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.

Familial homozygous hypercholesterolemia: effective long-term treatment with cascade double filtration plasmapheresis.

BACKGROUND: Homozygous familial hypercholesterolemia (FH) is a rare disease with an incidence of 1 in 1 million births. It is characterized by blood cholesterol levels over 600 mg/dl and the development of extensive cutaneous xanthomata before the age of 5. Severe premature coronary artery disease results in fatal myocardial infarctions within the first two decades of life. The absence of LDL receptors makes homozygous FH resistant to treatment with most HMG-CoA reductase inhibitors, and alternative methods of removing cholesterol have been employed. METHODS: We used the ASAHI plasauto-IQ double filtration cascade plasmapheresis to treat 2 young brothers aged 14 and 11 years and 6 months for 5 years and 1 month and 3 years and 10 months, respectively. The elder brother has received 136 double filtration treatments at 2-week intervals and the younger brother 100 such treatments without complications. RESULTS: During the period of treatment the average pretreatment total serum cholesterol level for patient 1 was 442 mg/dl. The average posttreatment value was 163 mg/dl. The average fall in total serum cholesterol with each treatment was 63.2%. The mean total serum cholesterol for all the periods of treatment was calculated at 303 mg/dl. For the 2nd patient, the average pretreatment value of total serum cholesterol was 435 mg/dl. The posttreatment value was 124 mg/dl and the average fall 71.5%. The calculated mean total serum cholesterol for all periods of treatment was 280 mg/dl. CONCLUSIONS: Double filtration cascade plasmapheresis at 2-week intervals provides an effective and safe long-term treatment for patients with homozygous FH. The achieved reduction in serum cholesterol allows complete resolution of cutaneous xanthomata, arrests previous atherosclerosis, and prolongs normal life.

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.

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.

Geographical clustering of low density lipoprotein receptor gene mutations (C292X; Q363X; D365E & C660X) in Cyprus.

In Cyprus, no data are yet available on the frequencies of clinically diagnosed FH patients. Further, until now, familial hypercholesterolaemia in Cyprus had not been studied at the molecular level to determine the nature or frequency of LDLR gene mutations. Being a relatively homogeneous population, we anticipated that a few founder mutations would predominate on the island. In the present study, three previously identified LDLR gene mutations were found to cosegregate with high LDL cholesterol levels in 23 unrelated, clinically diagnosed families with FH. Geographical clustering of each of these LDLR gene mutations was indicated, a phenomenon arising from low migration rates and high inbreeding. The latter cultural practices account for the discovery of a homozygous FH sib pair whose parents are carriers of the same mutation. Microsatellite and intragenic haplotype analysis in this FH population, suggested that the families which shared the same LDLR gene mutation have a common origin. This is supported by their relative geographical distribution. Thirty young FH individuals were also offered presymptomatic diagnosis which should facilitate the prevention of premature coronary artery disease. Finally, results from this study support the suggestion that the formation of tendon xanthomata in FH patients may be under environmental influence. Hum Mutat 15:380, 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.