ALBERT

Description: The genetic diagnosis of inherited anaemias is an important aspect of the diagnostic pathway for patients with haematological disorders, allowing discrimination between conditions of overlapping phenotypes therefore enabling more effective clinical treatment. Next Generation sequencing platforms are now in widespread use in diagnostic settings and are facilitating more rapid, accurate and cost-effective molecular diagnosis. The Red Cell Gene Panel developed by the Viapath Molecular Pathology laboratory based at King's College Hospital, London has harnessed this technology with the aim of identifying genetic diagnoses of rare inherited causes of anaemia. Although originally setup to diagnose inherited red cell disorders, clinical demand has led to the inclusion of inherited bone marrow failure syndromes and other related conditions such that the panel now consists of 194 genes, divided into 16 subpanels (see table 1). Here we present the data from the first 1000 diagnostic cases reported under the following referral groups: 462 cases of unexplained anaemia (including haemolytic anaemia, sideroblastic anaemia, congenital dyserythropoietic anaemia, Diamond-Blackfan Anaemia), 232 cases of inherited bone marrow failure syndromes (including thrombocytopenia and neutropenia), 163 cases of congenital erythrocytosis and 143 other cases (including but not limited to iron regulation, haemophagocytic lymphohistiocytosis (HLH) and Criggler-Najjar ). Of these 1000 cases, we have achieved an overall diagnostic yield of approximately 25%. A diagnosed case is defined here as one in which a clear pathogenic or likely pathogenic variant that explains the phenotype has been detected. The unexplained anaemia cases have achieved the highest percentage of cases diagnosed with 47% diagnostic yield and data will be presented outlining the gene-by-gene breakdown of diagnoses made. Our bespoke bioinformatics pipeline has also allowed the detection of novel disease-causing structural variants in 20 cases, contributing 2% of our diagnostic yield. These are detected using three different methods; read-depth analysis, split-read mapping and discordant insert-size analysis. All reported structural variants have been confirmed with a second method, either breakpoint mapping or dosage-sensitive PCR. A significant proportion of cases (28%) have been reported with variants of uncertain clinical significance, highlighting the need for family studies and functional characterisation to be able to accurately ascertain the significance of these variants. Future developments of the service include functional characterisation of membrane disorders using next generation ektacytometry and preliminary data from this work will be presented here. Disclosures Kulasekararaj: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Pagliuca:Gentium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: β thalassemia carriers are usually symptom free with microcytic hypochromic red cells and a raised HbA2 level. However, an increased output of α globin through co-inheritance of extra α globin genes, converts a typically asymptomatic β thalassemia carrier state to that of thalassemia intermedia. We describe 3 families with 3 unique head-to-tail duplications of the a-globin cluster in which all the probands have thalassemia intermedia ranging from moderately severe anemia with splenomegaly, to transfusion-dependence. In Family 1, both father (Chinese) and son (Chinese and Anglo-Saxon English) were heterozygous for the HBB:c.316-197C〉T β thalassemia variant but had moderately severe anemia (Hb 67 to 91 g/L) with splenomegaly; they were both transfusion-independent. In Family 2, the father (Syrian) had normal hematology, while mother (Iraqi) had a hematologic profile (Hb 110 g/L, RBC 5.68x1012/L, MCH 19.4 pg, MCV 58.9 fL and HbA2 4.8%) typical of heterozygous β0 thalassemia. The proband presented at age 5 years with severe anemia precipitated by an infection, that needed a blood transfusion. She continued to need intermittent blood transfusion while an older sister with a hematologic profile of Hb 75 g/L, RBC 3.04x1012/L, MCH 19.6 pg, MCV 65.6 fL, and mild spenomegaly, remained transfusion-independent. In Family 3 (Greek Cypriot), both parents were asymptomatic; the father was heterozygous for the HBB:c.93-21G〉A β thalassemia variant with a normal a globin genotype (aa/aa), and the mother had normal HbA2 levels. In contrast, both their daughter and son who had inherited father's β thalassemia, had moderately severe anemia and needed intermittent blood transfusion. In all 3 families, MLPA suggested duplication of the whole alpha globin cluster but could not differentiate the different duplications. Next generation sequencing using Agilent SureSelect bait capture, targeted sequence analysis to the two globin loci. Sequence alignment to the reference sequence was performed using NextGene (SoftGenetits, USA). Analysis of the β globin gene sequence identified the β thalassemia-causing variant in each family. Comparison of the sequence coverage across the a loci on chromosome 16 between each case and normal controls, showed that where duplications had occurred, there was proportionally more sequence captured, similar to SNP or CGH array analysis. At the point where the sequence coverage increased, a duplication breakpoint was suspected, and the aligned sequence reads were examined in detail. In Family 1, individual sequence reads matched part of the reference sequence, BLAT query in UCSC showed that the two halves of the read aligned at either end of the duplication, indicating they were sequences that spanned the head-to-tail breakpoint. This was confirmed by gap-PCR and Sanger sequence analysis. In the other two families, breakpoints were identified within repetitive regions which could not be captured by the baits and were therefore not covered by the captured sequence. The high resolution of the coverage map allowed precise characterisation of the duplications by gap-PCR and Sanger sequencing analysis of the breakpoint amplicons. Duplication of the a globin cluster was encompassed in 188.7 kb in family 1, 120.5 kb in family 2, and 274 kb in family 3 (Figure 1). Both father and son in Family 1 were heterozygous for the duplicated a globin cluster (aa/aa, aa) and HBB:c.316-197C〉T mutation. Both siblings in Family 2 were heterozygous for mother's β thalassemia c.135delC variant and father's duplicated a globin cluster. In family 3, the mother had a 3.7 kb a deletion variant and a duplicated a globin cluster (-a3.7/aa, aa), a total of 5 a globin genes. Both the daughter and son had inherited mother's duplicated a globin cluster with father's β thalassemia variant. These families clearly show that a duplicated a globin cluster does not have a discernible phenotype on its own but is readily detectable when co-inherited with a β thalassemia variant. In all 3 cases, the a globin cluster duplications are in a head-to-tail orientation and occurred in repeats. These have most likely formed by non-homologous recombination events involving repetitive Alu sequences interspersed throughout the region. Whether these events occur more commonly in this region or if the region tolerates these changes better, allowing them to accumulate, remains to be resolved. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Congenital anemia is difficult to diagnose once common causes have been excluded; for example 80% cases of congenital non-spherocytic hemolytic anemia are undiagnosed once pyruvate kinase and G6PD deficiencies have been excluded using phenotypic analysis. We describe a next generation sequencing strategy, targeting 147 genes, to facilitate the diagnosis of these conditions. The coding regions, splice sites and 200 bp into the untranslated regions were examined in each gene. All clinically significant variants were confirmed by Sanger sequencing, including confirmation in any appropriate family members. Illumina MiSeq data was analysed using a bespoke bioinformatics pipeline, which has been validated to a UK certified standard. The pipeline implements detection of genetic variants using multiple base callers and discovery of copy number variants based on sequencing depth. Variants are annotated with information from ClinVar, and population frequency data from ExAC and 1000 genomes project. All genes are sequenced in every individual but data analysis can easily be restricted to virtual subpanels, excluding analysis of genes not requested. Here we present three cases, highlighting the diagnostic utility of the panel as well as the underlying bioinformatics analysis. Case 1. A male Caucasian child of A; p.Arg28His and c.[5572C〉G; 6531-12C〉T]; p.[Leu1858Val;?] in the SPTA1 gene, suggesting the diagnosis of hereditary pyropoikilocytosis. The c.83G〉A; p.Arg28His mutation was inherited from the father and the c.[5572C〉G; 6531-12C〉T]; p.[Leu1858Val;?] low expression allele was inherited from the mother, who was homozygous. Case 2. The post mortem report from a hydropic still birth (36/40) showed extensive extramedullary hematopoiesis and severe anemia. A DNA sample was sent to the laboratory accompanied by blood samples from both parents whose hematology was normal. The DNA sample from the proband was relatively small so only the parental samples were analyzed using the red cell panel. Sequence analysis identified the mother to carry the c.3173dupG; p.Gln1659fs pathogenic variant and the father carried the c.2867_2868+1dupCCG pathogenic variant in the CDAN1 gene. Sanger Sequencing showed that the child had inherited both mutations from the parents. Variants in CDAN1 are associated with CDA type 1 which is documented to be a rare form of anemia which can be lethal. Case 3. An Italian girl carrying a paternally inherited c.118C〉T β0 thalassemia variant presented with a severe form of microcytic anemia (FBC, Hb 86g/L, RBC 4.87 x1012/L, MCV 55.2, MCH 17.7 and HbA2=5%). The severity of her anemia (not transfused) and palpable spleen suggested she had an additional pathogenic variant that had not been identified. Her mother had normal hematology FBC, Hb 133g/L, RBC 4.82x1012/L, MCV 79.9, MCH 27.6. After sequencing, Exome Depth analysis of the proband's LCR identified a novel deletion which removed the 5' HS1 and HS2 sites but left HS3-5 intact (confirmed by MLPA in the mother and proband). The combination of this mild down regulation of the beta globin locus in combination with the c.118C〉T β0 thalassemia variant caused her phenotype to be more severe than just a beta thalassemia carrier. Identifying pathogenic variants in these families is important as it facilitates prognosis and treatment, and allows prenatal diagnosis to be offered in future. To date the panel has assessed 10 cases of anemia with unknown cause and has made a definitive diagnosis in 8 (80%). Of the two undiagnosed, one was a child that died at 3 weeks and received multiple intrauterine and neonatal transfusions and had severe anemia and the other was a suspected case of CDA with little associated phenotype. Disclosures No relevant conflicts of interest to declare.