A rare gene variation cap +1 (A>C) (HBB: c. −50A>C) associated with codon 5 (‐CT) (HBB: c.17_18delCT) mutation in Syrian family

ABSTRACT Background CAP+1 [A>C] (HBB:c.‐50A>C) is a rare silent β‐thalassemia (β‐thal) mutation. Carrier individuals of this mutation show borderline hemoglobin (Hb), mean corpuscular volume (MCV) and Hb A2 levels. This mutation was previously reported in combination with different β‐thalassemia mutations, leading to variable phenotypes. Case presentation Here, we describe for the first time the combination of silent CAP+1 [A>C] (HBB:c.‐50A>C) mutation with β0 codon 5 [‐CT] (HBB:c.17_18delCT) mutation in a Syrian proband, leading to beta thalassemia intermedia (TI). Conclusions The compound heterozygotes of the silent CAP+1 (A>C) together with another severe beta gene mutation, are phenotypically severe enough to present at an early age and require appropriate therapeutic modalities.


| INTRODUCTION
Beta thalassemia (β-thal) is the most common blood disease in Syria Murad et al., 2014). It described by reduction or absence of beta globin chain synthesis (Galanello & Origa, 2010). Most beta thalassemia mutations are caused by point mutations, insertions or small deletions within the coding regions, the exon-intron junctions and substitution within the TATA box of the β-globin gene promoter (Cao et al., 2011;Ropero et al., 2017). There are certain "silent" β-thalassemia mutations in which redcell indices are normal and Hb A2 is not raised in carriers (Aslan, 2016;Vinciguerra et al., 2018). CAP+1 (A>C) transversion is one of these mutations which was previously found in Asian Indians (Wong et al., 1987). This mutation was described in a homozygous individual who presented a β-thalassemia trait with a Hb A2 level of 4.1%. While, presence of this variant with different β-thalassemia mutations as a compound heterozygous state, showed a heterogeneous clinical presentation from nontransfusion-dependent to transfusion-dependent thalassemia patients (Garewal et al., 2007).

| Patient
A 12-year-old female Syrian proband, was referred to our center AECS-in Damascus for confirmation of her status for β-thalassemia. The parents were nonconsanguineous. Her history revealed pallor and anemia. The electropherogram revealed a low level of (Hb A1) 52.6%, high level of (Hb F) 42.5% and (Hb A2) was 4.9%. The proband received erythrocyte transfusions at the age of 2 years. She underwent splenectomy at 8 years. She requires blood transfusion every 40 days. Her father had a classic clinical picture of β-thalassemia trait. Her mother had normal indices with a borderline of (Hb A2) 3.5%. Both parents had no splenomegaly and hepatomegaly and had never been transfused. All hematological data have been summarized in Table 1.

| Analysis of hematological parameters and hemoglobin components
An automated differential cell counter (ABX Micros ES60; HORIBA ABX SAS, Montpellier, France) was used to obtain the hematological parameters of the parents and proband. A Capillarys 2 system (Sebia, Lisses, France) was used to analyse Hemoglobin electrophoresis (Hb).

| DNA sample preparation & Sequencing
QIAamp DNA Blood Mini kit (Qiagen, Germany) was used to isolate the genomic DNA from peripheral the blood of both parents and proband according to the manufacturer's instructions. Purified gDNA was run on a 0.8% agarose gel.
Polymerase chain reaction (PCR) method was used to determine the genotype of HBB gene. The appropriate primers used for exon 1, exon 2 and exon 3 of β-globin gene included the promoter, first intron, 5` and 3` untranslated region (UTR) sequences as previously reported (Yassin et al., 2013). The entire human HBB gene was sequenced using an ABI PRISM 310-DNA Analyzer (Applied Biosystem, Foster City, CA, USA) as reported previously (Murad et al., , 2018.

| Reverse hybridization assay
Reverse hybridization assay (ViennaLab Diagnostics Gmb Vienna, Austria) used to to detect α-globin gene deletions. To investigate of the increased level of Hb F in the proband, PCR-RFLP (restriction fragment length polymorphism) was used for the detection of -158Gγ (C→T) Xmn1 polymorphism using the Xmn1 restriction enzyme with specific primers (Rujito et al., 2016).

| DISCUSSION
CAP+1 (A>C) is known as a silent HBB variation that was first reported in an Asian Indian patient (Wong et al., 1987). The carriers of this variation CAP+1 (A>C) have been shown to have borderline hematologic profiles (Garewal et al., 2007;Rangan et al., 2011). In our case, the mother was T A B L E 1 The hematological and molecular data of the family. found to be carrier of the CAP+1 [A>C] variation, and her hematologic parameters were (MCV) 77.1 fl, (MCH) 24 pg, and Hb A2 level was 3.5%. Compound heterozygosity of the silent CAP+1 (A>C) variation with different β-thalassemia mutations showed a heterogeneous clinical presentation (Garewal et al., 2007). The combination of the silent CAP+1 (A>C) variation with different mutations has been previously reported. The combination of CAP+1 (A>C) variation with β 0 IVS-I-5 (G>C) mutation was relatively leading at 85% of cases to the beta thalassemia major (TM). In the other hand, 50% of cases with CAP+1 (A>C) / codons 41/42 (-TTCT) mutation had beta thalassemia intermedia (TI), while, the remains combinations with other mutations had beta thalassemia major (TM). Our proband had silent CAP+1 [A>C] variation and β 0 codon 5 [-CT] mutation in a heterozygous state. We propose that, this combination leads to beta thalassemia intermedia (TI), wherein, the proband requires blood transfusion every 40 days.

Parameters
Complete inhibition of β-chain synthesis by the β 0 Codon 5 [-CT] mutation was known by the creation of a premature termination signal at codon 21 (Kollia et al., 1989). In our case, the combination of a silent HBB mutation CAP+1 (A>C) with β 0 codon 5 [-CT] mutation was reported for the first time, and it led to transfusion-dependent thalassemia in the patient.

| CONCLUSIONS
In this case, we report for the first time the silent CAP+1 (A>C) (HBB: c.-50A<C) variation in a Syrian family. The compound heterozygotes of the silent CAP+1 (A>C) variation and another severe beta gene mutation, are phenotypically severe enough to present at an early age and require appropriate therapeutic modalities.