Allele In Trans Research Articles

Allele-Specific Impairment of GJB2 Expression by GJB6 Deletion del(GJB6-D13S1854)

Mutations in the GJB2 gene, which encodes connexin 26, are a frequent cause of congenital non-syndromic sensorineural hearing loss. Two large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854), which truncate GJB6 (connexin 30), cause hearing loss in individuals homozygous, or compound heterozygous for these deletions or one such deletion and a mutation in GJB2. Recently, we have demonstrated that the del(GJB6-D13S1830) deletion contributes to hearing loss due to an allele-specific lack of GJB2 mRNA expression and not as a result of digenic inheritance, as was postulated earlier. In the current study we investigated the smaller del(GJB6-D13S1854) deletion, which disrupts the expression of GJB2 at the transcriptional level in a manner similar to the more common del(GJB6-D13S1830) deletion. Interestingly, in the presence of this deletion, GJB2 expression remains minimally but reproducibly present. The relative allele-specific expression of GJB2 was assessed by reverse-transcriptase PCR and restriction digestions in three probands who were compound heterozygous for a GJB2 mutation and del(GJB6-D13S1854). Each individual carried a different sequence variant in GJB2. All three individuals expressed the mutated GJB2 allele in trans with del(GJB6-D13S1854), but expression of the GJB2 allele in cis with the deletion was almost absent. Our study clearly corroborates the hypothesis that the del(GJB6-D13S1854), similar to the larger and more common del(GJB6-D13S1830), removes (a) putative cis-regulatory element(s) upstream of GJB6 and narrows down the region of location.

Molecular Basis of the Rare Gene Complex, D(C)-, Which Encodes Four Low Prevalence Antigens In the Rh Blood Group System.

AbstractAbstract 1117 Background:Over 40 years ago, the investigation of a case of fatal HDN in the third child of Madame Nou, a native of Ivory Coast, revealed that Madame Nou's RBCs had an unusual phenotype in the Rh blood group system denoted DIVa(C)-/DIVa(C)-. Initially, her RBCs were shown to express a partial D, a weak form of C, and Goa (RH30) [Salmon, et al., Rev Franc Transf 1969;12:239]. Later her RBCs were shown to also express RH33, Riv (RH45), and FPTT (RH50) [Bizot, et al., Transfusion 1988;28:342; Delehanty, et al., Transfusion 1983;23:410, abstract]. R0Har and CeVA phenotypes are encoded by hybrid RHCE-D(5)-CE alleles (respectively, c+ and C+) and the RBCs express RH33 and FPTT antigens but not Goa or Riv [Noizat-Pirenne, et al. Transfusion 2002;42:627]. RHD*DIVa.2 encodes a partial D and the Goa antigen and frequently travels with RHCE*ce(1025T) (RHCE*ceTI) (Vege, et al., Transfusion 2007;47:159A). The purpose of this study was to determine the molecular basis associated with the rare DIVa(C)- complex. Material and Methods:Blood samples were obtained from three donors previously identified as having the DIVa(C)- haplotype. Molecular analyses were performed by standard methods and included AS-PCR, PCR-RFLP, genomic sequencing of specific exons, and cloning and direct sequencing of cDNA. Results:At the RHD locus all donors were heterozygous for RHD and RHD*DIVa.2 and at the RHCE locus all had a compound hybrid allele, which contains exons 2 and 3 from RHD*DIVa.2 (based on RHD*186G/T, RHD*410C/T, RHD*455A/C), and exon 5 from RHD. The altered RHCE is presumed to be in cis to RHD*DIVa.2. In all three probands RHCE*48 in exon 1 is G/C; presumably the G belonging to the in trans RHCE and the nt48C to the hybrid allele, and this assumption favors exon 1 of the hybrid being from RHCE. Thus, the RHCE allele is likely RHCE*CE-DIVa.2(2,3)-CE-D(5)-CE. The in trans allele in Proband 1 is RH*cE, in Proband 2 it is RHCE*ce 254C, 733G, and in Proband 3 it is RHCE*ce. Conclusions:The compound hybrid provides an explanation for the expression of the four low prevalence antigens on RBCs with the DIVa(C)- phenotype. RHD*DIVa.2 encodes the Goa antigen. The flanking of RHD exon 5 by RHCE exons in the compound hybrid likely results in RH33 and FPTT antigen expression because R0Har and CeVA RBCs express these two antigens. It is possible that the junction of RHD exon 3 to RHCE exon 4 is involved in the expression of Riv. The weak C expression could be a consequence of exons 2 and 3 from RHD*DIVa.2 in the compound hybrid because exon 2 of the wild type RHD is identical in sequence to exon 2 of RHCE*C. The three probands in our study had RHCE nt1025C/C (wild type) and thus, are not RHCE*ce(1025T). This is the first report of RHD*DIVa.2 being involved in a hybrid gene at the RHCE locus. Such a hybrid is not unprecedented in that RHD*DIIIa is involved in the RHD*DIIIa-CE(4-7)-D hybrid [(C)ceS type 1 in the r'S haplotype] As only one example of anti-Riv has been described, our findings provide a tool by which to predict the expression of Riv. Disclosures:No relevant conflicts of interest to declare.

Hemoglobin Q-Thailand related disorders: Origin, molecular, hematological and diagnostic aspects

We describe the molecular and hematological profiles of thalassemia syndromes caused by interactions of hemoglobin (Hb) Q-Thailand [α74(EF3) Asp-His] and various hemoglobinopathies found in 52 unrelated adult Thai subjects. Ten genotypes including several previously undescribed conditions were observed, which were classified into 4 groups. Group I included 26 Hb Q-Thailand heterozygotes and a homozygotous subject. Group II included subjects with Hb Q-Thailand and other α-thalassemia alleles in trans including 1 compound Hb Q-Thailand/α +-thalassemia (− α 3.7), 2 Hb Q-Thailand/Hb Constant Spring disease and 6 Hb H/Q-Thailand disease. The average levels of Hb Q-Thailand were found to be 29.8%, 82.3%, 34.7%, 49.2–49.3% and 79.4%, respectively. Both Hbs Bart's and H were observed in addition to Hb Q-Thailand in all 6 cases with Hb Q-H disease but not in a homozygous Hb Q-Thailand. Group III included 7 double heterozygotes for Hb Q-Thailand/Hb E, 3 Hb Q-Thailand/Hb E/α +-thalassemia (−α 3.7), 3 heterozygous Hb Q-Thailand/homozygous Hb E and 1 triple heterozygote for Hb Q-Thailand/Hb Constant Spring/Hb E. In this group, Hbs E (α A 2β E 2), Q-Thailand (α QT 2β A 2) and QE (α QT 2β E 2) were observed on both HPLC and capillary electrophoresis. The Hb QE, rather than Hb Q-Thailand, was detected in all 3 cases with heterozygous Hb Q-Thailand and homozygous Hb E. The remaining two cases in group 4 were double heterozygotes for Hb Q-Thailand and β 0-thalassemia in which Hb Q-Thailand, elevated Hb A 2 (α A 2δ 2), and Hb QA 2 (α QT 2δ 2) were detected. DNA analysis identified the Hb Q-Thailand mutation (α74: GAC- CAC) and the linked (− α 4.2) in all cases. Analysis of α-globin gene haplotype provided the first evidence of a single origin of this Hb variant in Thai population.

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.

Non‐classic cystic fibrosis associated with D1152H CFTR mutation

Limited knowledge exists on phenotypes associated with the D1152H cystic fibrosis transmembrane conductance regulator (CFTR) mutation. Subjects with a D1152H allele in trans with another CFTR mutation were identified using the French Cystic Fibrosis Registry. Phenotypic characteristics were compared with those of pancreatic insufficient (PI) and pancreatic sufficient (PS) cystic fibrosis (CF) subjects in the Registry (CF cohort). Forty-two subjects with D1152H alleles were identified. Features leading to diagnosis included chronic sinopulmonary disease (n = 25), congenital absence of the vas deferens (n = 11), systematic neonatal screening (n = 4), and genetic counseling (n = 2). Median age at diagnosis was 33 [interquartile range (IQR, 24-41)] years in D1152H subjects. Median sweat chloride concentrations were 43.5 (39-63) mmol/l in D1152H subjects and were markedly lower than in PI and PS CF subjects (p < 0.05). Bronchiectasis was present in 67% of D1152H subjects, but Pseudomonas aeruginosa colonization and pancreatic insufficiency were present in <30% of subjects. Estimated rates of decline in forced expiratory volume in 1 s (FEV(1)) were lower in D1152H subjects vs PI CF subjects (p < 0.05). None of the D1152H subjects identified since 1999 had died or required lung transplantation. When present in trans with a CF-causing mutation, D1152H causes significant pulmonary disease, but all subjects had prolonged survival.

Many genetically defined ABO subgroups exhibit characteristic flow cytometric patterns

A flow cytometric method for detection of low levels of A/B antigen had been developed previously in our laboratory. The aim of this study was to investigate if this approach could be utilized to characterize different ABO subgroups and constitute a useful tool in a reference laboratory. Blood samples causing ABO discrepancies (n = 94) by routine serology were further analyzed by ABO genotyping and flow cytometry. To verify the specificity of the monoclonal anti-A and -B reagents and to establish normal flow cytometric patterns, samples from 80 blood donors with common phenotypes were also assessed. Distinguishable flow cytometric patterns were detected for several subgroups but were more apparent for A(weak) (n = 80) samples than B(weak) (n = 14). Two subgroups, A(finn) (n = 11) and A(3) (n = 10) displayed diagnostic features and were used to establish reproduciblity over time and between donors. In general, the consistency within subgroups was remarkable. The allelic enhancement phenomenon was clearly visualized among A(x) samples (n = 10) where different alleles in trans resulted in high, low, or no A antigen expression. Nonsubgroup samples including O/A and O/B chimeras or A(h) and B(h) para-Bombay phenotypes displayed clearly distinguishable histograms. Samples from pregnant women (n = 10) displayed acquired A antigen loss, apparently accentuated during the third trimester. Genetically defined ABO subgroups and other anomalous phenotypes displayed flow cytometric profiles that may contribute valuable information to the investigation of ABO discrepancies. We conclude that the presented assay may complement traditional serology and genetic analysis in the reference laboratory setting.

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization

Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti-C has not been investigated. We sought partial C status and anti-C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided. We constituted a cohort of 177 randomly selected SCD patients expressing C antigen. We screened for (C)ce(s) and R(N) haplotypes, presumably associated with partial C antigen in Afro-Caribbeans, and we recorded the number of transfused C+ red blood cell (RBC) units, immunization status, and extended phenotype. Forty-nine patients carried abnormal C antigen, deduced from the presence of (C)ce(s) and/or R(N), not compensated by a normal RHC allele in trans. Among patients with partial C phenotype exposed repeatedly to C+ RBCs, 30% produced anti-C. Two patients experienced hemolysis. In our hospital, with 22% of SCD patients expressing C, prevention of anti-C immunization for all individuals with partial C antigen would require a 7% increase in the use of C- RBC units. These RBCs are already in short supply for SCD patients who are C-. This study demonstrates the need to detect partial C within C+ SCD patients and to prevent immunization. A larger number of Afro-Caribbeans donors is needed to provide these patients with C- RBCs.

The digenic hypothesis unraveled: The GJB6 del( GJB6-D13S1830) mutation causes allele-specific loss of GJB2 expression in cis

Connexin 26 and connexin 30 are the major connexins expressed in the cochlea, where they are co-localized and form heteromeric gap junctions. Mutations in the GJB2 gene, which encodes connexin 26, are the most common cause of prelingual non-syndromic sensorineural hearing loss. The large del( GJB6-D13S1830) mutation which involves GJB6 (connexin 30), causes hearing loss in homozygous individuals, or when compound heterozygous with a GJB2 mutation. Until now, it remained unresolved whether this phenomenon results from digenic inheritance or because of lack of GJB2 mRNA expression. After RNA extraction from buccal epithelium, a tissue known to express connexin 26 as well as connexin 30, allele-specific expression of GJB2 was investigated by reverse-transcriptase PCR and restriction digestions in three unrelated individuals compound heterozygous for a GJB2 mutation and del( GJB6-D13S1830). Each proband carried a different sequence change in GJB2. The mutated GJB2 allele in trans with del( GJB6-D13S1830) was expressed in all three individuals whereas the GJB2 allele located in c is with the deletion was not expressed at all. Thus, mutations in these two genes do not cause hearing loss through a digenic mechanism of inheritance alone, as was postulated previously, but instead GJB2 expression is abolished through an effect in cis with the deletion. Our study provides unequivocal support for the hypothesis that del( GJB6-D13S1830) eliminates a putative cis-regulatory element located within the deleted region.

Laboratory Exercises To Examine Recombination &amp; Aneuploidy inDrosophila

[ILLUSTRATION OMITTED] Chromosomal aneuploidy, a deviation from an exact multiple of an organism's haploid chromosome number, is a difficult concept for students to master. Aneuploidy arising from chromosomal non-disjunction (NDJ) is particularly problematic for students, since it arises in the context of meiosis, itself a challenging subject. Students learning NDJ are forced to actively apply their knowledge of normal meiosis, often revealing previously unapparent deficits in their understanding. Teaching NDJ is thus an important opportunity for students to review and apply their skills in modeling meiosis. Despite this opportunity, standard methods for teaching NDJ are passive. While experiments illustrating recombination mapping, sex-linkage, and other core genetics concepts are readily available for high school and undergraduate genetics courses, simple exercises demonstrating NDJ are not. This is regrettable, for non-disjunction is not only medically important but also historically significant as the original proof for the chromosomal theory of inheritance (Bridges, 1916). Presented here is a straightforward method to detect NDJ of the sex chromosomes in Drosophila in the context of laboratory exercises to examine recombination of sex-linked loci. The exercises presented here allow for detecting maternal and paternal NDJ events within a single cross, an advantage since the use of standard markers obscures paternal NDJ events (Bridges, 1916). An additional advantage of this method is that it easily allows for recombination mapping with recessive alleles in trans as well as in cis. This method allows students to simultaneously investigate recombination and aneuploidy with live organisms, and as such is complementary to theoretical methods of teaching recombination and NDJ. The exercises presented here are based on a Drosophila virginizing system that has recently been developed as a method to perform teaching crosses without the need for collecting virgins manually (Venema, 2006). This system employs a modified Y chromosome that harbors a conditional, dominant lethal mutation: a regulatory sequence that is activated in response to heat connected to a protein called head involution defective, or hid that activates programmed cell death. Since this modified Y chromosome (abbreviated as Y{hs-hid}) is present only in males, temporarily raising the temperature of a Y{hs-hid} Drosophila culture during larval stages will cause all males to undergo systemic apoptosis and die. Female larvae, however, survive the heat shock and remain virgin after hatching since there are no surviving males to mate with (Venema, 2006). The use of this system is of great advantage for educators in that large numbers of virgins can be obtained without scoring or segregating young female flies. This ability to use large numbers of flies in pedagogical crosses also facilitates finding the results of rare genetic events in the progeny. (1) Venema (2006) describes a scheme for recombination mapping between autosomal loci using this system; however, recombination mapping of sex-linked loci in Drosophila is more convenient for high school or introductory-level university courses. Additionally, examining recombination between sex-linked loci allows for the simultaneous detection of chromosomal NDJ events in the progeny, since some Drosophila sex chromosome aneuploids are viable (Table 1). Four possible exercises are presented: two versions of a dihybrid testcross and two versions of a three-point testcross. The dihybrid testcross exercises map sex-linked loci with the recessive mutant alleles either together as a parental genotype (i.e., recessive alleles in cis) or with recessive mutations inherited from both parents (recessive alleles in trans). Similarly, the options for the three-point testcross are mapping the three loci with mutant alleles all in cis, or with two in cis, and the third in trans. The dihybrid or three-point testcross options allow for instructors to choose the exercises of appropriate complexity for their particular grade level and learning objectives. …

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G&gt;A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the delta-globin gene is relevant to the prevention of beta-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and the double heterozygosis for alleles of delta- and beta-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the delta- and beta-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the delta-globin alleles was detected; seven known alleles in trans to the beta-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a beta-thalassemia allele was detected in one family. Moreover, the relative frequency of the delta-mutants was quite different from that found among heterozygotes. The new allele delta-cod 5 CCT>ACT, in cis to the allele beta(+) thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant delta5(A2)Pro-->Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the beta(+) thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

RipA, a Cytoplasmic Membrane Protein Conserved amongFrancisellaSpecies, Is Required for Intracellular Survival

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

Outcome of intracytoplasmic sperm injection for a couple in which the man is carrier of CFTR p.[R74W;V201M;D1270N] and p.P841R mutations and his spouse a heterozygous carrier of p.F508del mutation of the cystic fibrosis transmembrane conductance regulator gene

To document the phenotype associated with the p.[R74W;V201M;D1270N] and p.P841R mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. Case report. Biology and medicine of reproduction in a university hospital. A couple in which the man is carrier of the triple mutant p.[R74W;V201M;D1270N] allele in trans to p.P841R mutation and his spouse a heterozygous carrier for the severe p.F508del mutation of the CFTR gene, who became pregnant after intracytoplasmic sperm injection (ICSI) with twins. Genetic counseling; CFTR gene sequencing; ICSI; children's follow-up. First report of a male phenotype associated with the p.P841R mutation. The triple mutant p.[R74W;V201M;D1270N] allele associated with the unknown p.P841R mutations were detected in this man with congenital bilateral absence of the vas deferens, which may presume p.P841R as a severe mutation. After genetic counseling, the couple preferred prenatal diagnosis after ICSI than preimplantation genetic diagnosis, which revealed that the boys were both carriers of p.[R74W;V201M;D1270N] and p.F508del mutations. They are now 4 years old and show normal growth without nutritional deficiency. This case report documents for the first time a male phenotype associated with the p.P841R mutation and underlines the difficulties in counseling a man with congenital bilateral absence of the vas deferens carrying uncommon mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene before ICSI.

Use of BRCA1 protein:protein interactions to classify cancer risk

Germline loss-of-function mutations in BRCA1 are associated with a high lifetime risk of breast and ovarian cancer. Most mutations in the gene are 'truncating': in the main these induce premature termination codons, resulting in nonsense-mediated decay, loss of the transcript and/or the entire protein. The improved screening methods now in use across the UK will identify many carriers of unclassified BRCA1 variants. These are chiefly missense mutations, introducing an amino acid change in the context of an expressed protein. Indeed more than one-quarter of entries recorded in the Breast Cancer Information Core dataset of BRCA1 sequence variants collected from patients worldwide are unclassified missense alterations http://research.nhgri.nih.gov/bic/. Currently, discovery of the majority of missense variants leaves both variant carriers and their families in an ambiguous position. These variants remain unclassified because in the majority of cases it is not possible to follow variants by cosegregation analysis, and the number of appropriate controls required to be certain that a variant is absent in unaffected individuals is prohibitive. Currently, in silico algorithms try to distinguish between missense substitutions that are likely to be pathogenic and those that are not. These algorithms compile a multicomponent likelihood ratio that integrates assessment methods ranging from conservation analysis, co-occurrence of a deleterious allele in trans, and immuno-histochemical analysis [1-3]. What is missing from these analyses is the relationship between loss of protein function and detriment to patient health. We have focused on the N-terminal region of BRCA1. This region has a high density of missense substitutions, including those of known pathogenic status, and many currently unclassified variants. We have shown that experimental missense variants, generated randomly and selected for loss of interaction with the BRCA1 ubiquitin ligase components, BARD1 and the E2 enzyme UbcH5, identify variants reported within the Breast Cancer Information Core database of individuals with a personal or family history of breast cancer [4]. The E2 component is particularly sensitive to missense alteration in BRCA1, with the majority of currently unclassified variants in the region inhibiting interaction, whereas the BARD1 component is disrupted by a smaller, but overlapping, subset restricted to substitution of the structurally detrimental zinc-ligation residues. Variants that inhibited the E2 also prevented the enzymatic activity. These data strongly suggest that the ligase activity of BRCA1, through interaction with E2 and BARD1, is related to breast cancer predisposition. Using yeast two-hybrid analysis for BRCA1:BARD1 and BRCA1:E2 interaction, we have tested the most chemically different substitutions achievable by single nucleotide change in all of the most highly conserved amino acids of the region (invariant from human to sea urchin), and have also tested all currently identified patient missense variants. These data have been combined with Grantham variation and Grantham deviance scores (a measure of how conserved an amino acid is, together with how different the protein change is) to assess the relationship between protein:protein interaction and measures of disease risk. Risk measures were based on the results of full sequence tests of BRCA1 and BRCA2 from 68,000 BRAC Analysis subjects (Myriad Genetics, Salt Lake City, UT, USA), and used estimates of the odds of developing breast cancer for a carrier of a BRCA1 missense substitution [2], together with enrichment ratios achieved by comparing the variants observed in the dataset with the variants expected on the basis of known substitution rates. Classification methods in the past have attempted to place variants in either the pathogenic or the little-clinical significance categories. The results of this analysis suggest that some classes of variant may confer an intermediate risk. If so, these data have considerable implications for the counselling and clinical management of women found to be positive for missense variants in future.

Segmental duplications involving the α-globin gene cluster are causing β-thalassemia intermedia phenotypes in β-thalassemia heterozygous patients

We describe two cases of simple heterozygosity for the common β°-thalassemia mutation β39 (C → T), both presenting with a thalassemia intermedia phenotype. In both cases synergic effect deriving from membrane defects or red cell enzyme deficiencies were excluded. In one case a triplication of the α-globin genes was found which did not justify the severity of the transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) analysis of the α-globin gene cluster revealed two new rearrangements, consisting of a full duplication of the α-globin genes locus including the upstream regulatory element. In one case the duplication was in the presence of the common anti-α 3.7 triplication in trans, resulting in a total of 7 active α-globin genes. In the other case the duplicated allele and the normal allele in trans resulted into a total of 6 active α-globin genes. We report the clinical and hematological data and the molecular analysis and discuss the occurrence of α-globin genes duplication defects in cases of β-thalassemia heterozygotes with thalassemia intermedia phenotypes.

Deletion of the core- H region in mice abolishes the expression of three proximal odorant receptor genes in cis

We have previously reported that a 2.1-kb homology (H) sequence, conserved between mouse and human, regulates the odorant receptor (OR) gene MOR28 in transgenic mice. Here, we narrowed down the essential sequences of the H to a core of 124 bp by using a transient expression system in zebrafish embryos. Transgenic experiments in mice demonstrated that the core-H sequence is sufficient to endow expression of the MOR28 minigene. Deletion and mutation analyses of the core-H region revealed two homeodomain sequences to be essential for the H enhancer activity. Targeted deletion of the core-H abolished expression of three proximal OR genes, MOR28, MOR10, and MOR83, in cis, indicating the presence of another locus control region/enhancer in the downstream region, that regulates four distal OR genes in the same MOR28 cluster. In the heterozygous mice, the H(-) phenotype of the mutant allele was not rescued by the wild-type H(+) allele in trans.

Intragenic Cis and Trans Modification of Genetic Susceptibility in DYT1 Torsion Dystonia

A GAG deletion in the DYT1 gene is a major cause of early-onset dystonia, but clinical disease expression occurs in only 30% of mutation carriers. To gain insight into genetic factors that may influence penetrance, we evaluated three DYT1 single-nucleotide polymorphisms, including D216H, a coding-sequence variation that moderates the effects of the DYT1 GAG deletion in cellular models. We tested DYT1 GAG-deletion carriers with (n=119) and without (n=113) clinical signs of dystonia and control individuals (n=197) and found the frequency of the 216H allele to be increased in GAG-deletion carriers without dystonia and to be decreased in carriers with dystonia, compared with the control individuals. Analysis of haplotypes demonstrated a highly protective effect of the H allele in trans with the GAG deletion; there was also suggestive evidence that the D216 allele in cis is required for the disease to be penetrant. Our findings establish, for the first time, a clinically relevant gene modifier of DYT1.

YscU recognizes translocators as export substrates of the Yersinia injectisome

YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

Tethering RITS to a Nascent Transcript Initiates RNAi- and Heterochromatin-Dependent Gene Silencing

In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4+ gene silences ura4+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4+ siRNAs to silence a second ura4+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4+, or ura4+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism.

Allele-specific transcription of fetal genes in primary erythroid cell cultures from Lepore and δβ° thalassemia patients

Autonomous gene silencing and gene competition by globin promoters for locus control region (LCR) function have been proposed as mechanisms in developmental regulation of beta-like genes. deltabeta degrees thalassemias are syndromes presenting an increased production of fetal hemoglobin in adult life; the majority of them are due to various deletions in beta-globin gene cluster. We studied samples from double heterozygotes for beta-thalassemia and for Lepore or Sicilian deltabeta degrees deletions, both lacking beta-promoter sequence. Our goal was to address the question of whether the allele carrying the deltabeta degrees deletion is responsible for high level of fetal hemoglobin (HbF) production. We analyzed the globin gene transcription in human erythroid cell cultures from peripheral blood stem cells, using primary transcript in situ hybridization. We performed primary erythroid cultures from patients with the following genotypes: Lepore/beta degrees 39, Sicilian deltabeta degrees /beta degrees 39, and, as controls, two thalassemia patients with nondeletional mutations (IVS1,6/IVS1,6; IVS1,6/beta degrees 39), and one normal individual. The cells where it is possible to unambiguously assign gamma genes transcription in cis with the deletion (gamma:beta) are strongly represented with respect to the nine other combinations of gamma and beta hybridization signals. These cells are at least nine times more represented than those expressing the gamma allele in trans to the deletion. The allele-specific transcription of fetal genes in cis with the deletion is favored in both deletional genotypes. The absence of the adult promoter may influence LCR recruitment by fetal promoter, supporting the hypothesis that competition mechanism and gene silencing can coexist in regulating human globin gene transcription.