In haemophilia A, linkage analysis with coagulation factor VIII (F8) intragenic and/or neighbouring extragenic short tandem repeats (STRs) enables indirect tracking F8 pathogenic allelic variant-carriers. Even where DNA sequencing is available, linkage analysis still has a role if no causative or candidate mutation is unveiled. The cumulative heterozygosity rate of the available multiplexed STRs haplotyping assays rarely reaches 100%. This means that in a proportion of women these loci are uninformative. The norm-referenced assessment is based on at least one informative marker criterion. We reasoned that by typing a dense market set, spanning a small fraction of recombination, we should be able to improve assessment. The aim of this study was to improve criterion-referenced assessment in polymorphism segregation analyses using a low-recombination fraction and dense informative STRs set. The multiplex quantitative fluorescence PCR assay comprises four novel tetranucleotide and pentanucleotide STRs distant < or = 0.15 cM from the F8 gene, and three F8 intragenic dinucleotide STRs, mapped to a 0.23 cM interval spanning the F8 on human chromosome band Xq28. We determined heterozygosity rates and allele frequencies from 100 unrelated healthy females. To investigate about segregation stability, we typed 50 true trios (mother, daughter and father) and 50 true mother-and-son duos from the general population. The heterozygosity rates for the extragenic markers ranged 0.49-0.76. The 0.23 cM-resolution heptaplex rendered a cumulative heterozygosity of 0.89 for a minimum of two informative markers, with at least one F8 intragenic. The heptaplex assay enabled improving the criterion-referenced assessment in indirect carrier-detection.

Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host.

Linkage analysis using multiple F8 (Factor VIII) intragenic (type I) and/or closely linked extragenic (type II) short tandem repeats (STRs) DNA multiallelic loci is the indirect method of choice for carrier-tracking the defective F8 gene within haemophilia A at-risk families [1,2]. The most commonly used F8 intragenic markers are the dinucleotide loci F8Int13 and F8Int22, and the heterozygosity rates vary considerably among worldwide populations (0.46-0.91 for F8Int13; 0.40-0.79 for F8Int22) [3]. Importantly, the cumulative heterozygosity rate of these two intragenic markers does not reach 100% [4]. This means that in a proportion of women, these loci are uninformative (i.e. homozygous profiles), constituting a major drawback to diagnosis in some families. We aimed at characterizing a novel (TG) n dinucleotide STR located on F8 intron 25 (termed F8Int25.2), earlier identified in silico by us as polymorphic [3]. We determined heterozygosity and allele frequencies for F8Int25.2, F8Int13 and F8Int22 markers in 100 unrelated females by quantitative fluorescence polymerase chain reaction using published primer sequences (Supplementary Online Material Table S1). In addition, we typed four mother-and-son duos from our cohort of haemophilia A families. We observed five F8Int25.2 alleles: four in healthy females and a fifth (150 bp), exclusively found in a haemophilia family. In healthy females, the heterozygosity for F8Int25.2 (0.52) was comparable to the rate for F8Int22 (0.57), both of which were lower than the rate for F8Int13 (0.74), Table 1. All three genotype frequency distributions were in Hardy-Weinberg equilibrium (P ‡ 0.15175).Even with a small number of alleles, F8Int25.2 exhibited a heterozygosity rate higher than that reported for F8Int1 [5] and F8Int25 markers [4,5], either one reported with at least five alleles. This is due to a homogenous frequency distribution of the two most frequent alleles. Typing with both F8Int13 and F8Int22 enabled the identification of X Table 1. Allele frequency distribution and statistical parameters for F8Int25.2, F8Int13 and F8Int22 markers in 100 unrelated healthy females. F8Int13F8Int22F8Int25.2

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ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains.