JAK2 allele burden is correlated with a risk of venous but not arterial thrombosis.

BACKGROUND: Thrombosis is the main complication in myeloproliferative neoplasms (MPN). A JAK2V617F mutation has been shown to be a risk factor for thrombosis. The implication of other risk factors alongside a mutation allele burden needs to be clarified (Trifa et al., 2018; Borowczyk et al., 2015). OBJECTIVE: Our aim was to investigate the role of the JAK2 mutation allele burden in the risk of cardiovascular events (CVE) and/or venous thrombosis (VTE) in a cohort of patients with confirmed MPN, as well as in patients without confirmed MPN. METHODS: We restrospectively included all consecutive patients who were positive for JAK2V617F seen by our unit between December 2008 and September 2016. Inclusion criteria were a positive test for the JAK2V617F mutation, with at least 1% allele burden, with or without confirmed MPN. RESULTS: We included 239 patients of median age 71 years [60-81], followed-up for a median of 82.8 months [41.08-146.88]. For JAK2V617F positive patients having an allele burden superior to 50% the cumulative incidence of VTE was significantly higher than for those with an allele burden inferior to 50% (HR 3.11 95% CI [1.10-8.76] p = 0.031). The cumulative incidence of VTE was also higher in patients with obesity (HR 4.58 95% CI [1.33-15.8] p = 0.016). There was no significant association between a JAK2V617F allele burden and arterial thrombosis (manifesting as CVE). Previous VTE was also associated with a higher cumulative incidence of recurrence during follow-up HR 3.22 95% CI [1.17-8.81] p = 0.0231. CONCLUSION: We show that a JAK2V617F allele burden is associated with risk of VTE but not with CVE.

New intragenic rearrangements in non-Finnish mulibrey nanism.

Prenatal growth is a complex dynamic process controlled by various genetic and environmental factors. Among genetic syndromes characterized by growth restriction, MULIBREY nanism represents a rare autosomal recessive condition presenting with severe pre- and post-natal growth failure, characteristic dysmorphic features but normal neurological development. The phenotype of MULIBREY nanism is variable and overlaps with others such as the Silver-Russell syndrome. We report here three patients in two distinct non-Finnish families from North France who were first suspected to have Silver-Russell syndrome which failed to be confirmed on molecular analyses. Clinical features in the three patients led us to also consider the diagnosis of MULIBREY nanism. Sequencing of the TRIM37 gene showed the three patients shared a novel nonsense mutation (c.181 C>T p.Arg61*) in a heterozygous state. Quantitative fluorescent multiplex PCR identified a new deletion of exons 15 and 16 in TRIM37 in one isolated patient and another deletion of exon 9 in two siblings. Breakpoints of both the deletions were localized in Alu sequences. Given the high number of Alu repeats, which predispose to gene rearrangements, one should always consider such genetic rearrangements in the molecular diagnosis of non-Finnish MULIBREY nanism patients. Early diagnosis of the disease would prompt careful cardiac follow up of such patients as cardiological complication is a characteristic feature of the MULIBREY nanism as described in this report.

A New Intergenic α-Globin Deletion (α-αΔ125) Found in a Kabyle Population.

We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a ß-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients.

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.

Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants.

BACKGROUND: Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6. DESIGN AND METHODS: We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies. RESULTS: We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345 > LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G > A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T > C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

[Flowcharts for the diagnosis and the molecular characterization of hemoglobinopathies]. / Arbres décisionnels pour le diagnostic et la caractérisation moléculaire des hémoglobinopathies.

The diagnosis of the main hemoglobinopathies (HbS, HbC, HbE, heterozygous beta-thalassemia) is easy for laboratories using Hb electrophoresis and/or cation-exchange high performance liquid chromatography (CE-HPLC) methods. However, the diagnosis of alpha-thalassemias and of rare Hb variants is much more complex. Six French laboratories, forming together the "Pathologie héréditaire de l'érythrocyte" network, routinely practice the molecular diagnosis of hemoglobinopathies. These laboratories have shared their experiences to propose flowcharts for the phenotypical diagnosis and the molecular characterization of the main hereditary Hb pathologies. These flowcharts are applicable to any single patient with an adult erythropïesis (more than two-years-old), that is to say after the fetal to adult (gamma>beta) Hb commutation, when the HbF level is stabilized.

First identification of a point mutation at position -83 (G>A) of the beta-globin gene promoter.

We report the first identification of a point mutation located within the promoter region of the beta-globin gene at position -83 (G>A) and associated with the common heterozygous deletional alpha-thalassemia (alpha-thal) (-alpha(3.7)/alphaalpha). The patient was an adult male from Gabon belonging to the Obamba sub ethnic group, who was referred to our clinics for a mild microcytic anemia with a Hb A(2) level at the upper limit of the normal value (3.5%). This observation is a new example of alpha- and beta-thal co-inheritance with a normal Hb A(2) level, and illustrates a potential source of pitfall in screening for alpha- and beta-thal carriers.

An unusual case of hemochromatosis due to a new compound heterozygosity in HFE (p.[Gly43Asp;His63Asp]+[Cys282Tyr]): structural implications with respect to binding with transferrin receptor 1.

Most adults affected with HFE hereditary hemochromatosis (HH type 1, MIMmusical sharp 235200) are homozygous for the p.Cys282Tyr mutation in HFE (NC_000006.10, region 26195427 to 26205038). The aim of this study was to investigate the molecular basis of iron overload in a patient presenting with severe clinical HH with one c.845G>A (p.Cys282Tyr) allele only. Molecular and pedigree studies demonstrated the presence of the c.845G>A (p.Cys282Tyr) mutation in one allele whereas the other carried the c.187C>G (p.His63Asp) mutation plus a new c.128G>A (p.Gly43Asp) substitution in cis. A molecular modeling study of the p.[Gly43Asp;His63Asp] and p.His63Asp variants versus the wild type was carried out using molecular dynamics (MD) simulation in presence of implicit solvent. We found that the c.187C>G (p.His63Asp) mutation does not introduce any major change in the 1- domains of HFE whereas the c.128G>A (p.Gly43Asp) substitution is responsible for a modification of the dynamics and the structure of the Gln40-Ser45 loop, a critical region for HFE-TfR1 interaction thus impairing HFE-TfR1 normal contact. We conclude that the occurrence of complex alleles may be an alternative explanation for the variability of the phenotype in individuals who are compound heterozygous for c.[187C>G]+[845G>A] (p.[His63Asp]+[Cys282Tyr]).

A novel beta-Thalassemic allele due to a two nucleotide deletion: beta76 (-GC).

We have identified and characterized a novel beta-thalassemic mutation in a North African adult. The molecular defect consists of a two nucleotide (nt) deletion in the beta-globin gene at codon 76 [beta76 (-GC), c.229-230delGC]. This frameshift mutation generates a TGA stop codon at position 89. The carrier presented with mild microcytic anemia (Hb 12.8 g/dL, MCV 60 fL), no detectable Hb F, an elevated Hb A2 level (5.5%) with no other mutation in the beta-globin gene and none of the more common known deletions in the alpha-globin cluster. No abnormal hemoglobin (Hb) was present in routine electrophoresis or in high performance liquid chromatography (HPLC) analyses. Pathologic inclusions were absent in both mature red cells and in reticulocytes. This observation reinforces the hypothesis that nonsense and frameshift mutations that result in a premature stop codon in exon 1 or exon 2 inherited in the heterozygous state do not generate dominant beta-thalassemia (thal). This is the first example of a premature stop codon at position 89.

Juvenile hemochromatosis caused by a novel combination of hemojuvelin G320V/R176C mutations in a 5-year old girl.

During a screening program we identified a 5-year old girl with elevated iron parameters. The child was found to have a combination of a novel R176C mutation together with the G320V mutation in the juvenile hemochromatosis gene (HJV). The girl was also homozygous for the H63D mutation in HFE. The possibility of detecting juvenile hemochromatosis before the onset of clinical manifestations raises questions about the management of such young children in order to prevent iron overload.

Digenic inheritance of mutations in HAMP and HFE results in different types of haemochromatosis.

Haemochromatosis (HH) is a clinically and genetically heterogeneous disease caused by inappropriate iron absorption. Most HH patients are homozygous for the C282Y mutation in the HFE gene. However, penetrance of the C282Y mutation is incomplete, and other genetic factors may well affect the HH phenotype. Ferroportin and TFR2 mutations also cause HH, and two HAMP mutations have recently been reported that causes juvenile haemochromatosis (JH) in the homozygous state. Here, we report evidence for digenic inheritance of HH. We have detected two new HAMP mutations in two different families, in which there is concordance between severity of iron overload and heterozygosity for HAMP mutations when present with the HFE C282Y mutation. In family A, the proband has a JH phenotype and is heterozygous for C282Y and a novel HAMP mutation Met50del IVS2+1(-G). This is a four nucleotide ATGG deletion which causes a frameshift. The proband's unaffected mother is also heterozygous for Met50del IVS2+1(-G), but lacks the C282Y mutation and is heterozygous for the HFE H63D mutation. Met50del IVS2+1(-G) was absent from 642 control chromosomes. In family B, a second novel, less severe HAMP mutation, G71D, was identified. This was detected in the general population at an allele frequency of 0.3%. We propose that the phenotype of C282Y heterozygotes and homozygotes may be modified by heterozygosity for mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload corresponding to the severity of the HAMP mutation.

Novel mutation in ferroportin 1 gene is associated with autosomal dominant iron overload.

We report a family affected with dominant autosomal iron overload related to a new mutation in ferroportin 1, a transmembrane protein involved in the export of iron from duodenal enterocytes and likely from macrophages. The originality of this family is represented by the nature of the mutation consisting in the replacement of glycine 490 with aspartate. Clinicians should be aware of this novel iron overload entity, which corresponds to a particular phenotypic expression (high serum ferritin values contrasting with relatively low transferring saturation, and important Kupffer cell iron deposition as compared to hepatocytic iron excess) with poor tolerance of venesection therapy and a dominant pattern of inheritance. Given this dominant transmission, the mixed Causasian-Asian origin of our Asian proband leaves open the issue of the ethnic origin of the new mutation.

Hyperhomocysteinaemia, folate and vitamin B12 in unsupplemented haemodialysis patients: effect of oral therapy with folic acid and vitamin B12.

BACKGROUND: Hyperhomocysteinaemia, a risk factor for atherosclerosis, is common in dialysis patients and particularly in those homozygous for a common polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T transition). B-complex vitamin supplements have been shown to lower plasma total homocysteine (tHcy) concentrations, but the respective effectiveness of folate and oral vitamin B12 is not yet known. Our objectives were: (i) to determine the status of folate and vitamin B12 in a cohort of unsupplemented dialysis patients (ii) to assess the homocysteine-lowering effect of a folate supplement and then of a folate supplement with added vitamin B12. The responses were analysed for the C677T genotypes of MTHFR. METHODS: Plasma tHcy, folate and vitamin B12 were measured in 51 haemodialysis patients genotyped for the C677T MTHFR mutation (homozygotes, TT; heterozygotes, CT; without mutation, CC). All patients were then given daily supplements of 15 mg of folic acid for 2 months. They were given daily supplements of 1 mg of vitamin B12 in addition to the folate supplements for a further 2 months. Plasma tHcy, folate and vitamin B12 were monitored after each intervention. RESULTS: At baseline folate and vitamin B12 deficiencies were found in 10% and 6% of the patients. Initial plasma tHcy concentrations were high in all patients (mean 38.1+/-15 micromol/l). CC patients tended to have a lower tHcy concentration than pooled CT and TT patients. After 2 months of folate therapy, tHcy concentration decreased significantly to 20.2+/-7 micromol/l (P<0.001) and no significant differences were observed between the different genotype subgroups (19.4+/-6 for CC, 21.3+/-8 for CT, 18.5+/-4 for TT). A significant positive relationship was found between the reduction of tHcy and its initial value (rho=0.615, P<0.0001). The impact of the added vitamin B12 was negligible since tHcy concentrations did not change for the patients as a whole (19.8+/-7 micromol/l, NS) or in any subgroup (19.1+/-5 for CC, 20.3+/-9 for CT and 20+/-7 micromol/l for TT). CONCLUSIONS: (i) Folate and vitamin B12 deficiencies were observed in 10% and 6% respectively of our unsupplemented dialysis patients. (ii) After folate therapy, tHcy levels decreased significantly in all patients and were identical between the three C677T MTHFR genotype subgroups. (iii) Vitamin B12 supplements are useful in folate treated patients to prevent cobalamin deficiency and its neurological consequences but they did not lower tHcy plasma levels for the patients as a group or for any of the MTHFR subgroups.