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•ERCC6L2 disease causes BMF that requires timely stem cell transplantation before progressing to a high-risk myeloid malignancy.•Frequent somatic TP53mutation screening and BM examination are essential for surveillance of patients with ERCC6L2-related disease.

Inherited bone marrow (BM) failure syndromes are a diverse group of disorders characterized by BM failure, usually in association with ≥1 extrahematopoietic abnormalities. BM failure, which can involve ≥1 cell lineages, often presents in the pediatric age group. Furthermore, some children initially labeled as having idiopathic aplastic anemia or myelodysplasia represent cryptic cases of inherited BM failure. Significant advances in the genetics of these syndromes have been made, identifying more than 100 disease genes, giving insights into normal hematopoiesis and how it is disrupted in patients with BM failure. They have also provided important information on fundamental biological pathways, including DNA repair: Fanconi anemia (FA) genes; telomere maintenance: dyskeratosis congenita (DC) genes; and ribosome biogenesis: Shwachman-Diamond syndrome and Diamond-Blackfan anemia genes. In addition, because these disorders are usually associated with extrahematopoietic abnormalities and increased risk of cancer, they have provided insights into human development and cancer. In the clinic, genetic tests stemming from the recent advances facilitate diagnosis, especially when clinical features are insufficient to accurately classify a disorder. Hematopoietic stem cell transplantation using fludarabine-based protocols has significantly improved outcomes, particularly in patients with FA or DC. Management of some other complications, such as cancer, remains a challenge. Recent studies have suggested the possibility of new and potentially more efficacious therapies, including a renewed focus on hematopoietic gene therapy and drugs [transforming growth factor-β inhibitors for FA and PAPD5, a human poly(A) polymerase, inhibitors for DC] that target disease-specific defects.

Standardized variant curation is essential for clinical care recommendations for patients with inherited disorders. Clinical Genome Resource (ClinGen) variant curation expert panels are developing disease-associated gene specifications using the 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines to reduce curation discrepancies. The ClinGen Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) was created collaboratively between the American Society of Hematology and ClinGen to perform gene- and disease-specific modifications for inherited myeloid malignancies. The MM-VCEP began optimizing ACMG/AMP rules for RUNX1 because many germline variants have been described in patients with familial platelet disorder with a predisposition to acute myeloid leukemia, characterized by thrombocytopenia, platelet functional/ultrastructural defects, and a predisposition to hematologic malignancies. The 28 ACMG/AMP codes were tailored for RUNX1 variants by modifying gene/disease specifications, incorporating strength adjustments of existing rules, or both. Key specifications included calculation of minor allele frequency thresholds, formulating a semi-quantitative approach to counting multiple independent variant occurrences, identifying functional domains and mutational hotspots, establishing functional assay thresholds, and characterizing phenotype-specific guidelines. Preliminary rules were tested by using a pilot set of 52 variants; among these, 50 were previously classified as benign/likely benign, pathogenic/likely pathogenic, variant of unknown significance (VUS), or conflicting interpretations (CONF) in ClinVar. The application of RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the benefit of gene-specific criteria and sharing internal laboratory data.

Standardized variant curation is essential for clinical care recommendations for patients with inherited disorders. Clinical Genome Resource (ClinGen) variant curation expert panels are developing disease-associated gene specifications using the 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines to reduce curation discrepancies. The ClinGen Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) was created collaboratively between the American Society of Hematology and ClinGen to perform gene- and disease-specific modifications for inherited myeloid malignancies. The MM-VCEP began optimizing ACMG/AMP rules for RUNX1because many germline variants have been described in patients with familial platelet disorder with a predisposition to acute myeloid leukemia, characterized by thrombocytopenia, platelet functional/ultrastructural defects, and a predisposition to hematologic malignancies. The 28 ACMG/AMP codes were tailored for RUNX1variants by modifying gene/disease specifications, incorporating strength adjustments of existing rules, or both. Key specifications included calculation of minor allele frequency thresholds, formulating a semi-quantitative approach to counting multiple independent variant occurrences, identifying functional domains and mutational hotspots, establishing functional assay thresholds, and characterizing phenotype-specific guidelines. Preliminary rules were tested by using a pilot set of 52 variants; among these, 50 were previously classified as benign/likely benign, pathogenic/likely pathogenic, variant of unknown significance (VUS), or conflicting interpretations (CONF) in ClinVar. The application of RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the benefit of gene-specific criteria and sharing internal laboratory data.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.

The effectiveness of intermittent montelukast for wheeze in young children is unclear. We aimed to assess whether intermittent montelukast is better than placebo for treatment of wheeze in this age group. Because copy numbers of the Sp1-binding motif in the arachidonate 5-lipoxygenase (ALOX5) gene promoter (either 5/5, 5/x, or x/x, where x does not equal 5) modifies response to montelukast in adults, we stratified by this genotype.

Four reports have been published on an association between acute myeloid leukaemia AML and primary lymphedema, with or without congenital deafness. We report seven new cases, including one extended family, confirming this entity as a genetic syndrome. The lymphedema typically presents in one or both lower limbs, before the hematological abnormalities, with onset between infancy and puberty and frequently affecting the genitalia. The AML is often preceded by pancytopenia or myelodysplasia with a high incidence of monosomy 7 in the bone marrow five propositi and two relatives. Associated anomalies included hypotelorism, epicanthic folds, long tapering fingers andor neck webbing four patients, recurrent cellulitis in the affected limb four patients, generalized warts two patients, and congenital, high frequency sensorineural deafness one patient. Children with lower limb and genital lymphedema should be screened for hematological abnormalities and immunodeficiency. © 2010 WileyLiss, Inc.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and a predisposition to cancer. The genetic basis of DC remains unknown in more than 60% of patients. Mutations have been identified in components of the telomerase complex (dyskerin, TERC, TERT, NOP10, and NHP2), and recently in one component of the shelterin complex TIN2 (gene TINF2). To establish the role of TINF2 mutations, we screened DNA from 175 uncharacterised patients with DC as well as 244 patients with other bone marrow failure disorders. Heterozygous coding mutations were found in 33 of 175 previously uncharacterized DC index patients and 3 of 244 other patients. A total of 21 of the mutations affected amino acid 282, changing arginine to histidine (n = 14) or cysteine (n = 7). A total of 32 of 33 patients with DC with TINF2 mutations have severe disease, with most developing aplastic anaemia by the age of 10 years. Telomere lengths in patients with TINF2 mutations were the shortest compared with other DC subtypes, but TERC levels were normal. In this large series, TINF2 mutations account for approximately 11% of all DC, but they do not play a significant role in patients with related disorders. This study emphasises the role of defective telomere maintenance on human disease.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and a predisposition to cancer. The genetic basis of DC remains unknown in more than 60% of patients. Mutations have been identified in components of the telomerase complex (dyskerin, TERC, TERT, NOP10, and NHP2), and recently in one component of the shelterin complex TIN2 (gene TINF2). To establish the role of TINF2mutations, we screened DNA from 175 uncharacterised patients with DC as well as 244 patients with other bone marrow failure disorders. Heterozygous coding mutations were found in 33 of 175 previously uncharacterized DC index patients and 3 of 244 other patients. A total of 21 of the mutations affected amino acid 282, changing arginine to histidine (n = 14) or cysteine (n = 7). A total of 32 of 33 patients with DC with TINF2mutations have severe disease, with most developing aplastic anaemia by the age of 10 years. Telomere lengths in patients with TINF2mutations were the shortest compared with other DC subtypes, but TERC levels were normal. In this large series, TINF2mutations account for approximately 11% of all DC, but they do not play a significant role in patients with related disorders. This study emphasises the role of defective telomere maintenance on human disease.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC(telomerase RNA component) and TERT(telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERTmutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERTmutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERTmutations or hemizygous DKC1mutations, these 2 homozygous TERTpatients were observed to have higher-than-expected TERClevels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERTmutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.

Dyskeratosis congenita (DC) is a rare inherited multi-system disorder. Although DC is classically characterized by mucocutaneous features, the vast majority of patients develop hematologic abnormalities, and in its occult form the disease can present as aplastic anemia. The gene responsible for the X-linked form of the disease encodes a protein involved in ribosome biogenesis and in stabilizing the telomerase complex, while the autosomal dominant form is caused by mutations in the core RNA component of telomerase. It has been suggested that DC is primarily a disease of defective telomere maintenance. Premature shortening of telomeres resulting in a limited proliferative potential of stem cells would explain the pathology observed in DC, as the affected tissues are those that require constant renewal.

The two genes mutated in the bone marrow failure syndrome dyskeratosis congenita (DC) both encode components of the telomerase complex responsible for maintaining the ends of chromosomes in stem cells and in the germ line. In reviewing the mutation profile that is found in DC, we describe 9 novel mutations in the DKC1 gene and 3 novel TERC mutations responsible for the X-linked and autosomal dominant forms of the disease, respectively, but find that two thirds of the families do not have mutations in either of these genes. In a significant subset of these uncharacterized families, the index case presents with severe disease previously defined as the Hoyeraal Hreidarsson (HH) syndrome. The diverse clinical phenotype seen in patients with X-linked DC is not explained by the different amino acid substitutions: Presentation of the recurrent A353V substitution ranges from classic DC to the severe HH variant. However, we do see that patients with HH have significantly shorter telomeres than those with a relatively mild presentation. In the new families described with TERC mutations, there is further evidence of disease anticipation associated with shorter telomeres in the younger generations. This study highlights the considerable genetic and phenotypic diversity of DC.

The two genes mutated in the bone marrow failure syndrome dyskeratosis congenita (DC) both encode components of the telomerase complex responsible for maintaining the ends of chromosomes in stem cells and in the germ line. In reviewing the mutation profile that is found in DC, we describe 9 novel mutations in the DKC1gene and 3 novel TERCmutations responsible for the X-linked and autosomal dominant forms of the disease, respectively, but find that two thirds of the families do not have mutations in either of these genes. In a significant subset of these uncharacterized families, the index case presents with severe disease previously defined as the Hoyeraal Hreidarsson (HH) syndrome. The diverse clinical phenotype seen in patients with X-linked DC is not explained by the different amino acid substitutions: Presentation of the recurrent A353V substitution ranges from classic DC to the severe HH variant. However, we do see that patients with HH have significantly shorter telomeres than those with a relatively mild presentation. In the new families described with TERCmutations, there is further evidence of disease anticipation associated with shorter telomeres in the younger generations. This study highlights the considerable genetic and phenotypic diversity of DC.

Mutations in TERC, encoding the RNA component of telomerase, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia (AA). Several polymorphisms also exist in the TERC gene, making functional testing of potential pathogenic mutations essential. Here, we have tested normal and mutant TERC molecules in 2 telomerase reconstitution assays, 1 in vitro and 1 in transfected telomerase-negative cells. We find that 2 polymorphic mutations G58A and G228A have no effect on telomerase activity in these assays, whereas 6 mutations found in DC and AA cause reduction or abolition of telomerase activity. Mutations in the pseudoknot region of the TERC molecule, C72G, 96-7ΔCT, GC107-8AG and 110-3ΔGACT reduce the catalytic activity of reconstituted telomerase, whereas mutations in the 3′ portion of the molecule C408G and a deletion of the 3′ 74 bases have normal activity in vitro but reduced intracellular activity. By analyzing second site mutations that recreate regions of secondary structure but retain the pathogenic mutations we show that mutations C72G, GC107-8AG, and C408G act by disrupting the secondary structure or folding of TERC. Finally, experiments reconstituting telomerase with both normal and mutant TERC molecules suggest the mutations act via haploinsufficiency rather than by a dominant-negative mechanism. (Blood. 2004;104:3936-3942)

Mutations in TERC, encoding the RNA component of telomerase, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia (AA). Several polymorphisms also exist in the TERCgene, making functional testing of potential pathogenic mutations essential. Here, we have tested normal and mutant TERC molecules in 2 telomerase reconstitution assays, 1 in vitro and 1 in transfected telomerase-negative cells. We find that 2 polymorphic mutations G58A and G228A have no effect on telomerase activity in these assays, whereas 6 mutations found in DC and AA cause reduction or abolition of telomerase activity. Mutations in the pseudoknot region of the TERC molecule, C72G, 96-7ΔCT, GC107-8AG and 110-3ΔGACT reduce the catalytic activity of reconstituted telomerase, whereas mutations in the 3′ portion of the molecule C408G and a deletion of the 3′ 74 bases have normal activity in vitro but reduced intracellular activity. By analyzing second site mutations that recreate regions of secondary structure but retain the pathogenic mutations we show that mutations C72G, GC107-8AG, and C408G act by disrupting the secondary structure or folding of TERC. Finally, experiments reconstituting telomerase with both normal and mutant TERC molecules suggest the mutations act via haploinsufficiency rather than by a dominant-negative mechanism. (Blood. 2004;104:3936-3942)

Clinical, pathological, and X-ray findings of two brothers with features resembling congenital intrauterine infection-like syndrome are presented. Extensive screening for intrauterine infection was performed. Nevertheless all confirmatory tests were normal. Both brothers showed extensive intra- and extra-cranial calcifications, thrombocytopenia, a septum pellucidum cyst, one-sided paresis of the diaphragm, and metaphyseal changes on X-ray scans resembling intrauterine infection. Within the first days of life, they developed seizures and died from severe cerebral hemorrhage. The MRI scan of the brain showed cerebellar hypoplasia in one of the boys, while the cerebellum had normal size in the other. No indication of a metabolic disorder, especially in calcium metabolism, was identified. Due to the clinical overlap with Hoyeraal-Hreidarsson syndrome, mutations in the DKC1 gene (Xq28) and the hTR gene (RNA component of telomerase on chromosome 3q) have been excluded. The parents are non-consanguineous and further family history was unremarkable. The findings in these boys overlap with features described in congenital intrauterine infection-like syndrome (pseudo-TORCH syndrome). © 2003 Wiley-Liss, Inc.

ABSTRACT

Glucose-6-phosphate dehydrogenase (G6PD) is expressed in all tissues, where it catalyses the first step in the pentose phosphate pathway. G6PD deficiency is prevalent throughout tropical and subtropical regions of the world because of the protection it affords during malaria infection. Although most affected individuals are asymptomatic, there is a risk of neonatal jaundice and acute haemolytic anaemia, triggered by infection and the ingestion of certain drugs and broad beans (favism). A rare but more severe form of G6PD deficiency is found throughout the world and is associated with chronic non-spherocytic haemolytic anaemia. Many deficient variants of G6PD have been described. DNA sequence analysis has shown that the vast majority of these are caused by single amino acid substitutions. The three-dimensional structure of G6PD shows a classical dinucleotide binding domain and a novel β+α domain involved in dimerization.

Inherited bone marrow (BM) failure syndromes are a diverse group of disorders characterized by BM failure, usually in association with one or more extra-hematopoietic abnormalities. BM failure, which can involve one or more cell lineages, often presents in the pediatric age group. Furthermore, some children initially labeled as having idiopathic aplastic anemia or myelodysplasia represent cryptic cases of inherited BM failure. Significant advances in the genetics of these syndromes have been made, identifying more than 100 disease genes, giving insights into normal hematopoiesis and how this is disrupted in patients with BM failure. They have also provided important information on fundamental biological pathways: DNA repair: Fanconi anemia (FA) genes; telomere maintenance: dyskeratosis congenita (DC) genes; and ribosome biogenesis: Shwachman-Diamond syndrome and Diamond-Blackfan anemia genes. Additionally, as these disorders are usually associated with extra-hematopoietic abnormalities and increased cancer risk, they have provided insights into human development and cancer. In the clinic, genetic tests stemming from the recent advances facilitate diagnosis, especially when clinical features are insufficient to accurately classify a disorder. Hematopoietic stem cell transplantation using fludarabine-based protocols has significantly improved outcomes, particularly for patients with FA and DC. Management of some other complications, such as cancer, remains a challenge. Recent studies have suggested the possibility of new and potentially more efficacious therapies, including a renewed focus on hematopoietic gene therapy and drugs (transforming growth factor (TGF)-beta inhibitors for FA and PAPD5, a human poly(A) polymerase, inhibitors for DC) that target disease-specific defects.

Biallelic mutations in ERCC6L2were first reported to cause bone marrow failure (BMF). Additionally, we recently described a strong predisposition to erythroid lineage-restricted acute myeloid leukemia (AML-M6). Today, 31 ERCC6L2-mutated cases have been reported. This study aims to further explore the clinical and molecular features, as well as outcomes, of the ERCC6L2 patients.

Siitonen: celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Brystol Myers Squibb: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Honoraria.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C?T mutation, predicting a Pro172?Ser replacement. Analysis of G6PD RNA from the patient’s leukocytes and fibroblasts showed only transcripts with the 514C?T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient’s daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

ABSTRACT: Using the published protein sequence from a rabbit microsomal glucose-6-phosphate dehydrogenase G6PD we have isolated and sequenced a cDNA clone coding for its human equivalent, which is also known as hexose-6-phosphate dehydrogenase (H6PD) and glucose dehydrogenase. The corresponding genomic sequence is in the databases enabling its localization to chromosome 1p36. The gene spans 37 kb and consists of 5 exons, the fifth of which codes for more than half of the 89kDa protein. The first intron is a 10kb insertion in the 5′ untranslated sequence. The predicted mRNA has an exceptionally long (6.5kb) 3′ untranslated sequence. The predicted protein shows extensive homology with X-linked G6PD, suggesting the two genes share a common ancestor but no intron positions are conserved between the two genes suggesting the gene duplication was an ancient event. The C-terminal portion of the protein is not homologous with G6PD but shows limited homology with proteins of unknown function found throughout evolution and encoded next to G6PD in various micro-organisms. Intriguingly this C-terminal portion has some homology with the N-terminal sequence ofPlasmodium falciparumG6PD.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C→T mutation, predicting a Pro172→Ser replacement. Analysis of G6PD RNA from the patient’s leukocytes and fibroblasts showed only transcripts with the 514C→T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient’s daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coliexpression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

The electrophoretic mobility and level of enzyme activity of glucose-6-phosphate dehydrogenase (G6PD) was established in 100 unrelated Algerian males with G6PD deficiency. DNA from these subjects was analysed for the presence of certain known G6PD mutations by the appropriate restriction enzyme digestion of fragments amplified by the polymerase chain reaction. Where the mutation could not be identified in this way, the samples were subjected to single-strand conformation polymorphism analysis and abnormal fragments were sequenced. In this way, eight different mutations have been identified, of which five are polymorphic and account for 92% of the samples. The most common variants are G6PD A-(46%) and G6PD Mediterranean (23%), both of which were associated with favism. A new polymorphic variant, G6PD Aures, has been identified during the course of this study, whereas another, G6PD Santamaria, has now been established as a polymorphic variant (11%). Thus, G6PD deficiency in Algeria is heterogeneous, suggesting that there has been significant gene flow, both from sub-Saharan Africa and from other parts of the Mediterranean.

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3′-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3′ UTR, over 3 kb, accounts for one-half of the mature mRNA.

X-linked recessive dyskeratosis congenita (DKC) is a rare bone-marrow failure disorder linked to Xq28. Hybridization screening with 28 candidate cDNAs resulted in the detection of a 3′ deletion in one DKC patient with a cDNA probe (derived from XAP101). Five different missense mutations in five unrelated patients were subsequently identified in XAP101, indicating that it is the gene responsible for X-linked DKC (DKC1). DKC1 is highly conserved across species barriers and is the orthologue of rat NAP57 and Saccharomyces cerevisiae CBF5. The peptide dyskerin contains two TruB pseudouridine (Ψ) synthase motifs, multiple phosphorylation sites, and a carboxy-terminal lysine-rich repeat domain. By analogy to the function of the known dyskerin orthologues, involvement in the cell cycle and nucleolar function is predicted for the protein.

This communication reports a case of T-cell acute lymphoblastic leukaemia (T-ALL) which, after treatment and remission induction, relapsed 17 months after apparent disease-free remission as acute myeloblastic leukaemia (AML). Extensive immunophenotypic, analysis an initial presentation revealed a typical T-ALL phenotype (HLA-Dr-, TdT+, CD2+, CD3+, CD4-, CD5+, CD7+, CD8-, CD19-, CD13/33-) that was unusual in that the majority of blasts expressed membrane TCR γδ chains but not TCR αβ chains. Similarly, the morphological (Auer rods +), cytochemical (MPO+ and SBB+) and phenotypic characteristics at relapse were unequivocably consistent with a diagnosis of AML. In contrast to previous case studies of "phenotypic switch" or "metachronous bilineal leukaernia", Southern blot analysiis of TCR and Ig gene rearrangements at presentation and relapse were able, because of an unusual genotypic pattern (TCR β +, TCR γ +, TCR δ + and JH), to confirm the common clonal origin of the two leukaemias. It is suggested that the transition from T-ALL to AML may not be a "random" phenomenon but may rather reflect subclonal domination by mycloid blasts with relative drug resistance (i.e. synchronous bilineal differentiation with an undetectable myeloid component), or therapy-induced alterations in the balance of (myelosuppressive) factors.

Gene expression profiling has long been used in understanding the contribution of genes and related pathways in disease pathogenesis and susceptibility. We have performed whole blood transcriptomic profiling in a subset of inherited bone marrow failure (IBMF) cases that are clinically and genetically characterised as Fanconi anemia (FA), dyskeratosis congenita (DC) and Shwachman Diamond syndrome (SDS). We hypothesized that annotating whole blood transcripts genome wide will aid in understanding the complexity of gene regulation across these IBMF subtypes. Initial analysis of these blood derived transcriptomes revealed significant skewing towards upregulated genes in FA cases when compared to controls. Both DC and SDS cases also showed similar skewing profiles in their transcriptional status revealing a common pattern across these different IBMF subtypes. Gene set enrichment analysis revealed shared pathways involved in protein translation and elongation (ribosome constituents), RNA metabolism (nonsense mediated decay) and mitochondrial function (electron transport chain). We further identified a discovery set of 26 upregulated genes at stringent cut-off (FDR<0.05) that appeared as a unified signature across the IBMF subtypes. Subsequent transcriptomic analysis on genetically uncharacterised BMF cases revealed a striking overlap of genes, including 22 from the discovery set indicating a unified transcriptional drive across the classic (FA, DC and SDS) and uncharacterised BMF subtypes. This study has relevance in disease pathogenesis, for example in explaining the features (including the BMF) common to all IBMF cases and suggests harnessing this “transcriptional signature” for patient benefit.

Cell-surface markers are useful in identifying and studying different cell types in the nervous system. For example, the major cell types in rat dorsal root ganglion (DRG) cultures have been identified using a combination of tetanus toxin and antibodies to the cell-surface antigens Ran-1 and Thy-1. Neurones bind tetanus toxin and express Thy-1, Schwann cells express only Ran-1 and fibroblasts express only Thy-1 (ref. 1). A natural extension of this approach was to use antibodies to distinguish between different neuronal subpopulations on the basis of their cell-surface antigens. We therefore immunized mice with cells from rat DRG cultures, and then used the hybridoma technique of Köhler and Milstein2to produce monoclonal antibodies. We report here an antibody that recognizes a surface antigen which is present on all rat peripheral neurones we have studied and absent from neurones derived from the central nervous system. An antigen with reciprocal distribution, expressed only by neurones of the rat central nervous system, has been defined by Cohen and Selvendran3.

DiNardo: Karyopharm: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Abbvie: Honoraria. Nichols:Incyte: Research Funding; Alpine Immune Sciences: Research Funding. Plon:Baylor Genetics: Membership on an entity's Board of Directors or advisory committees.

Natsoulis: Imago BioSciences, Inc.: Consultancy, Equity Ownership. Guzman:Cellectis: Research Funding. DiNardo:Bayer: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Abbvie: Honoraria. Borate:Novartis: Consultancy; Agios: Consultancy. Wei:Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau. Dokal:MRC, Bloodwise, Telomerase Activator Sciences: Research Funding; The Gary Woodward Dyskeratosis Congenita Trust: Membership on an entity's Board of Directors or advisory committees; Action Medical Research, European School of Haematology: Membership on an entity's Board of Directors or advisory committees; Barts and The London School of Medicine and Dentistry, Queen Mary University of London,: Employment, Research Funding; Telomerase Activator Sciences: Research Funding; Barts and The London, Queen Mary University of London: Employment; Gary Woodward Dyskeratois Congenita Trust: Membership on an entity's Board of Directors or advisory committees. Hiwase:Novartis: Research Funding; Celgene: Research Funding. Branford:Cepheid: Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Owen:Merck: Honoraria; Teva: Honoraria; AbbVie: Research Funding; Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Research Funding; AstraZeneca: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding. Fitzgibbon:Epizyme: Consultancy, Research Funding; Gilead: Consultancy. Rienhoff:Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kurokawa:Astellas Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Eizai: Research Funding; MSD: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Pfizer: Research Funding; Takeda Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Teijin Pharma: Research Funding; Chugai Pharmaceutical: Research Funding.

Clinical Genome Resource (ClinGen) is an NIH/NHGRI-funded effort dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for use in precision medicine and research. ClinGen has developed both gene and variant expert panels to adapt the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for consistent and accurate variant classification of specific genes and diseases. Here, we describe a new effort initiated in 2018 and supported by the American Society of Hematology (ASH) in collaboration with ClinGen to develop expert panels. This effort was motivated by the increasing use of genomics in clinical hematology and the lack of resources containing expert interpretation of germline variation. This panel, named the ClinGen Myeloid Malignancy Variant Curation Expert Panel is focused on the curation and annotation of variants in genes associated with familial/inherited risk for myeloid malignancies. Our team consists of expert clinicians, clinical laboratory diagnosticians, and researchers interested in developing and implementing standardized protocols for sequence variant specific annotations of genes in inherited myeloid malignancies. The optimization of the ACMG/AMP guidelines encompasses disease-/gene-informed specifications or strength adjustments of existing rules, including defining gene-specific population frequency cutoffs, and specifying recommendations for the use of computational/predictive data, as supported by published functional and clinical data in addition to guidance on ACMG/AMP variant interpretation provided by the ClinGen effort. Our initial focus has been to organize sub-groups of teams to develop approaches for evaluating ACMG/AMP codes to interpret germline variants of the RUNX1gene. Once the curation of RUNX1variants is underway, we will extend our focus to include CEBPA, DDX41, ETV6, and GATA2. These efforts will be bolstered by encouraging submission of existing variant interpretations to ClinVar or other public variant databases by the Hematology community. In summary, the ClinGen Myeloid Malignancy Variant Curation Expert Panel aims to develop recommendations to optimize ACMG/AMP criteria for standardization of variant interpretation in myeloid leukemia genes and make expert-reviewed and interpreted variants available to the hematology community through ClinVar and the ClinGen website (www.clinicalgenome.org) to support patient care and research.

Background:It has been known for approximately 19 years that germline mutations in RUNX1, lead to familial platelet disorder with predisposition to myeloid malignancy (FPD-MM, OMIM 601399). Since that time researchers have identified a broad range of different RUNX1mutations, in over 100 families. In large families, the diagnosis of malignancy shows variable penetrance among family members with the same mutation; some carriers of RUNX1mutations do not develop malignancy. The causes of this heterogeneity are currently not known, complicating counselling and risk analysis for individual carriers. Recent advances in genetic sequencing technology applied by many FPD-MM research groups around the world have highlighted their value in understanding the somatic genetic changes that are associated with development of malignancies in germline RUNX1mutation carriers. Collectively this information could lead to powerful insights essential for more precise risk assessment, monitoring, and therapeutic intervention. Specifically, a growing catalogue of somatic mutations associated with germline RUNX1malignancy offers the opportunity for informed monitoring of asymptomatic RUNX1carriers for additional high-risk somatic mutations, in turn providing the possibility for early therapeutic intervention to arrest the leukemic process. The challenge in advancing these goals for FPD-MM is the relative rarity of the disorder in individual populations. Global data sharing in a highly interactive FPD-MM research community offers a solution to this problem that benefits all patients world-wide.

Cavenagh: Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

The bone marrow failure syndromes are a diverse group of rare genetic conditions. Mutations in telomere maintenance genes cause a large proportion of cases of dyskeratosis congenita. Our aim was to determine whether TA65, marketed as a telomerase activator, can correct this defect in vitro. We also investigated danazol, which improves peripheral blood counts in dyskeratosis congenita and Fanconi's anaemia by an unknown mechanism.