Extreme Genotype/Phenotype Heterogeneity of Double Heterozygous Sickle β-Thalassemia in a Family: Implications in Antenatal Diagnosis

Dear Sir,

A 12-year-old boy from North India with transfusion-dependent anemia for 3 years presented with recurrent episodes of chest and abdominal pain. Examination revealed mild pallor, icterus, and splenomegaly (12 cm below costal margin). No positive family history was found. On investigation, hemoglobin (Hb) was 83 g/L with reticulocytosis (8.0%). Peripheral blood film revealed hypochromic microcytosis with few target cells and drepanocytes. Cation-exchange high-performance liquid chromatography (CE-HPLC, Variant II instrument, Beta Thal Short program, BioRad Laboratories, USA) revealed a double heterozygous state for HbS + β-thalassemia with HbS (61.2%), HbF (31%) with HbA2 (4.8%), and near absence of HbA₀. Hemoglobin electrophoresis (pH-8.4) showed slow-migrating bands of HbF and HbS. Laboratory findings are summarized in Table 1. Hb-HPLCs of his father and two sisters showed β-thalassemia trait (Table 1, Fig. 1), while his mother’s Hb-HPLC also showed double heterozygous HbS + β-thalassemia (HbS 62.1%, HbF 8.3%, HbA2 6.5%) but with 20.1% HbA₀. The asymptomatic mother was 8 weeks pregnant and had mild pallor. In a detailed and leading history she denied any prior anemia, blood transfusions (including her three prior pregnancies), jaundice, chronic/episodic pain, leg ulcers or chronic medication use. Physical examination did not reveal organomegaly.

Table 1.

Laboratory findings in the family members

Parameters	Index case	Mother	Father	Sister 1	Sister 2
Age (years)/Sex	12/M	34/F	36/M	5/F	4/F
Hemoglobin (g/l)	83	100	101	109	101
RBC (× 1012/l)	3.8	4.18	5.77	5.07	4.81
MCV (fl)	69.4	72.6	57.01	66.7	64.5
MCH (pg)	21.9	23.9	17.5	21.5	21.1
MCHC (gm %)	31.5	33	30.6	32.2	32.7
RDW (%)	21	15.4	21.4	14.5	15.1
Reticulocyte (%)	8	5	6	2.0	1.5
HbA0/S/F/A2 (%)	1.9/61.2/31/4.8	20.1/62.1/8.3/6.5	83.2/0/0.3/5	79.1/0/2.6/5.9	80.2/0/3.2/5.8
Test for sickling	Positive	Positive	Negative	Negative	Negative
HBB mutation	HBB:c.126_129delCTTT [Fr 41/42 (-CTTT)]	HBB:c.-138C > T [-88(C > T)]	HBB:c.126_129delCTTT [Fr 41/42 (-CTTT)]	ND	ND
Multiplex PCRs for α-thalassemia and α-triplications	Negative	Negative	ND	ND	ND
Diagnosis	Sickle β-thalassemia	Sickle β-thalassemia	β-thalassemia trait	β-thalassemia trait	β-thalassemia trait

F female; M male; ND not done

Fig. 1.

CE-HPLC chromatograms of the five family members and a pedigree chart

Since both the mother and son showed double heterozygous HbS + β-thalassemia but with completely divergent phenotypes, genetic studies were undertaken. Both were heterozygous for the sickle cell mutation [HBB:c.20A > T] by PCR–RFLP with DdeI. Amplification-refractory mutation system PCR testing in the patient and his father revealed heterozygosity for HBB:c.126_129delCTTT [Fr 41/42 (–CTTT)]. The mother was heterozygous for HBB:c.−138C > T [−88(C > T)]. Multiplex GAP PCR for α-globin gene deletions [−α^3.7, −α^4.2, –^SEA, –^MED, –^SA, –^THAI, –^FIL and –(α) ^20.5] and gene triplications ααα^anti−3.7 and ααα^anti−4.2 were negative in the mother and son. Xmn-1^Gγ polymorphism by PCR–RFLP displayed a +/− pattern in both. Subsequently, chorionic villus biopsy, revealed that the fetus was compound heterozygous for severe β⁰ Fr 41/42 (–CTTT) and mild β⁺⁺ −88(C > T) mutation. The family was counseled about a likely transfusion-dependant thalassemia phenotype in the fetus and termination of the pregnancy.

Sickle-beta thalassemia, despite its monogenic origins, can display genotype–phenotype heterogeneity as shown by our cases. Major implicating factors are the type of HBB allele (β⁺⁺, β⁺or β⁰), the HbS haplotype, co-inheritance of alpha thalassemia (ameliorating) or alpha gene triplication (aggravating), and factors modulating HbF production (e.g., Xmn1^Gγ)[1]. In our mother-son duo, the co-inheritance of HbS, alpha-globin status, and Xmn1^Gγ status were identical. The clinical heterogeneity was attributable to the different HBB mutations which led to a significant difference in the production of HbA₀.

A similar observation was documented by Serjeant et al. who demonstrated that Jamaican Sβ patients with higher HbA₀ (Sβ⁺ cases) performed clinically better compared to lower HbA₀ (Sβ⁰-thalassemia). They also documented that the −88(C > T) mutation was associated with higher hemoglobin and preserved red blood cell indices within the Sβ⁺ group [2]. The −88 (C > T) mutation has been reported with high frequency in Jat Sikhs. In the homozygous state, it has a milder clinical course; however, the compound heterozygous states can give rise to thalassemia intermedia/thalassemia major phenotypes [3]. Atweh et al reported a case in which co-inheritance of the milder ‘Cap’ site mutation did not prevent a severe sickling phenotype. They postulated that this was due to reduced hemolysis that leads to increased red cell mass and blood viscosity which are risk factors for vaso-occlusive crises [4]. Molecular diversity of HbS-β thalassemia in western India has been studied by Mukherjee et al. in a limited cohort (n = 19) with IVS 1–5 (G > C) 52.7%, Codon 15 (G > A) 31.5%, Codon 30 (G > C) 10.5% and Codon 8/9 (+ G) 5.3% [5].

In conclusion, this family, with the apparently asymptomatic HbS + β-thalassemia mother and her severely affected son, illustrates the major influence that the degree of β-globin underproduction exerts on the phenotype of HbS + β-thalassemia.

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None of the authors have any conflict(s) of interest.

Human Participants and/or Animals

This descriptive report involved no human or animal experimentation. All laboratory testing procedures followed were by the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.

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All subjects gave informed consent.