lost allele is always the functional one, thereby rendering a phenotypically dominant disease of recessive character at the cellular level. A disease more recently shown to be associated with LOH is polycystic kidney disease type 1, a systemic disorder characterized by significant pleiotropy. The main pathology is from renal cyst formation that eventually leads to end-stage renal failure during adult life. We describe the identification of a missense mutation in the repeated part of the PKD1 gene, exon 31, that substitutes valine for methionine. The mutation, M3375V, cosegregates with the disease phenotype in a large Cypriot family. During transplantation of one patient, one of the polycystic kidneys was removed and DNA was isolated from cystic epithelial cells. In 3 of 17 cysts examined with intragenic and flanking polymorphic markers on chromosome 16 we detected LOH, since the wild-type allele was lost, thereby rendering the affected kidneys of mosaic character. The degree of LOH was extensive and varied among the three cysts, supporting the multiplicity of expression of the phenomenon on different occasions. No LOH was detected for other selected loci examined. Our work further supports the hypothesis that the rate-limiting step in cyst formation may be the occurrence of a second somatic hit, although other factors may be also involved. The high frequency of mutations at this locus may, to a great extent, explain the variability in phenotype observed among patients in the same families, and the relatively high frequency of the disease worldwide.

A translation frameshift mutation induced by a cytosine insertion in the polycystic kidney disease 2 gene (PDK2).

Mutations in the PKD2 gene on the long arm of chromosome 4 are responsible for approximately 15% of cases of polycystic kidney disease. Perhaps the only difference from the more common ADPKD1 cases is the rate of progression of cystic changes, and the age of onset, which is 10-15 years later for the ADPKD2 form. In Cyprus there are at least three large families, documented by molecular linkage analysis, that map to the PKD2 locus. For two of them the defects were recently shown to be nonsense mutations at positions arginine 742 and glutamine 405. In this report, we describe the mutation in the third family, CY1602. For this, the entire coding sequence was systematically screened by single strand conformation analysis and heteroduplex formation. A novel mutation was identified in exon 2 where a new cytosine residue was inserted immediately after codon 231 (231insC). It causes a translation frameshift and is expected to lead to the introduction of 37 novel amino acids before the translation reaches a new STOP codon. It is the most amino terminal mutation reported to date, and based on the protein's modeled structure, is predicted to be within the first transmembrane domain. It is the fourth PKD2 mutation reported thus far, and the first which is not a nonsense mutation.

New amino acid polymorphism, Ala/Val4058, in exon 45 of the polycystic kidney disease 1 gene: evolution of alleles.

The PKD1 gene, which is responsible for the most common form of autosomal dominant polycystic kidney disease, has recently been cloned and sequenced. Many disease-causing mutations have been characterized in this gene, most of them resulting in premature protein termination. However, mutation analysis not routinely implemented for family investigations in a clinical setting, because of the large size and complexity of the gene. Instead, genetic linkage analysis using highly polymorphic CA dinucleotide repeats that map around the gene is still the method of choice. Recently, a few intragenic polymorphisms have been described that are also useful for linkage studies. Here, a new diallelic polymorphism is described for amino acid residue 4058, Ala/Val4058, with allelic frequencies of 0.88 and 0.12, respectively, and a heterozygosity of 0.23, in the Greek and Greek-Cypriot populations. Interestingly, this polymorphism and Ala4091-A/G, which has previously been described in Caucasians, were not detected in DNA from 44 Japanese samples tested. This is particularly important when allelic frequencies in a particular population are used for linkage analysis of families of different ethnic origin. Also, observation of the two polymorphisms together as haplotypes suggests that the Ala/Val4058 polymorphism occurred more recently than the establishment of the Ala4091-A/G polymorphism, and specifically on the G allele.

Cytogenetic and fragile X molecular testing of individuals with mental retardation of unknown etiology.

The aim of this program was to investigate the patients with Mental Retardation Of Unknown Etiology (MROUE), on the island of Cyprus. The MROUE patients were examined cytogenetically for gross chromosomal abnormalities, and by molecular methods for the Fragile X syndrome pathology. Specialized physicians examined all institutionalized or non institutionalized patients throughout Cyprus. Cytogenetic analysis was carried out on 105 individuals, six of which showed various chromosomal aberrations. PCR and Southern blot analysis were carried out on 170 patients referred for exclusion of the Fragile X syndrome. Three patients had positive findings. Although the number of cases elucidated with this general approach was not spectacular, it allowed the resolution of a few clinically equivocal cases, to the satisfaction of the clinicians and, most importantly, the relatives involved. We believe that such screening programs should continue until all cases are thoroughly examined, thus providing definite genetic counseling and psychological support, at least in those cases that are clearly resolved. Equally important is the prospect for prevention through prenatal diagnostic programs, that are already available for such conditions.

Detection of a novel nonsense mutation and an intragenic polymorphism in the PKD1 gene of a Cypriot family with autosomal dominant polycystic kidney disease.

Mutations in the PKD1 gene on the short arm of chromosome 16 account for 85%-90% of polycystic kidney disease patients in the Caucasian population. After the recent characterization of the gene, we started a search for mutations in its 3 -end unique portion in Cypriot patients, by using the method of single-strand conformation polymorphism (SSCP). In one large family, we identified a nucleotide substitution at position 12258 of the cDNA; this substitutes cysteine-4086 by a premature termination codon (C4086X). It has been inherited by every affected family member but not by unaffected members, nor by patients from 13 other Cypriot families. A new polymerase chain reaction (PCR) primer has been designed to engineer a novel DdeI recognition site upon PCR amplification, thereby allowing easy detection of the mutation by PCR-restriction digestion. The premature STOP codon is expected to remove 217 residues from the putative C-terminal intracellular domain of the gene product, polycystin and thus identifies this part as being critical to the production of the disease phenotype, possibly by interfering with the transmission of signals from the extracellular matrix to the cytoplasm. We also describe the identification of the first polymorphism within the encoding region of the gene. It is at alanine 4091, which is encoded by either GCA or GCG. With a heterozygosity of 35%, it should be extremely useful in informative families, especially because the gene lies in an unstable region and is prone to rearrangements. This polymorphism is readily detectable by PCR-restriction digestion with Bsp 12861.

Description of a symptomless cystic fibrosis L346P/M348K compound heterozygous Cypriot individual.

During the past few years we have been testing the hypothesis that Cyprus may have been spared many severe cystic fibrosis (CF) cases but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We have been analysing by molecular methods patients with atypical mild phenotypes where CF enters the differential diagnosis. With this approach we identified a mutation, L346P, which in association with the severe mutation delta F508 or 1677delTA, confers a mild and atypical presentation. Recently, we identified another entirely symptomless 48-year-old individual, with genotype L346P/M348K. The fact that M348K was initially identified in a severely affected Italian patient strengthens the hypothesis that L346P, a putative mild mutation, is dominant over severe ones. One other explanation is that M348K is not a causative defect but a rare polymorphism. These findings have important implications for genetic counselling, especially when the counselling is sought by concerned couples for prenatal diagnostic purposes.

Presymptomatic molecular diagnosis of autosomal dominant polycystic kidney disease using PKD1- and PKD2-linked markers in Cypriot families.

Autosomal dominant polycystic kidney disease (ADPKD), is a heterogeneous disorder, primarily characterized by the formation of cysts in the kidneys, and the late development in life of progressive chronic kidney failure. Three genes are implicated in causing ADPKD. One on chromosome 16, PKD1, accounts for 85-90% of all cases, and the PKD2 gene on chromosome 4 accounts for the remainder. A very rare third locus is still of unknown location. We used PKD1- and PKD2-linked polymorphic markers to make the diagnosis of ADPKD in young presymptomatic members in affected families. We showed that in young members of families where clinical diagnosis cannot be definitively established, molecular linkage analysis can assist clinicians in the diagnosis. In one family a 24-year old had one cyst on the right kidney; however, molecular analysis showed clearly that he had inherited the normal haplotype. In another family, in one part of the pedigree there was co-inheritance of the disease with a PKD1-linked haplotype which originated in a non-affected 78-year-old father. Analysis with PKD2-linked markers excluded this locus. The data can be explained in one of two ways. Either this family phenotype is linked to a third locus, or the proband was the first affected person, most probably because of a novel mutation in one of her father's chromosomes. In conclusion, the combined use of markers around the PKD1 and the PKD2 locus provides more definitive answers in cases where presymptomatic diagnosis is requested by concerned families.

PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.