Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation

The methylation-based PCR assay at the polymorphic (CAG)n repeat in exon 1 of the human androgen receptor _AR_ gene (HUMARA) is a standard method for determination of the methylation state of alleles in active X (Xa) and inactive X (Xi) chromosomes. HUMARA assay is endowed with heterozygosity rates ~85% worldwide. This means that in a proportion of females it is uninformative. The HUMARA genotype is not neutral, being linked to Kennedy´s disease. Moreover, allele designation and quantification from trinucleotide repeats demand normalizing for minor (stutter) products differing from the original template by multiples of the repeat unit. Here, we report on the identification of a highly polymorphic tetranucleotide repeat (named DXpS) mapping to within a CpG island on Xp. This island is 191 bp downstream from the start of the repeat element, and contains sites for the HhaI, HpaII and BstUI methyl-sensitive restriction enzymes. We developed the DXpS and the HUMARA markers into a biplex methylation-based quantitative fluorescent PCR assay. For DXpS we observed twelve alleles with negligible stuttering. DXpS exhibited a heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined informativeness of the biplex assay was 98%. Random and nonrandom X-inactivation patterns inferred with DXpS in phenotypically normal females and haemophiliac females carrying a defective F8 gene were highly concordant (r^2^ = 0.96) with HUMARA patterns. DXpS represents a notable advancement in detecting X-chromosome inactivation due to the observed high rate of heterozygosity, negligible stuttering, concordance with HUMARA, and the apparent neutrality of allelic variants. Financial support: FAPESP (09/10615-7), FAPERJ, CNPq

Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence— Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems

Genetic testing in bleeding disorders

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.Fil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: El Maarri, O.. Universitat Bonn; AlemaniaFil: Perry, D. J.. Universitat Bonn; AlemaniaFil: Oldenburg, J.. Universitat Bonn; AlemaniaFil: Pezeshkpoor, B.. Universitat Bonn; AlemaniaFil: Goodeve, A.. Sheffield Children’s NHS Foundation Trust; Reino Unido. Sheffield University Medical School; Reino Unid

CTLA4 p.Thr17Ala (c.49A>G) polymorphism associates with inhibitor development in argentine patients with severe hemophilia A

El desarrollo de inhibidores de FVIII en hemofilia A (HA) es considerado un rasgo complejo, ya que involucra factores genéticos y ambientales, siendo el genotipo del gen del factor VIII (F8) el factor condicionante más importante, seguido por factores genéticos secundarios, más débiles. Se estimaron los riesgos de inhibidor asociados a cada genotipo-F8 mediante un estudio de casos/ controles en pacientes HA-severos (n=390), que caracteriza especialmente el estrato con la Inv22 (inversión del intrón 22). El cálculo de la prevalencia de inhibidor (IP) y odds-ratio (OR) con intervalos de confianza del 95% (IC95%) permitió clasificar tres grupos de riesgo (alto, medio y bajo) asociado a cada genotipo-F8. La asociación de factores genéticos secundarios se investigó mediante el análisis de concordancia/discordancia en el estatus de inhibidor en 17 pares de hermanos con la Inv22 vs pares al azar mostrando un OR(IC95%) de 1,83(0,69-4,85) (p=0,33), sugiriendo una tendencia a coincidir con el estatus de inhibidor del hermano. Se realizaron estudios tipo caso/control del riesgo de inhibidor en polimorfismos de IL10, TNFA y CTLA4 en los estratos Inv22-positivo (n=140-148). IL10 c.- 1117A>G, TNFA c.-488G>A y CTLA4 c.-319C>T mostraron tendencias de riesgo o protección similares a las reportadas, con ORs no significativos. CTLA4 c.49A>G p.Thr17Ala mostró un incremento significativo del riesgo de inhibidor con OR de 2,61(1,27-5,36) (p=0,0096). Nuestros resultados concuerdan con especulaciones teóricas previas acerca de diferencias regionales en los factores secundarios no modificables. CTLA4 p.Thr17Ala contribuye a aumentar el riesgo de inhibidor en nuestra población de pacientes con HA-severa.FVIII inhibitor development in hemophilia A (HA) is considered a complex trait as it involves genetic and environmental factors; the causative factor VIII gene (F8) genotype has been established as the most important determining factor, followed by weaker secondary genetic risks factors. Risks of inhibitor development associated with each F8- genotype were estimated by a case/control study in HA-severe patients (n=390), which especially characterizes strata with Inv22 (intron 22 inversion). Inhibitor prevalence (IP) and odds ratio (OR) with confidence intervals of 95% (95%CI) calculations allowed classifying three risk groups (high, medium and low) associated with each F8-genotype. The association of genetic factors was investigated by analyzing concordance/discordance inhibitor-status in 17 pairs of brothers with Inv22 vs random pairs showing 1.83(0.69 to 4.85) (p=0.33), suggesting a trend to agree the inhibitor status between siblings. Case/control studies associated risk inhibitor development with IL10, TNFA and CTLA4 polymorphisms in Inv22-positive strata (n=140- 148) were performed and in IL10 c.-1117A>G, TNFA c.-488G>A & CTLA4 c.-319C>T trends of risks or protection, similar to reported ones with not significant ORs. CTLA4 c.49A>G p.Thr17Ala showed significantly increased inhibitor risks with OR of 2.61(1.27-5.36) (p=0.0096). Our findings agree with previous theoretical speculations about regional differences in non-modifiable secondary factors. CTLA4 p.Thr17Ala, contributes to an increased risk of inhibitor in our population of patients with HA-severFil: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Primiani, L.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: Neme, D.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: Candela, M.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: de Tezanos Pinto, M.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Genética; Argentina. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Genética; ArgentinaFil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Phenotype–genotype correlations in hemophilia A carriers are consistent with the binary role of the phase between F8 and X-chromosome inactivation

Background: The recessive X-linked disorder hemophilia A (HA) is rarely expressed in female carriers, most of whom express about half of normal factor VIII activity (FVIII:C). Objective: To propose an integrative assessment model for the binary role of the phase between the mutated F8 and the active X-chromosome (Xa) in FVIII:C in HA carriers. Methods: We studied 67 females at risk of severe HA, comprising five symptomatic females (FVIII:C < 1.5 IU dL−1) and 14 controls. A correlation study between FVIII:C (observed vs. expected) and X-chromosome inactivation (XCI) patterns (XIPs; androgen receptor gene [AR] system) in blood leukocyte DNA was performed in carriers, by comparison of a model correlating FVIII:C and XIP with arbitrary models devoid of biological significance, and with FVIII:C levels in non-carriers (mean model) as a proxy from background data dispersion not influenced by XIP. Results: We provide proof-of-concept example from a family presenting with extremely skewed XIPs in which the severe HA phenotype appeared in a heterozygous carrier of a crossover between AR and F8 loci that phased the mutated F8 with the maternally inherited Xa. Furthermore, four cases of severe HA affected women who had a combination of a heterozygous F8 mutation and extremely skewed XIPs in leukocytes or oral mucosa are presented. Correlation analyses between FVIII:C levels and XIPs in carriers (n = 38) but not in non-carriers (n = 20) showed highly significant differences between the proposed correlation model and models without biological significance. The data support a binary influence of XCI, either increasing or decreasing the FVIII:C, subject to the underlying phase set between the F8 mutation and XCI. Conclusions: Our evidence suggests that the phase between XCI and mutated F8 acts as a molecular switch conditioning FVIII:C levels and HA expression in carriers.Fil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Tetzlaff, T.. Universidad Nacional de General Sarmiento. Instituto de Ciencias; ArgentinaFil: Candela, M.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Neme, D.. Fundación de la Hemofilia "Alfredo Pavlovsky"; ArgentinaFil: Sciuccati, G.. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Bonduel, M.. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Medina Acosta, E.. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; BrasilFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: De Tezanos Pinto, M.. Fundación de la Hemofilia "Alfredo Pavlovsky"; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants. We describe a neurodevelopmental disorder associated with de novo gain-of-function variants in PIP5KIγ kinase. The variants cause perturbed endosomal function resulting from increased production of phosphatidylinositol 4,5 bisphosphate and enhanced association of F-actin at endosomes. Moreover, mutant zebrafish larvae recapitulate the phenotypes observed in affected individuals from our cohort