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Carrier detection in families with properdin deficiency by microsatellite haplotyping.

ABSTRACT: Human properdin deficiency is an X-linked disorder strongly predisposing to meningococcal disease which has been recorded in over 50 cases of various ethnic origins. Immunochemically, total deficiency (type I), partial deficiency (type II), and deficiency due to a dysfunctional molecule (type III) can be differentiated. It is therefore most likely that the causative molecular defects will show considerable genetic heterogeneity. Analysis of the properdin locus at Xp11.3-Xp11.23 has led to the characterization of two polymorphic (dC-dA)n.(dG-dT)n repeats located approximately 15 kb downstream from the structural gene. Three families (two Scottish Caucasoid, one Tunisian Sephardic) with seven deficient individuals were investigated immunochemically and using a nonradioisotopic polymerase chain reaction-based method for microsatellite detection. Probable and definite carriers frequently showed properdin levels which were in the normal range. No recombinants between the microsatellite loci and properdin deficiency were detected, thus allowing identification of the defective allele through the generations in all three pedigrees. Haplotyping for these highly polymorphic microsatellites in close physical linkage to the properdin gene can provide rapid and nonradioactive detection of carrier status and prenatal diagnosis without extensive sequencing analysis.

SUBMITTER: Kolble K

PROVIDER: S-EPMC330001 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Carrier detection in families with properdin deficiency by microsatellite haplotyping.

Kölble K K Cant A J AJ Fay A C AC Whaley K K Schlesinger M M Reid K B KB

The Journal of clinical investigation 19930101 1

Human properdin deficiency is an X-linked disorder strongly predisposing to meningococcal disease which has been recorded in over 50 cases of various ethnic origins. Immunochemically, total deficiency (type I), partial deficiency (type II), and deficiency due to a dysfunctional molecule (type III) can be differentiated. It is therefore most likely that the causative molecular defects will show considerable genetic heterogeneity. Analysis of the properdin locus at Xp11.3-Xp11.23 has led to the ch ...[more]

PMID: 8423238

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Project description:C3 glomerulopathy (C3G) is an umbrella classification for severe renal diseases characterized by predominant staining for complement component C3 in the glomeruli. The disease is caused by a dysregulation of the alternative pathway (AP) of the complement system. In more than half of C3G patients C3 nephritic factors (C3NeFs) are found. These autoantibodies bind to the AP C3 convertase, prolonging its activity. C3NeFs can be dependent or independent of the complement regulator properdin for their convertase-stabilizing function. However, studies to determine the properdin-dependency of C3NeFs are rare and not part of routine patient workup. Until recently, only supportive treatments for C3G were available. Complement-directed therapies are now being investigated. We hypothesized that patients with properdin-dependent C3NeFs may benefit from properdin-inhibiting therapy to normalize convertase activity. Therefore, in this study we validated two methods to distinguish between properdin-dependent and properdin-independent C3NeFs. These methods are hemolytic assays for measuring convertase activity and stability in absence of properdin. The first assay assesses convertase stabilization by patient immunoglobulins in properdin-depleted serum. The second assay measures convertase stabilization directly in patient serum supplemented with the properdin-blocking agent Salp20. Blood samples from 13 C3NeF-positive C3G patients were tested. Three patients were found to have properdin-dependent C3NeFs, whereas the C3NeF activity of the other ten patients was independent of properdin. The convertase-stabilizing activity in the samples of the patients with properdin-dependent C3NeFs disappeared in absence of properdin. These data indicate that inhibition of properdin in patients with properdin-dependent C3NeFs can normalize convertase activity and could represent a novel therapy for normalizing AP hyperactivity. Our assays provide a tool for identifying C3G patients who may benefit from properdin-inhibiting therapy and can be incorporated into standard C3G laboratory investigations.

Project description:Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known and novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. Keywords: detection, pathogen, virulence mechanism In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.

Project description:BackgroundDeveloping lepidopteran microsatellite DNA markers can be problematical, as markers often exhibit multiple banding patterns and high frequencies of non-amplifying "null" alleles. Previous studies identified sequences flanking simple sequence repeat (SSR) units that are shared among many lepidopteran species and can be grouped into microsatellite-associated DNA families. These families are thought to be associated with unequal crossing-over during DNA recombination or with transposable elements (TEs).ResultsWe identified full-length lepidopteran non-LTR retrotransposable elements of the RTE clade in Heliconius melpomene and Bombyx mori. These retroelements possess a single open reading frame encoding the Exonuclease/Endonuclease/Phosphatase and the Reverse Transcriptase/nLTR domains, a 5' UTR (untranslated region), and an extremely short 3' UTR that regularly consists of SSR units. Phylogenetic analysis supported previous suggestions of horizontal transfer among unrelated groups of organisms, but the diversity of lepidopteran RTE elements appears due to ancient divergence of ancestral elements rather than introgression by horizontal transfer. Similarity searches of lepidopteran genomic sequences in GenBank identified partial RTE elements, usually consisting of the 3' terminal region, in 29 species. Furthermore, we identified the C-terminal end of the Reverse Transcriptase/nLTR domain and the associated 3' UTR in over 190 microsatellite markers from 22 lepidopteran species, accounting for 10% of the lepidopteran microsatellites in GenBank. Occasional retrotransposition of autonomous elements, frequent retrotransposition of 3' partial elements, and DNA replication slippage during retrotransposition offers a mechanistic explanation for the association of SSRs with RTE elements in lepidopteran genomes.ConclusionsNon-LTR retrotransposable elements of the RTE clade therefore join a diverse group of TEs as progenitors of SSR units in various organisms. When microsatellites are isolated using standard SSR enrichment protocols and primers designed at complementary repeated regions, amplification from multiple genomic sites can cause scoring difficulties that compromise their utility as markers. Screening against RTE elements in the isolation procedure provides one strategy for minimizing this problem.

Dataset Information

Carrier detection in families with properdin deficiency by microsatellite haplotyping.

Publications

Carrier detection in families with properdin deficiency by microsatellite haplotyping.

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