Next generation sequencing for diagnosis of hereditary anemia: Experience in a Spanish reference center.

BACKGROUND AND AIMS: Hereditary anemia (HA) encloses a wide group of rare inherited disorders with clinical and hematologic overlaps that complicate diagnosis. MATERIALS AND METHODS: A 48-gene panel was developed to diagnose HA by Next Generation Sequencing (NGS) in a large cohort of 165 patients from 160 unrelated families. RESULTS: Patients were divided in: A) patients who had a suspicion of a specific type of HA (n = 109), and B) patients who had a suspicion of HA but with no clear type (n = 56). Diagnostic performance was 83.5% in group A and a change of the initial diagnosis occurred in 11% of these patients. In group B, 35.7% of patients achieved a genetic diagnosis. NGS identified 6 cases of xerocytosis, 6 of pyruvate kinase (PK) deficiency, 4 of G6PD, and 1 case of phytosterolemia with no initial suspicion of these pathologies, which is clinically relevant since they have specific treatment. Five patients were found to carry variants associated to two different pathologies (4 of them combining a metabolic deficiency and a membrane defect), and 44 new variants were identified in 41 patients. CONCLUSION: The use of NGS is a sensitive technique to diagnose HA and it shows better performance when patients are better characterized.

Mutations in XLF/NHEJ1/Cernunnos gene results in downregulation of telomerase genes expression and telomere shortening.

NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.

Red cell pyruvate kinase deficiency in Spain: A study of 15 cases. / Déficit de piruvato cinasa eritrocitaria en España: estudio de 15 casos.

BACKGROUND AND OBJECTIVE: Pyruvate kinase deficiency (PKD) is a rare, inherited disease causing chronic hemolysis and anemia of varying intensity. The genetic heterogeneity of PKD is high and, to this day, over 240 different mutations have been identified. PATIENTS AND METHODS: 15 unrelated patients affected by PKD have been studied. PKLR gene sequencing was performed by SANGER, including the determination of promoter regions, exonic, intronic flanking and 3'UTR. RESULTS: Patients were classified into 3 groups based on the intensity of their clinical symptoms: I) severe and very severe (8 patients); II) moderate (2 patients), and III) mild (5 patients). Six out of the 18 alleles found were new mutations which had not been described previously, with the PKLR c.721G>T mutation being the most prevalent (26.67%), followed by the PKLR c.1456C>T mutation (13.33%). CONCLUSIONS: In Spain, the genetic heterogeneity of PKLR is still high but differs from that observed in the previous study carried out in 1998. Total PKLR gene sequencing is necessary for the characterization of all patients with PKD and for genetic counseling.

Missense SLC25A38 variations play an important role in autosomal recessive inherited sideroblastic anemia.

BACKGROUND: Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved. DESIGN AND METHODS: In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene. RESULTS: Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders. CONCLUSIONS: Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein.