ABSTRACT
MYH9‐related disease (MYH9‐RD) is a rare autosomal‐dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC‐IIA). MYH9‐RD is characterized by a ...considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end‐stage renal disease (ESRD). We searched for genotype–phenotype correlations in the largest series of consecutive MYH9‐RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9‐RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early‐onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC‐IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C‐terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9‐RD.
MYH9‐related disease (MYH9‐RD), the complex syndromic disorder deriving from mutations in MYH9, is characterized by a considerable variability in clinical evolution. This paper identifies significant genotype–phenotype correlations in the largest series of consecutive MYH9‐RD patients collected so far. These data allow us to predict the evolution of the disease associated to genotypes responsible for 85% of MYH9‐RD cases, providing an essential tool for patients' clinical management and genetic counseling and suggesting new mechanisms for molecular pathogenesis of the disease.
MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth ...with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease. Hum Mutat 29(3), 409-417, 2008.
MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of non-muscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the ...hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicating a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.
Crohn's disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights ...the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application.
T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors' MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes.
A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4(+)CD25(+) subset and increase of the CD3(+)CD69(+) population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used.
MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.
Mutations decreasing function of the Fas death receptor cause the autoimmune lymphoproliferative syndrome (ALPS) with autoimmune manifestations, spleen/lymph node enlargement, and expansion of ...CD4/CD8-negative T cells. Dianzani Autoimmune Lymphoproliferative Disease (DALD) is a variant lacking this expansion. Perforin is involved in cell-mediated cytotoxicity and its biallelic mutations cause familial hemophagocytic lymphohistiocytosis (HLH). We previously described an ALPS patient carrying heterozygous mutations of the Fas and perforin genes and suggested that they concurred in ALPS. This work extends the analysis to 14 ALPS, 28 DALD, and 816 controls, and detects an N252S amino acid substitution in 2 ALPS, and an A91V amino acid substitution in 6 DALD. N252S conferred an OR = 62.7 (P = .0016) for ALPS and A91V conferred an OR = 3 (P = .016) for DALD. Copresence of A91V and variations of the osteopontin gene previously associated with DALD conferred an OR = 17 (P = .0007) for DALD. In one N252S patient, NK activity was strikingly defective in early childhood, but became normal in late childhood. A91V patients displayed lower NK activity than controls. These data suggest that perforin variations are a susceptibility factor for ALPS/DALD development in subjects with defective Fas function and may influence disease expression.
Abstract 2533
MYH9-Related Disease (MYH9-RD) is one of the less rare forms of inherited thrombocytopenia. It derives from mutations of the gene MYH9 for the heavy chain of nonmuscle myosin IIA and is ...characterized by congenital macrothrombocytopenia variably associated with young-adult onset of hearing loss, cataract, and a severe proteinuric nephropathy.
Only platelet transfusions are available for increasing platelet counts in this condition, but they expose to the risks of acute reactions, transmission of infectious diseases, and refractoriness to subsequent platelet transfusions. Moreover, this treatment suffers from scarceness of blood donors.
Novel thrombopoiesis-stimulating agents have been developed and 2 of them, eltrombopag and romiplostin, have been approved for increasing platelet count in a few forms of acquired thrombocytopenia. Since it has been recently shown that megakaryocytes of patients with MYH9-RD respond in vitro to TPO stimulation, we reasoned that TPO-mimetics could be effective also in this condition and decided to test the effect of eltrombopag.
Therefore, we performed a phase II, multicentre, open-label, dose escalation trial. Twelve adult patients with a platelet count lower than 50×10e9/L and a diagnosis of MYH9-RD confirmed by identification of the causative MYH9 mutations received orally eltrombopag 50 mg daily for 21 days (Revolade®, GSK). Patients with platelet counts lower than 100×10e9/L at day 21 increased eltrombopag to 75 mg daily for 21 additional days. Patients with platelet counts between 100 and 150×10e9/L at day 21 continued eltrombopag 50 mg daily for the following 21 days, while patients with more than 150×10e9 platelets/L stopped therapy.
The primary endpoints were the achievement of a platelet count over 100×10e9/L or at least three times the baseline value (major response), or at least twice the baseline value but less than major response (minor response). Secondary end points included safety and tolerability, and the reduction of bleeding tendency.
After 3 weeks at the eltrombopag dose of 50 mg daily, 3 patients achieved platelet counts of 150×10e9/L or more and stopped therapy. Two had a platelet counts between 100 and 150×10e9/L and continued treatment at the same dosage, while 7 had less than 100×10e9 platelets/L and received eltrombopag 75 mg daily for 3 weeks.
A major response was obtained in 8 patients (67%), in 5 of them after 3 weeks of eltrombopag 50 mg daily, and in 3 cases after 3 additional weeks at the dose of 75 mg. Three patients (25%) achieved a minor response, 1 after 3 weeks at 50 mg daily, and 2 after 3 additional weeks at 75 mg. In one patient the treatment resulted in no response.
Mean platelet count at the end of treatment was significantly higher than at baseline (105 versus 31×10e9 platelets/L, p=0.0022). In the 11 patients that achieved major or minor responses, mean platelet count was still higher than baseline 15 days after discontinuation of the drug, while it returned to levels near baseline 15 days later.
Platelet size did not change either during treatment or after its cessation, and this indicates that the increases in platelet count were paralleled by corresponding increases in total platelet mass.
The extent of platelet aggregation was within the normal range after all tested agonists in 5 of the 7 patients that achieved platelet counts higher than 100×10e9/L, while it was slightly reduced after ADP and collagen in 2 patients.
Mean serum TPO level was higher than the normal range and this figure did not change neither during treatment with eltrombopag nor after its discontinuation.
Ten of 12 patients had grade 1 or 2 bleeding symptoms, as measured by the WHO bleeding scale, at baseline, while 2 were asymptomatic. Upon treatment, bleeding diathesis quickly ameliorated and disappeared in 8 cases, while it remained unchanged in the only patient with no response to eltrombopag and in another one with a minor response. The benefit in terms of bleeding diathesis lasted well beyond treatment discontinuation.
Treatment was well tolerated in all cases, with only two patients reporting mild and transient headache and one patient suffering from transient dry mouth at the beginning of treatment.
In conclusion, 50–75 mg of eltrombopag per day increased platelet count and reduced bleeding tendency in most patients with MYH9-RD. Further research is required to ascertain whether TPO mimetics are effective also in other forms of inherited thrombocytopenia.
Off Label Use: Eltrombopag for MYH9-related disease.
MYH9‐related disease (MYH9‐RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non‐muscle myosin IIA. All patients present congenital ...macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end‐stage renal failure. We report four families, each with a novel mutation: two missense mutations, in exons 31 and 32, and two out of frame deletions in exon 40. They were associated with no bleeding diathesis, normal, or only slightly reduced platelet count and no extra‐hematological manifestations, confirming that alterations of the tail domain cause a mild form of MYH9‐RD with no clinically relevant defects.
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