Abstract
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 <1 year, presented with haemolysis (LDH 539 IU/L, total bilirubin 39 umol/L), haematology (Hb 92g/L, MCV 84.4, MCH 28.9, absolute retic count 313.8x109/L); his film showed marked anisopoikilocytes, microspherocytes and polychromasia. He had frontal bossing and a palpable spleen and had suffered several infections, the child was transfused once. His father's film showed elliptocytes, FBC (Hb 127g/L, MCV 89.6, MCH 30.6, absolute retic count 230.5x109/L) but he had never been transfused. The mother's FBC was normal (Hb 113g/L, MCV 87.0, MCH 29.2, absolute retic count 48.4x109/L) but her film also showed elliptocytes. Analysis using the red cell panel found the child to be compound heterozygous for c.83G>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.
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