HAEMATOLOGY EQA
and delta genotyping is not currently assessed; however, two of the 48 laboratories did notify us that delta gene testing was performed, which revealed the presence of the c.179delA mutation.
Results
For alpha-globin genotyping, 35/47 participants (74%) returned the expected result: α3.7/
αIVS1(-5nt) α. Twelve reported the
3.7 kb alpha globin gene deletion but did not report the IVS1(-5nt) mutation. One failed to detect it by Sanger sequencing and received an adverse score. The remaining 12 did not detect it because they used assays that detect deletional mutations only (eg MLPA or GAP PCR).
For beta-globin genotyping, 44/48 participants (92%) returned the expected result: βA/
βCodon27(GCC>TCC) . Four did not
detect Hb Knossos due to the limitations of the assay used. None of these laboratories performed Sanger or next- generation sequencing; two of them used reverse dot blot, one used the Vienna laboratory strip assay and one used PCR plus reverse hybridisation.
Discussion and conclusions This case highlights the importance of interpreting Hb genotyping with the clinical details in mind. Initial review of the details would lead to suspicion of alpha- thalassaemia only. However, a key detail was that the child’s father has a diagnosis of beta-thalassaemia intermedia, implying that the child is likely to have inherited a beta-thalassaemia mutation from his father. It also showed that delta- globin gene sequencing was the key to explaining the normal range Hb A2 seen in this child.
Carriers of Hb Knossos are almost undetectable on HPLC/CE because the abnormal variant is hidden within the Hb A peak, and the Hb A2 peak is normal, although they do have thalassaemic indices. This mutation typically results in beta-thalassaemia intermedia when homozygous or inherited in combination with other beta-thalassaemia mutations, so it is important to be able to detect it in cases of anaemia or to establish reproductive risks. When discussing reproductive options or family testing, it is worthwhile mentioning that this beta-thalassaemia can be missed or misdiagnosed as alpha-thalassaemia using conventional screening tests, so it might be worth considering using genetic testing to obtain an unambiguous carrier diagnosis in these families.
This is the first time that this atypical beta-thalassaemia mutation has been included within this molecular EQA
scheme and it is encouraging that the majority of laboratories detected and interpreted it correctly.
Reference 1 Nasouhipur H, Banihashemi A, Youssefi
Kamangar R, Akhavan-Niaki H. Hb Knossos: HBB:c.82G>T Associated with HBB:c.315+1G>A Beta Zero Mutation Causes Thalassemia Intermedia. Indian J Hematol Blood Transfus. 2014 Sep; 30 (Suppl 1): 243–5. doi: 10.1007/s12288- 014-0343-y.
Alpha genotype
Sanger sequencing (21) MLPA (19)
Multiplex gap PCR (18) Vienna labs strip assay (8) Gap PCR (8) NGS (5)
Reverse dot blot (1) ARMS PCR (1)
PCR + reverse hybridisation (1) Multiplex triple PCR (1)
Beta genotype
Sanger sequencing (35) MLPA (12)
Vienna labs strip assay (3) NGS (5)
Reverse dot blot (2)
PCR + reverse hybridisation (1) Multiplex gap PCR (2) PCR-other (1)
Real-time PCR assay (discrimination of alleles S and C) (1)
Methods used by UK NEQAS participants for alpha- and beta-globin genotyping (distribution 2203DN2; some laboratories used more than one method).
908 No further action necessary 907 Iron studies recommended
906 Suggest refer to a consultant haematologist
905 Refer for genetic counselling, including information on prenatal diagnosis
903 Parental testing indicated 904 Family studies indicated
901 Partner testing recommended 902 Genetic counselling recommended
0 0
5 5
10 10 15 15 Number Recommendations made by UK NEQAS participants for a child with Hb Knossos (distribution 2203DN2). 20 20 25 25 30 30
Bashori Rahman and Barbara De la Salle, UK NEQAS Haematology, PO Box 14, Watford WD18 0FJ, UK.
UK NEQAS Haematology is operated by the West Hertfordshire Teaching Hospitals NHS Trust.
This article is based on a poster presented at the IBMS Congress 2023 event, held last September at the International Convention Centre in Birmingham.
G G T G G T G A G G C C C T G G G C A G G Hb Knossos
Codon 27 GCC>TCC: Hb Knossos as detected by DNA Sanger sequencing (from Nasouhipur et al.1
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