Thrombocytopenia 4 (THC4): Six novel families with mutations of the cytochrome c gene.

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants.

A Novel Mutation in GP1BB Reveals the Role of the Cytoplasmic Domain of GPIbß in the Pathophysiology of Bernard-Soulier Syndrome and GPIb-IX Complex Assembly.

Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.

Renin Angiotensin System, COVID-19 and Male Fertility: Any Risk for Conceiving?

The current knowledge concerning the connection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the renin-angiotensin system (RAS) system in the male reproductive apparatus is still limited, so dedicated studies are urgently required. Concerns about the male fertility consequences of SARS-CoV-2 infection have started to emerge, since epidemiologic studies observed that this coronavirus affects male patients more frequently and with increased severity, possibly because of the hormone-regulated expression of angiotensin-converting enzyme 2 (ACE2) receptor. A disturbance in fertility is also expected based on studies of the previous SARS-CoV infection, which targets the same ACE2 receptor when entering the host cells. In addition, bioinformatics analyses reveal the abundant expression of ACE2 receptor in the male reproductive tissues, particularly in the testis. It has been proposed that pharmacological intervention favoring the angiotensin-(1-7)/ACE2/Mas receptor pathway and increasing ACE2 expression and activity could greatly prevent inflammatory lesions in this area. Finally, in laboratories performing assisted reproductive technologies it is recommended that more attention should be paid not only to sperm quality but also to safety aspects. Data about the potential infectivity of seminal fluid are in fact conflicting and do not exclude risks for both personnel and patients. The potential infectivity of SARS-CoV-2 in reproductive male tissues should be strongly considered and further investigated for the proper management of in vitro fertilization procedures.

MYH9-Related Thrombocytopenia: Four Novel Variants Affecting the Tail Domain of the Non-Muscle Myosin Heavy Chain IIA Associated with a Mild Clinical Evolution of the Disorder.

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease.

Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia.

Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I-III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA-/- cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria.

Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim.

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of MPL, the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease-causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the THPO gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO-mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with THPO mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression.

Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia.

ETV6-related thrombocytopenia is an autosomal dominant thrombocytopenia that has been recently identified in a few families and has been suspected to predispose to hematologic malignancies. To gain further information on this disorder, we searched for ETV6 mutations in the 130 families with inherited thrombocytopenia of unknown origin from our cohort of 274 consecutive pedigrees with familial thrombocytopenia. We identified 20 patients with ETV6-related thrombocytopenia from seven pedigrees. They have five different ETV6 variants, including three novel mutations affecting the highly conserved E26 transformation-specific domain. The relative frequency of ETV6-related thrombocytopenia was 2.6% in the whole case series and 4.6% among the families with known forms of inherited thrombocytopenia. The degree of thrombocytopenia and bleeding tendency of the patients with ETV6-related thrombocytopenia were mild, but four subjects developed B-cell acute lymphoblastic leukemia during childhood, resulting in a significantly higher incidence of this condition compared to that in the general population. Clinical and laboratory findings did not identify any particular defects that could lead to the suspicion of this disorder from the routine diagnostic workup. However, at variance with most inherited thrombocytopenias, platelets were not enlarged. In vitro studies revealed that the maturation of the patients' megakaryocytes was defective and that the patients have impaired proplatelet formation. Moreover, platelets from patients with ETV6-related thrombocytopenia have reduced ability to spread on fibrinogen. Since the dominant thrombocytopenias due to mutations in RUNX1 and ANKRD26 are also characterized by normal platelet size and predispose to hematologic malignancies, we suggest that screening for ETV6, RUNX1 and ANKRD26 mutations should be performed in all subjects with autosomal dominant thrombocytopenia and normal platelet size.

Somatic, hematologic phenotype, long-term outcome, and effect of hematopoietic stem cell transplantation. An analysis of 97 Fanconi anemia patients from the Italian national database on behalf of the Marrow Failure Study Group of the AIEOP (Italian Association of Pediatric Hematology-Oncology).

We analyzed 97 Fanconi anemia patients from a clinic/biological database for genotype, somatic, and hematologic phenotype, adverse hematological events, solid tumors, and treatment. Seventy-two patients belonged to complementation group A. Eighty percent of patients presented with mild/moderate somatic phenotype and most with cytopenia. No correlation was seen between somatic/hematologic phenotype and number of missense mutations of FANCA alleles. Over follow-up, 33% of patients improved or maintained mild/moderate cytopenia or normal blood count, whereas remaining worsened cytopenia. Eleven patients developed a hematological adverse event (MDS, AML, pathological cytogenetics) and three developed solid tumors. 10 years cumulative risk of death of the whole cohort was 25.6% with median follow-up 5.8 years. In patients eligible to hematopoietic stem cell transplantation because of moderate cytopenia, mortality was significantly higher in subjects transplanted from matched unrelated donor over nontransplanted subjects, whereas there was no significant difference between matched sibling donor transplants and nontransplanted patients. In patients eligible to transplant because of severe cytopenia and clonal disease, mortality risk was not significantly different in transplanted from matched unrelated versus matched sibling donor versus nontransplanted subjects. The decision to transplant should rely on various elements including, type of donor, HLA matching, patient comorbidities, impairment, and clonal evolution of hematopoiesis. Am. J. Hematol. 91:666-671, 2016. © 2016 Wiley Periodicals, Inc.

Clinical aspects of Fanconi anemia individuals with the same mutation of FANCF identified by next generation sequencing.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disease characterized by congenital malformations, aplastic anemia and increased risk of developing malignancies. FA is genetically heterogeneous as it is caused by at least 17 different genes. Among these, FANCA, FANCC, and FANCG account for approximately 85% of the patients whereas the remaining genes are mutated in only a small percentage of cases. For this reason, the molecular diagnostic process is complex and not always extended to all the FA genes, preventing the characterization of individuals belonging to rare groups. METHODS: The FA genes were analyzed using a next generation sequencing approach in two unrelated families. RESULTS: The analysis identified the same, c.484_485del, homozygous mutation of FANCF in both families. A careful examination of three electively aborted fetuses in one family and one affected girl in the other indicated an association of the FANCF loss-of-function mutation with a severe phenotype characterized by multiple malformations. CONCLUSION: The systematic use of next generation sequencing will allow the recognition of individuals from rare complementation groups, a better definition of their clinical phenotypes, and consequently, an appropriate genetic counseling.

Impaired immune response to Candida albicans in cells from Fanconi anemia patients.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. Several studies show alterations of the immunological status of FA patients including defects in peripheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines. However scanty information is available on the response of FA cells to specific infectious antigens. In this work we examined the response of FA cells to different immunological stimuli and found a defective response of IL-1ß, TNF-α and IL-17 to Candida albicans stimulation thus pointing to a potentially impaired response to fungal infections of FA patients.

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia.

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Identification of point mutations and large intragenic deletions in Fanconi anemia using next-generation sequencing technology.

Fanconi anemia (FA) is a rare bone marrow failure disorder characterized by clinical and genetic heterogeneity with at least 17 genes involved, which make molecular diagnosis complex and time-consuming. Since next-generation sequencing technologies could greatly improve the genetic testing in FA, we sequenced DNA samples with known and unknown mutant alleles using the Ion PGM (™) system (IPGM). The molecular target of 74.2 kb in size covered 96% of the FA-coding exons and their flanking regions. Quality control testing revealed high coverage. Comparing the IPGM and Sanger sequencing output of FANCA,FANCC, and FANCG we found no false-positive and a few false-negative variants, which led to high sensitivity (95.58%) and specificity (100%) at least for these two most frequently mutated genes. The analysis also identified novel mutant alleles, including those in rare complementation groups FANCF and FANCL. Moreover, quantitative evaluation allowed us to characterize large intragenic deletions of FANCA and FANCD2, suggesting that IPGM is suitable for identification of not only point mutations but also copy number variations.

Spectrum of the mutations in Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb-IX-V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbß), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Unusual splice site mutations disrupt FANCA exon 8 definition.

The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position -3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition.

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia.

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L.

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology.

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes.

Mutations of cytochrome c identified in patients with thrombocytopenia THC4 affect both apoptosis and cellular bioenergetics.

Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia.