Comprehensive genomic filtering algorithm to expose the cause of skewed X chromosome inactivation. The proof of concept in female haemophilia expression.

BACKGROUND: Exploring the expression of X linked disorders like haemophilia A (HA) in females involves understanding the balance achieved through X chromosome inactivation (XCI). Skewed XCI (SXCI) may be involved in symptomatic HA carriers. We aimed to develop an approach for dissecting the specific cause of SXCI and verify its value in HA. METHODS: A family involving three females (two symptomatic with severe/moderate HA: I.2, the mother, and II.1, the daughter; one asymptomatic: II.2) and two related affected males (I.1, the father and I.3, the maternal uncle) was studied. The genetic analysis included F8 mutational screening, multiplex ligation-dependent probe amplification, SNP microarray, whole exome sequencing (WES) and Sanger sequencing. XCI patterns were assessed in ectoderm/endoderm and mesoderm-derived tissues using AR-based and RP2-based systems. RESULTS: The comprehensive family analysis identifies I.2 female patient as a heterozygous carrier of F8:p.(Ser1414Ter) excluding copy number variations. A consistent XCI pattern of 99.5% across various tissues was observed. A comprehensive filtering algorithm for WES data was designed, developed and applied to I.2. A Gly58Arg missense variant in VMA21 was revealed as the cause for SXCI.Each step of the variant filtering system takes advantage of publicly available genomic databases, non-SXCI controls and case-specific molecular data, and aligns with established concepts in the theoretical background of SXCI. CONCLUSION: This study acts as a proof of concept for our genomic filtering algorithm's clinical utility in analysing X linked disorders. Our findings clarify the molecular aspects of SXCI and improve genetic diagnostics and counselling for families with X linked diseases like HA.

The hidden joint in children with haemophilia on prophylaxis.

Prophylaxis is the gold standard treatment for children with haemophilia (CWH). MRI studies revealed joint damage, even with this treatment; this suggests the presence of subclinical bleeding. In the case of children with haemophilia, it is relevant to detect early signs of joint damage, as this allows the medical team to provide the appropriate treatment and follow-up, in order to avoid arthropathy development and its consequences. The aim of this study is to detect the hidden joint in children with haemophilia on prophylaxis (CWHP) and analyse, by age group, which joint is the most affected. We define the hidden joint in CWH on prophylaxis as the joint that presents joint damage secondary to repetitive bleeding episodes and is detected in the joint evaluation, despite being asymptomatic or with mild symptoms. It is most commonly caused by repetitive subclinical bleeding. METHODS: This was an observational, analytical, cross-sectional study of 106 CWH on prophylaxis treated in our centre. Patients were divided according to age and type of treatment. Joint damage was defined as a HEAD-US score ≥ 1. RESULTS: Patients' median age was 12 years. All had severe haemophilia. The median age of onset of prophylaxis was 2.7. Forty-seven (44.3 %) patients received primary prophylaxis (PP) and 59 (55.7 %), secondary prophylaxis. Six hundred and thirty-six joints were analysed. Type of prophylaxis and joint involvement showed statistically significant differences (p < 0.001). However, patients on PP had a greater number of damaged joints at older ages. Twenty-two % (140) of the joints scored ≥1 on HEAD-US. Cartilage was most frequently involved, followed by synovitis, and bone damage. We observed a greater frequency and degree of arthropathy in subjects aged 11 and above. Sixty (12.7 %) joints showed a HEAD-US score ≥ 1, with no history of bleeding. The ankle was the most affected joint, representing the hidden joint according to our definition. CONCLUSION: Prophylaxis is the best treatment for CWH. However, symptomatic or subclinical joint bleeding may occur. The routine evaluation of joint health is relevant, particularly, of the ankle. In our study, early signs of arthropathy according to age and type of prophylaxis were detected by HEAD-US.

Haemophilia B, severe childhood obesity and other extra-haematological features associated with similar 4Mb-deletions on Xq27: Clinical findings, molecular insights and literature update.

INTRODUCTION: Haemophilia B (HB) is associated with pathogenic variants in F9. Hemizygous deletions encompassing the entire F9 and proximate genes may express extra-haematological clinical phenotypes. AIM: To analyse the genotype/phenotype correlations in two unrelated boys with severe early childhood obesity (SCO), global developmental delay (GDD) and similar bleeding phenotype associated with comparable Xq27 deletions spanning the entire F9 and proximate genes, and characterise the pathogenic events estimating the most likely mutational mechanism involved. METHODS: Entire F9-deletions were detected in three hemizygous unrelated probands with HB: two cases, C#1/C#2, presented SCO and GDD and a control patient (Co), who only had severe bleeding symptoms. Dense SNP-array and case-specific STS walking scan allowed characterisation of the deletion breakpoints. Extensive use of bioinformatics, statistics and clinical databases allowed the investigation of genotype-phenotype associations. RESULTS: Patients C#1/C#2 and Co resulted in a complete F9 and additional gene deletions of variable extensions on Xq26.3-Xq27.2 (C#1/C#2/Co: 4.3Mb/3.9Mb/160Kb). C#1/C#2 common deleted gene SOX3 is directly associated with SCO, GDD and pituitary hypothyroidism (PH) whilst C#2 extra-deleted gene MAGEC2 indirectly relates to anal atresia (AA). Breakpoint analysis revealed the involvement of the mechanisms of Alu/Alu recombination for the first time in HB and non-homologous or alternative end-joining. CONCLUSION: Our results represent the first report of unrelated patients with HB, SCO and GDD. This study and the literature update expand the spectrum of clinical findings and molecular insights observed in patients with HB caused by complete F9 and nearby SOX3 and MAGEC2 gene deletions, which may configure a contiguous gene syndrome.

How mild is mild haemophilia?

INTRODUCTION: People with mild haemophilia (PWMH) experience sporadic bleeds and are less likely to receive an early diagnosis, appropriate treatment and medical care. Arthropathy is a key determinant of health-related quality of life (QoL), producing pain, limitations in mobility and daily activities. The aim of this study is to evaluate the incidence, risk factors and QoL associated with arthropathy in PWMH. MATERIALS AND METHODS: Observational, cross-sectional cohort study. Data were collected in a single interview and evaluated by a physiotherapist and an orthopaedist and analysed on demographics; baseline factor levels; as well as clinical (Haemophilia Joint Health Score [HJHS]), ultrasound (Haemophilia Early Arthropathy Detection with Ultrasound [HEAD-US]), radiological (Pettersson score [PS]), pain (visual analogue scale [VAS]) and QoL evaluations. We defined arthropathy when at least one of the joints shown with a HEAD-US score ≥ 1. RESULTS: Eighty-five patients and 510 joints were included. Patients' mean age was 35.9 years-old. Median age was 44.2 in patients with arthropathy versus 14.9 in patients without; the difference was statistically significant (p < .001). In patients over 20 years old, 90.5% shown arthropathy. Only 24 (28%) patients had no joint damage (HEAD-US = 0), and 61 (72%) had at least one joint with a HEAD-US ≥ 1. The ankle was the most affected joint. Patient age was found to be the most important risk factor associated with the development of arthropathy. CONCLUSIONS: Joint damage as a result of prior hemarthrosis was the most relevant factor associated with lower QoL, and emphasised the importance of early diagnosis and appropriate management in this particular population.

A multicentre real-world data study to assess the bleeding rate and management of patients with haemophilia A and factor VIII inhibitors in Argentina.

The development of inhibitors against factor VIII (FVIII) concentrates is a severe complication of treatment for patients with haemophilia. We investigated annualized bleeding rates (ABRs) in patients in Argentina with haemophilia A with inhibitors and analysed potential differences between treatment strategies. This multicentre, retrospective, real-world data, cohort design study comprised ambulatory paediatric and adult patients with congenital haemophilia A and FVIII inhibitors treated according to standard clinical practice, with 12-months follow-up. Of 69 included patients, 39 (56.5%) received on-demand treatment, 13 (18.8%) received prophylactic treatment, and 17 (24.6%) received immune tolerance induction (ITI) therapy. The mean overall ABR was 7.68 ±â€Š8.18, with similar rates for on-demand (8.59 ±â€Š9.69), prophylaxis (5.54 ±â€Š4.71), and ITI (7.24 ±â€Š6.23) subgroups. In the negative binomial regression model, prophylactic treatment [incidence rate ratio (IRR) 0.41, 95% confidence interval (CI): 0.21-0.79, P < 0.01] and ITI (IRR 0.47, 95% CI: 0.27-0.81, P < 0.01) therapy were significantly associated with a decrease in the ABR compared with on-demand treatment. Age (IRR 0.96, 95% CI: 0.94-0.97, P < 0.01), number of target joints (IRR 1.21, 95% CI: 1.11-1.31, P < 0.001), and history of recurring bleeding (IRR 2.3, 95% CI: 1.19-4.57, P = 0.012) were significantly and independently associated with ABR. The ABR in standard clinical practice was lower than that reported in controlled clinical trials. Patients undergoing prophylaxis and ITI therapy showed reduced ABRs compared with on-demand treatment, after controlling for bleeding predictor variables.

Clinical and ultrasound evaluation of patients with haemophilia on prophylaxis.

INTRODUCTION: Primary prophylaxis is the current gold standard in haemophilia care for the prevention of bleeding and ensuing joint damage. Early detection of joint bleeding, whether symptomatic or subclinical, preferably during childhood, helps prevent joint deterioration and subsequent disability. The aim of this study is to evaluate the level of agreement between the Haemophilia Joint Health Score and the Haemophilia Early Arthropathy Detection with Ultrasound tools in children with severe haemophilia on primary and secondary prophylaxis. MATERIALS AND METHODS: All patients were followed up regularly at our centre. Elbows, knees and ankles were evaluated by physical examination using the Haemophilia Joint Health Score 2.1 (HJHS 2.1), and by ultrasound with HEAD-US score. RESULTS: A total of 80 children with haemophilia on prophylaxis were included in this study. Mean age was 10.8 years (range 4-18). We evaluated 480 joints, of which 423 (88.1%) were concordant with both tools, whereas 57 (11.9%) were discordant; 377 (78.5%) joints scored 0 on HJHS, 370 (77%) on HEAD-US and 345 (72%) on both tools. The overall Kappa concordance coefficient was .656. For elbows, knees and ankles the respective values were .783, .522 and .589. For HJHS scores greater than 3, all joints scored ≥1 on HEAD-US. CONCLUSION: HJHS and HEAD-US are used to assess joint health in children with haemophilia on prophylaxis. In this study, the level of agreement between both tools was consistent with literature values only for the elbow joint.

Molecular insights into the mechanism of nonrecurrent F8 structural variants: Full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A.

Hemophilia A (HA) provides excellent models to analyze genotype-phenotype relationships and mutational mechanisms. NhF8ld's breakpoints were characterized using case-specific DNA-tags, direct- or inverse-polymerase chain reaction amplification, and Sanger sequencing. DNA-break's stimulators (n = 46), interspersed repeats, non-B-DNA, and secondary structures were analyzed around breakpoints versus null hypotheses (E-values) based on computer simulations and base-frequency probabilities. Nine of 18 (50%) severe-HA patients with nhF8lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi-exons. NhF8lds range: 2-165 kb. Five (45%) nhF8lds involve F8-extragenic regions including three affecting vicinal genes (SMIM9 and BRCC3) but none shows an extra-phenotype not related to severe-HA. The contingency analysis of recombinogenic motifs at nhF8ld breakpoints indicated a significant involvement of several DNA-break stimulator elements. Most nhF8ld's breakpoint junctions showed microhomologies (1-7 bp). Three (27%) nhF8lds show complexities at the breakpoints: an 8-bp inverted-insertion, and the remnant two, inverted- and direct-insertions (46-68 bp) supporting replicative models microhomology-mediated break-induced replication/Fork Stalling and Template Switching. The remnant eight (73%) nhF8lds may support nonhomologous end joining/microhomology-mediated end joining models. Our study suggests the involvement of the retroposition machinery (e.g., Jurka-targets, Alu-elements, long interspersed nuclear elements, long terminal repeats), microhomologies, and secondary structures at breakpoints playing significant roles in the origin of the upmost severe phenotype in HA.

Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia.

Among other applications of long-distance haplotype phasing in clinical genetics, determination of linked DNA markers as surrogate for problematic structural variants (e.g., repeat-mediated rearrangements) is essential to perform diagnosis from low-quality DNA samples. We describe a next-of-kin-independent (physical) phasing approach based on inverse-PCR (iPCR) paired-end amplification (PI). This method enables typing the multialleles of the short tandem repeat (STR) F8Int21[CA]n at the F8-intron 21, as a surrogate DNA marker for the F8-intron 22 inversion (Inv22), the hemophilia A-causative hotspot, within the transmitted haplotype in informative carriers. We provide proof-of-concept by blindly validating the PI approach in 15 carrier mother/affected-son duos. Every F8Int21[CA]n STR allele determined in phase with the Inv22 allele in the female carriers from the informative duos was confirmed in the hemizygous proband (P = 0.00003). A second surrogate STR locus at the F8-IVS22 was obtained by the PI approach improving severe-HA preimplantation genetic diagnosis by augmenting heterozygosity in Inv22 carriers bypassing the requirement for family linkage analysis. The ability of the PI-assay to combine other marker pairs was demonstrated by haplotyping a SNV (F8:c.6118T > C) with a >28kb-distant F8-IVS22 STR. The PI approach has proven flexibility to target different marker pairs and has potential for multiplex characterization of iPCR products by massively parallel sequencing.

A Randomized Trial of Factor VIII and Neutralizing Antibodies in Hemophilia A.

BACKGROUND: The development of neutralizing anti-factor VIII alloantibodies (inhibitors) in patients with severe hemophilia A may depend on the concentrate used for replacement therapy. METHODS: We conducted a randomized trial to assess the incidence of factor VIII inhibitors among patients treated with plasma-derived factor VIII containing von Willebrand factor or recombinant factor VIII. Patients who met the eligibility criteria (male sex, age <6 years, severe hemophilia A, and no previous treatment with any factor VIII concentrate or only minimal treatment with blood components) were included from 42 sites. RESULTS: Of 303 patients screened, 264 underwent randomization and 251 were analyzed. Inhibitors developed in 76 patients, 50 of whom had high-titer inhibitors (≥5 Bethesda units). Inhibitors developed in 29 of the 125 patients treated with plasma-derived factor VIII (20 patients had high-titer inhibitors) and in 47 of the 126 patients treated with recombinant factor VIII (30 patients had high-titer inhibitors). The cumulative incidence of all inhibitors was 26.8% (95% confidence interval [CI], 18.4 to 35.2) with plasma-derived factor VIII and 44.5% (95% CI, 34.7 to 54.3) with recombinant factor VIII; the cumulative incidence of high-titer inhibitors was 18.6% (95% CI, 11.2 to 26.0) and 28.4% (95% CI, 19.6 to 37.2), respectively. In Cox regression models for the primary end point of all inhibitors, recombinant factor VIII was associated with an 87% higher incidence than plasma-derived factor VIII (hazard ratio, 1.87; 95% CI, 1.17 to 2.96). This association did not change in multivariable analysis. For high-titer inhibitors, the hazard ratio was 1.69 (95% CI, 0.96 to 2.98). When the analysis was restricted to recombinant factor VIII products other than second-generation full-length recombinant factor VIII, effect estimates remained similar for all inhibitors (hazard ratio, 1.98; 95% CI, 0.99 to 3.97) and high-titer inhibitors (hazard ratio, 2.59; 95% CI, 1.11 to 6.00). CONCLUSIONS: Patients treated with plasma-derived factor VIII containing von Willebrand factor had a lower incidence of inhibitors than those treated with recombinant factor VIII. (Funded by the Angelo Bianchi Bonomi Foundation and others; ClinicalTrials.gov number, NCT01064284; EudraCT number, 2009-011186-88.).

Secondary prophylaxis with rFVIIa in hemophilia and inhibitors: recommendations from an experts committee from Argentina.

Secondary prophylaxis with rFVIIa has been the subject of several publications in the past few years. However, there is no general consensus on how this treatment should be put into practice, as publications have been very heterogeneous in the dosing schedule they report. Furthermore, the mechanism of action of rFVIIa and its short half life have been used as arguments against its role in prophylaxis. There have been a series of recent publications that show that rFVIIa can traffic through the intact endothelium and be stored in the subendothelium of several organs for a prolonged period of time. In order to consensuate the role of rFVIIa in prophylaxis, a group of experts from Argentina, resumed available information regarding pharmacology and clinical experience with this treatment, and developed a series of recommendations to use this drug in the prophylaxis setting.

Cytogenetic, FISH, and molecular studies in a case of B-cell chronic lymphocytic leukemia with karyotypic evolution.

We report the clinical, cytogenetic, fluorescence in situ hybridization (FISH) and molecular findings in a 54-yr-old male patient diagnosed with B-cell chronic lymphocytic leukemia (B-CLL), who showed progression to a diffuse large B-cell lymphoma (Richter's syndrome). Genetic studies were performed at diagnosis and during the Richter's transformation (RT). A clonal karyotype with two dicentric chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was found. Both rearrangements were confirmed by FISH. Molecular cytogenetics analysis using p53 probe showed monoallelic loss of this tumor suppressor gene in 43.8% and 77.3% of cells for the first and the second studies, respectively). In both studies, deletions of D13S319 (18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at 13q14 were found. Polymerase chain reaction analysis showed the MBR/JH rearrangement of the bcl-2 gene. FISH studies using LSI bcl-2/IgH probe allowed quantifying the clonal cell population with this rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively). To our knowledge, this is the first case with a psu dic(12,21) described in B-CLL. The low percentage of cells with the 13q14 deletion and bcl-2/IgH rearrangement suggests that they were secondary events that resulted from clonal evolution. Our patient had a short survival (9 months) and a clear lack of response to several therapeutic agents, confirming the association of p53 gene deletion and karyotypic evolution with disease progression.