A copy of a complete blood count and hemoglobin electrophoresis indicative for an HBB-related hemoglobinopathy (affected or carrier) must be provided with all requests.
Diagnostic testing:
- Clinical suspicion of beta-thalassemia or sickle cell anemia
Carrier testing:
- Patient from ethnic background known for higher incidence of HBB-related hemoglobinopathies
- At least one affected or carrier relative
- Partner is affected or carrier
Depending on the hematology study results, this analysis may vary. If the study results indicate a risk for beta-thalassemia (affected or carrier) with no specific hemoglobin variant detected, this test will consist in sequencing of all HBB (NM_000518.4) coding exons and its flanking intronic regions. If the study results detect a specific hemoglobin variant (like HbS or HbC), this test will consist in genotyping the detected variant (like NM_000518.4:c.20A>T for HbS or NM_000518.4:c.19G>A for HbC).
In case sequencing of HBB is performed, targeted regions are enriched using hybridization probes (IDT) and sequencing libraries are prepared using the Illumina DNA Prep with Enrichment kit. Paired-end massively parallel sequencing of 150-bp fragments is performed with an Illumina instrument. Sequences are aligned and compared to reference genome GRCh37. Sample identity is confirmed in parallel using Applied Biosystems™ SNaPshot™ Multiplex Kit. Variants are interpreted as per standards and guidelines in the field (PMID: 25741868). Clinically relevant copy number variants are confirmed by an orthogonal method (gap-PCR, TaqMan, MLPA or microarray).
In case genotyping is performed, variants are tested by standard PCR amplification and Sanger sequencing. Positive results are confirmed by independent Sanger sequencing.
In case HBB sequencing is performed, it achieves >99% analytical sensitivity and specificity for SNVs, indels and CNVs. A negative result does not rule out the possibility that a rare variant not detected by this assay is present in the individual. This test does not detect all possible variants in the genes tested. Unless explicitly specified, only coding exons and flanking intronic regions are covered by this assay. Furthermore, technically challenging variant types, such as large indels, small CNVs, complex rearrangements, low-complexity repeat associated, segmental duplication associated, and postzygotic variants, may not be detected (PMID: 34007000). Interpretation of results is highly dependent on the clinical and demographic information provided.
In case genotyping is performed, it will not detect all possible variants in HBB. A negative result does not rule out the possibility that the individual harbors a rare variant not included in the assay. Analysis is dependent on accurate clinical diagnosis of affected individuals and on correct reporting of ethnicity and family relationships. DNA variants located in primer binding sites and rare genotyping errors can result in inaccurate diagnosis.
Only clinically relevant variants are reported. Benign/likely benign variants and variants of uncertain significance with limited evidence for pathogenicity are not reported. Only variants causing HBB-related hemoglobinopathies are reported for carrier testing.
Turnaround time: 4-6 weeks
Specimens accepted:
- 2 x 4 mL blood in EDTA tubes (purple top tube) – 2 mL for newborns
- DNA: min 10 ug
- Amniotic fluid: min 10 ml
- Direct CVS: min 10 mg direct villi
- Cultured amniocytes or CVS: 2 x T25 flasks (confluent)
- For any other sample type, contact the laboratory for testing availability
Submit your test request using our molecular genetics requisition.
A copy of a complete blood count and hemoglobin electrophoresis indicative for an HBB-related hemoglobinopathy (affected or carrier) must be provided with all requests.