Arseni Markoff

Incompletely Penetrant PKD1 Alleles Mimic the Renal Manifestations of ARPKD

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutation in PKD1 or PKD2, is usually an adult-onset disorder but can rarely manifest as a neonatal disease within a family characterized by otherwise typical ADPKD. Coinheritance of a hypomorphic PKD1 allele in trans with an inactivating PKD1 allele is one mechanism that can cause early onset ADPKD. Here, we describe two pedigrees without a history of cystic kidney disease that each contain two patients with onset of massive PKD in utero. The presentations were typical of autosomal recessive PKD (ARPKD) but they were not linked to the known ARPKD gene, PKHD1. Mutation analysis of the ADPKD genes provided strong evidence that both families inherited, in trans, two incompletely penetrant PKD1 alleles. These patients illustrate that PKD1 mutations can manifest as a phenocopy of ARPKD with respect to renal involvement and highlight the perils of linkage-based diagnostics in ARPKD without positive PKHD1 mutation data. Furthermore, the phenotypic overlap between ARPKD and these patients resulting from incomplete penetrant PKD1 alleles support a common pathogenesis for these diseases.

Functional Activation of the Formyl Peptide Receptor by a New Endogenous Ligand in Human Lung A549 Cells

The formyl peptide receptor (FPR), a heptahelical G protein-coupled receptor on phagocytic leukocytes, can be triggered by bacterially derived oligopeptides of the prototype fMLP. Although FPR expression and activation have been associated with cells of myeloid origin and bacterial inflammation, the receptor has recently been identified in nonmyeloid cells, thus suggesting additional physiological functions and the existence of an endogenous agonist. In this study, we demonstrate the presence and functional activation of the FPR in the human lung cell line A549, which represents an extrahepatic model for the regulation of acute-phase proteins. Activation of the FPR in A549 cells cannot only be triggered by fMLP, but also by an agonistic peptide of the recently identified endogenous FPR ligand, annexin 1. In addition to inducing changes in the F-actin content, annexin 1-mediated triggering of the FPR results in an increased expression of acute-phase proteins. Hence, activation of nonmyeloid FPR by its endogenous ligand annexin 1 could participate in the regulation of acute-phase responses, e.g., during inflammation and/or wound healing.

Comparison of conformation-sensitive gel electrophoresis and single-strand conformation polymorphism analysis for detection of mutations in the BRCA1 gene using optimized conformation analysis protocols

In order to develop a selective mutation screening strategy for BRCA1, one of the gene responsible for hereditary predisposition to breast cancer, we analysed by single-strand conformation polymorphism (SSCP) and conformation-sensitive gel electrophoresis (CSGE) a cohort of 20 Bulgarian breast cancer patients, prescreened for nonsense mutations by the protein truncation test. By assaying the complete coding sequence of the gene applying both methods, we were able to detect 12 sequence alterations: 11 nucleotide substitutions and one deletion. Two of the alterations are intronic polymorphisms, the rest are exon sequence variants. Of the 12 polymorphisms identified, 11 are described and one is new. All sequence changes were detected by CSGE and eight of them were also shown by SSCP analysis. There was no sequence alterations which could be detected by SSCP analysis only. We propose that because of the specificity of most sequence variants detected (nucleotide substitutions) and the comparatively high percentage of AT content of the BRCA1 gene (58.4%), CSGE turned out to be the more sensitive technique in our assay. This observation is in agreement with other accepted analysis strategies for BRCA1 and it may prove useful for mutation screening of AT-rich, multi-exon genes.

Reduced allele specific annexin A5 mRNA levels in placentas carrying the M2/ANXA5 allele

We aimed to trace the allele specific expression of ANXA5 mRNA in placentas carrying the M2 haplotype, conferring higher recurrent pregnancy loss risk, in order to verify directly the role of M2 in the relevant organ. The M2 allele in heterozygous placentas results in an average of 42% reduced ANXA5 mRNA levels as compared to the normal allele. Protein levels in these samples show considerable variations, impossible for statistical interpretation. The M2 allele of ANXA5 can be linked to reduced mRNA levels in heterozygous placentas and could result in more confined protein levels (lowered expression dynamics) of annexin A5.

Paternal and maternal carriage of the annexin A5 M2 haplotype are equal risk factors for recurrent pregnancy loss: a pilot study

OBJECTIVE To study the possible contribution of paternal, in addition to maternal, carriage of M2/ANXA5 as a risk factor for recurrent pregnancy loss (RPL). DESIGN Case-control study. SETTING Academic research center. PATIENT(S) Couples presenting themselves to the Fertility Center, Ludwig-Maximilians-University Munich with two or more consecutive, unexplained miscarriages were selected for this study. Fertile female controls were from the same center and also from the resource of the Institute of Human Genetics, Westfalian Wilhelms-University Muenster. Population controls were drafted from the PopGen biobank, University Clinic Schleswig-Holstein Kiel. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Incidence of M2 carriage was estimated in patient and control groups, odds ratios were calculated, and RPL risk was evaluated. RESULT(S) In comparison with female fertile controls, the risk for repeated abortion in the RPL group, associated with M2 carriage, was between 1.7 and 3.8, and it was 2.3 compared with population controls. Because of the equal genetic incidence of M2, with an allelic frequency of 0.167 in the female and male partner RPL subgroups, the haplotype confers approximately the same relative risk to carriers of both sexes. CONCLUSION(S) Paternal M2 carriage seems to confer an equal risk for recurrent miscarriages as M2 carriage in RPL mothers. This finding points to a role of ANXA5 and the M2 haplotype in the fetus and/or the extraembryonic membranes for pregnancy pathology. Prognostic RPL algorithms might be improved by testing the male partner for M2 carriage, and this may guide adequate therapeutic decisions.

Hereditary thrombophilic risk factors for recurrent pregnancy loss

This review summarizes current knowledge about the role of hereditary hypercoagulation factors predisposing to thrombophilia-associated recurrent fetal loss. Thrombophilias are a major cause of adverse pregnancy outcome, playing a role in the etiology of up to 40% of cases worldwide. Hereditary thrombophilic predispositions to recurrent pregnancy wastage include genetic lesions in blood coagulation factors II and V as well as natural anticoagulants antithrombin, protein C, and protein S. Furthermore, methylenetetrahydrofolate reductase gene variants conferring higher thrombophilia risk in combination with these mutations and the newly described annexin A5 gene M2 promoter allele are associated with repeated fetal loss. The review gives a concise description of the molecular defects arising from the genetic changes, of the role these factors play in the timing and definition of fetal loss, and risk estimates from available studies and meta-analysis. This knowledge is instrumental for a more precise assessment of individual risks for repeated fetal loss and should guide therapeutic strategies, where relevant. Since the average childbearing age increases in Western societies, the importance of a timely diagnosis of fetal loss predisposition is increasing.

Autosomal dominant polycystic kidney disease: Clinical and genetic aspects

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans. It accounts for 8–10% of the cases of end-stage renal disease worldwide, thus representing a serious medical, economical and social problem. ADPKD is in fact a systemic disorder, characterized with the development of cysts in the ductal organs (mainly the kidneys and the liver), also with gastrointestinal and cardiovascular abnormalities. In the last decade there was significant progress in uncovering the genetic foundations and in understanding of the pathogenic mechanisms leading to the renal impairment. This review will retrace the current knowledge about the epidemiology, pathogenesis, genetics, genetic and clinical heterogeneity, diagnostics and treatment of ADPKD.

Combined homology modelling and evolutionary significance evaluation of missense mutations in blood clotting factor VIII to highlight aspects of structure and function

Summary.  Most small lesions in the factor VIII (FVIII) gene that cause haemophilia A (HA) are single nucleotide substitutions resulting in amino acid replacing (missense) mutations and leading to various phenotypes, ranging from mild to severe. We took a combined approach of homology modelling and quantitative evaluation of evolutionary significance of amino acid replacing alterations using the Grantham Matrix Score (GMS) to assess their structural effects and significance of pathological expression. Comparative homology models of all amino acid substitutions summarized in the FVIII mutations database plus these identified and reported lately by us or by our collaborators were evaluated. Altogether 640 amino acid replacing mutations were scored for potential distant or local conformation changes, influence on the molecular stability and predicted contact residues, using available FVIII domain models. The average propensity to substitute amino acid residues by mutation was found comparable to the overall probability of de novo mutations. Missense changes reported with various HA phenotypes were all confirmed significant using GMS. The fraction of these, comprising residues apparently involved in intermolecular interactions, exceeds the average proportion of such residues for FVIII. Predicted contact residues changed through mutation were visualized on the surface of FVIII domains and their possible functional implications were verified from the literature and are discussed considering available structural information. Our predictive modelling adds on the current view of domain interface molecular contacts. This structural insight could aid in part to the design of engineered FVIII constructs for therapy, to possibly enhance their stability and prolong circulating lifetime.

Investigation of the Annexin A5 M2 haplotype in 500 white European couples who have experienced recurrent spontaneous abortion

Annexin A5 is a placental anti-coagulant protein that contains four nucleotide substitutions (M2 haplotype) in its promoter. This haplotype is a risk factor for recurrent spontaneous abortion (RSA). The influence of the M2 haplotype in the gestational timing of spontaneous abortions, paternal risk and relationships with known risk factors were investigated. European couples (n = 500) who had experienced three or more consecutive spontaneous abortions, and two fertile control groups, were selected for this study. The allele frequency of M2 was significantly higher among patients who had experienced early RSA than among controls (P = 0.002). No difference was found between controls and patients who had undergone late spontaneous abortions. No difference was found between patients who had experienced RSA who had a live birth or no live births, or between patients who were positive or negative for known risk factors. Male and female partners in each group had similar allele frequencies of M2. The M2 haplotype is a risk factor for early spontaneous abortions, before the 12th week of gestation, and confers about the same relative risk to carriers of both sexes. Having one or more M2 allele(s) in combination with other risk factors further increases the RSA risk.

Screening the 3′ region of the polycystic kidney disease 1 (PKD1) gene in 41 Bulgarian and Australian kindreds reveals a prevalence of protein truncating mutations

Screening for disease‐causing mutations in the unique region of the polycystic kidney disease 1 (PKD1) gene was performed in 41 unrelated individuals with autosomal dominant polycystic kidney disease. Exons 34‐41 and 43‐46 were assayed using PCR amplification and SSCP analysis followed by direct sequencing of amplicons presenting variant SSCP patterns. We have identified seven disease‐causing mutations of which five are novel [c.10634‐10656del; c.11587delG; IVS37–10C>A; c.11669‐11674del; c.13069‐13070ins39] and two have been reported previously [Q4010X; Q4041X]. Defects in this part of the gene thus account for 17% of our group of patients. Five of the seven sequence alterations detected are protein‐truncating which is in agreement with mutation screening data for this part of the gene by other groups. The two other mutations are in‐frame deletions or insertions which could destroy important functional properties of polycystin 1. These findings suggest that the first step toward cyst formation in PKD1 patients is the loss of one functional copy of polycystin 1, which indirectly supports the “two‐hit” model of cystogenesis where a second somatic mutation inactivating the normal allele is necessary to occur for development of the disease condition. Hum Mutat 16:166–174, 2000.