Genetic testing for haemoglobinopathies (R93, R94, R361, R362)

Mutations that reduce the level of globin proteins result in the thalassaemia syndromes (alpha thalassaemia is most commonly caused by partial or whole gene deletions).

Mutations in the globin genes that result in structurally abnormal haemoglobins (Hb variants) can cause thalassaemia-like syndromes, but may also cause sickling disorders, cyanosis or erythrocytosis.

Haemoglobinopathies are the commonest genetic defect worldwide (7 percent of the world's population has at least one haemoglobin mutation). Mutations of these genes are thought to provide a heterozygote advantage in terms of resistance to malarial infection so some of these mutations are extremely common in parts of the world where malaria is endemic and in countries with a high rate of migration population from these regions.

The thalassaemia syndromes and some of the Hb Variants disorders (such as sickle cell disease) are inherited as autosomal recessive conditions. Very rarely, β thalassaemia intermedia/major demonstrates an autosomal dominant inheritance pattern. Some Hb Variants also have an autosomal dominant inheritance pattern, while others can occur spontaneously (de novo). Thalassaemias are rarely reported to arise from de-novo mutations.

R93 (genetic testing for thalassaemia and other haemoglobinopathies) is the most commonly used test indication because it can be used to test for both thalassaemias and Hb variants. In addition to providing a molecular diagnosis for individuals affected with Hb H disease or beta thalassaemia intermedia, it can also be used to determine suitability for clinical trials by distinguishing between beta zero and beta plus thalassaemia mutations.

It can also distinguish between sickle cell disease and S/HPFH (sickle/hereditary persistence of fetal haemoglobin) in a young or mildly affected individual (test indication R94 involving targeted testing for the sickle mutation only is rarely used because it cannot distinguish between SS and S/HPFH, and could result in a transfused patient with sickle/beta zero thalassaemia being erroneously genotyped as a sickle carrier).

In addition, it can be used to confirm carrier status in individuals who are well but have a phenotype (also known as a haemoglobinopathy trait) such as persistent anaemia or an Hb variant incidentally identified during testing for diabetes, and can distinguish between significant carrier states such as delta-beta thalassaemia versus benign mutations such as HPFH (test indication R361 is currently usually reserved for referrals via the antenatal screening pathway, and test indication R362 is only used to confirm sickle carrier status in parents during prenatal diagnosis; NB confirmation of the sickle genotype is not usually required before arranging prenatal sampling as long as the parental haemoglobinopathy screening results are typical of sickle carriers).

It can also be used for investigation into fetal hydrops, although if both parents do not have thalassaemia trait, use of R21 (fetal anomalies with a likely genetic cause) may be more appropriate.

Patients with chronic anaemia that do not fit into one of the classical haemoglobinopathy phenotypes may be more appropriately tested under R92 (rare anaemia); the haemoglobinopathy genes are also included on this panel because rarer forms of haemoglobinopathies overlap phenotypically with some of these rarer anaemia conditions.

For erythrocytosis/methaemoglobinaemia referrals, use test code R405 (hereditary erythrocytosis).

Testing strategy

Clinically affected probands

• R93.1 - dosage analysis for copy number variants in the following genes: HBA1; HBA2; (and HBB; HBG1; HBG2 if appropriate)
• R93.2 - Sanger sequencing for the haemoglobinopathy genes: HBA1; HBA2; HBB (and HBG1& HBG2 if appropriate). The HBD gene is also sequenced; this is not currently routinely included in the funded NHS tests because genetic variants in this gene do not have any pathological effect, however, identification of variants can help to explain the haematological phenotype and therefore this result may be reported in some individuals.
• R94.1 - HbSS variant targeted mutation testing by pyrosequencing or ARMS in individuals with a likely HbSS sickle cell anaemia result on haemoglobin electrophoresis (in practice this test code is rarely used because R93 provides more clarity for genotyping of likely sickle cell anaemia patients; see above for more information).

Individuals identified as requiring molecular testing during sickle cell and thalassaemia antenatal screening

• R361.1 - Sanger sequencing for the haemoglobinopathy genes: HBA1; HBA2; HBB (and HBG1& HBG2 if appropriate). The HBD gene is also sequenced; this is not currently routinely included in the funded NHS tests because genetic variants in this gene do not have any pathological effect, however, identification of variants can help to explain the haematological phenotype and therefore this result may be reported in some individuals.
• R361.2 - dosage analysis for copy number variants in the following genes: HBA1; HBA2; (and HBB; HBG1; HBG2 if appropriate)
• R362.1 - HbS variant targeted mutation testing by pyrosequencing or ARMS in individuals with a likely sickle carrier genotype (in practice this is only used to confirm the sickle genotype in the parents of a fetus undergoing prenatal diagnosis for sickle cell anaemia)

Public Health England (PHE) have published comprehensive guidelines on when to refer for molecular testing (see Sickle Cell and Thalassaemia Antenatal Laboratory Handbook. This process is designed to identify couples at risk of severe haemoglobinopathies so that prenatal diagnosis (PND) can be performed in a timely fashion.

Targeted analysis for known / previously reported familial variants

• Prenatal diagnosis is available for severe conditions (R240).
• Carrier testing in relatives of clinically affected patients with an autosomal recessive condition (mutation known) (R244).
• Segregation studies in affected family members to aid variant interpretation (R375)
• Diagnostic confirmation in individuals at risk of inheriting a previously reported familial pathogenic variant and clinically suspected of having the familial condition (R240)
• Presymptomatic testing in clinically unaffected family members at risk of inheriting a previously reported familial pathogenic variant (R242)
  

Target reporting times

• Diagnostic test target reporting time is 42 days.
• Urgent diagnostic (typically neonatal testing) or urgent antenatal carrier testing target reporting time is 21 days.
• Prenatal diagnosis where one or both parental haemoglobinopathy mutations are unknown in advance has a target of reporting within 21 days (although most referrals are reported in considerably less time).
• Prenatal diagnosis when both familial variants are known in advance target reporting time is 3 days.

For other reporting times, please see turnaround times.

Sample requirements and referral information

All referrals should be accompanied by a completed diagnostic or carrier testing pre-referral form or a prenatal diagnosis pre-referral form. See referral forms and specimen requirements and referring samples.

Clinical guidance and advice is available to referring clinicians from:

Dr Noémi Roy
Consultant Haematologist
John Radcliffe Hospital

Email: noemi.roy@ouh.nhs.uk

Requesting specialties

• Haematology
• Fetal Medicine
• Clinical Genetics

Contact details

For enquiries relating to referral of samples for DNA testing please contact:

Red Cell Team scientists: molecularhaem.oxfordgenetics@ouh.nhs.uk
Tel: 01865 572769

For Haemoglobinopathy screening queries please contact:

Haemoglobinopathy screening laboratory: hbopathy.screening@ouh.nhs.uk
Tel: 01865 572768

For questions on screening policy or interpretation of results please go to www.sct.screening.nhs.uk. If your query is not covered further advice is available.

See the sickle cell and thalassaemia screening programme lab support service or:

Email: SCTlab.support@ouh.nhs.uk
Tel: 01865 572767

For other contact details, see contact us.

Price list for non NHSE referrals

Price list for rare disease services (pdf)