Premarital Counseling on the Alpha Thalassemia Allele HBA2 :c.*94A>G

: The mutation HBA2 :c.*94A>G (AATAAA>AATAAG; rs63751269) is a 3 (cid:48) -UTR (3 prime untranslated region) single-nucleotide substitution in the polyadenylation (PA) signal of HBA2 ( α PA:A → G ). This pathogenic (CADD score, 14.92) variant is sporadic in the Arabian Peninsula. It results in inefﬁcient mRNA processing, transcription termination, and possibly using an alternate cryptic downstream polyadenylation signal. As a result, the allele α T (or α T -Saudi) poses challenges in premarital counseling with respect to fetal risk of hemoglobin H disease. Homozygous HBA2 :c.*94A>G ( α T α / α T α ) results in moderate-to-severe microcytosis (mean red cell volume, MCV, 55 to 65 fL), reﬂecting markedly impaired hemoglobin synthesis (hemoglobin H disease). Homozy-gous rightward − α 3.7 (a 3804-neocleotide deletion allele, NM_000517.4:c.[-2_-3delAC; − α 3.7 ]), on the other hand, results in mild microcytosis (MCV, 70 to 75 fL, alpha-thalassemia trait). Thus, HBA2 :c.*94A>G is more damaging than − α 3.7 . Consistently, the value of MCV in compound heterozygosity, HBA2 :c.*94A>G and − α 3.7 , is 65 to 70 fL. We report here a healthy couple who presented for premarital counseling on their hemoglobinopathy. The man has homozygous HBA2 :c.*94A>G ( α T α / α T α ), and the woman has compound heterozygous ( − α 3.7 / α T α , also annotated as: − 3.7 α / α T α ). As a result, the genotype of their offspring would be that of the father ( α T α / α T α ) or the mother ( − α 3.7 / α T α ). The counseling was mainly based on the benign phenotypes of the parents. As both were asymptomatic and their anemia was clinically insigniﬁcant, they proceeded with the marriage.


Introduction
Alpha thalassemia (MIM#604131) is an inherited blood condition in which one or more of the HBA2 (hemoglobin-alpha locus 2, MIM#141850) or HBA1 (hemoglobin-alpha locus 1, MIM#141800) genes are defective.The normal genotype consists of two copies of HBA2 and two copies of HBA1, (one copy each on the two homologous chromosome 16).HBA2 and HBA1 share the same coding sequence but differ in the introns as well as the 5 and 3 UTR, thereby affecting gene expression [1].For example, HBA2 encodes 2-3 times more protein than HBA1.
The four forms of alpha thalassemia are as follows.
(1) The silent carrier (three functional genes), causing a slightly low MCV (75 fL to 80 fL).(2) The alpha thalassemia trait (two functional genes), causing a more pronounced microcytosis (MCV, 70 ± 5 fL).When HBA2 and HBA1 on a single chromosome are deleted (or inactivated) the designation is cis deletion (α • or null allele).This form is prevalent in Southeast Asia and the Mediterranean population, and carries fetal risk for severe alpha-thalassemia.When HBA2 or HBA1 on a single chromosome is deleted, the designation is trans deletion (α + ).There are two types of deletional alpha-thalassemia.The 3.7 kb deletion (rightward type; −α 3.7 ; HBVAR#1076) is found in the Arabian Peninsula, Africa, and the Mediterranean region (illustrated in Figure 1B).The 4.2 kb deletion (leftward type, −α 4.2 ) is found in Southeast Asia and Pacific Islands.(3) Hemoglobin H disease (one functional gene, e.g., offspring of one parent with α • thalassemia trait and one with silent carrier).Importantly, the unpaired β-chain aggregates, known as hemoglobin H (not to be confused with 'hemoglobin H disease'), are more soluble than the unpaired α-chain aggregates found in β-thalassemia.Thus, individuals with hemoglobin H disease have moderate rather than severe anemia.(4) Hemoglobin Bart's hydrops fetalis syndrome, i.e., four defective genes (e.g., in the offspring of parents with the α • thalassemia trait), is incompatible with life [2].The prevalence of α + thalassemia trait is high in the UAE.In one study, 49% of the newborns had HBA2 or HBA1 defects; the majority were −α 3.7 [3].In other studies, the prevalence of α + thalassemia trait ranged from 15% to 20% [4,5].Thus, −α 3.7 is the most common variant in the region, while the non-deletional variant HBA2:c.*94A>G is less frequent [6][7][8].
Premarital screening and counseling programs are well established in the UAE.They mainly aim to prevent beta-thalassemia major and sickle cell disease.Less frequently, α • thalassemia and HBA2:c.*94A>Galso produce challenges with respect to assessing fetal risk.There is no fetal risk with respect to α + thalassemia, which is most common.We present here a healthy couple with moderate-to-severe microcytosis associated with alpha-thalassemia.They came for counseling on fetal risk of hemoglobin H disease.

Discussion
Polyadenylation, or the addition of a poly(A)-tail, is an essential post-transcriptional modification of nearly all eukaryotic messenger RNAs (mRNA) [9][10][11].This step is vital for the stability of mRNA in the cytoplasm.In humans, the highly conserved sequence motif AATAAA, the polyadenylation site, ensures efficient cleavage of the primary transcript and subsequent addition of the poly(A)-tail [12].Variations in this signal sequence are rare and are typically associated with various disorders or the use of alternate polyadenylation sites [13].In HBA2, the canonical signal sequence is encoded in the third exon of the gene, 89 bases downstream of the translation stop codon (Figure 1).The mutation HBA2:c.*94A>Gresults in a change in the transcribed mRNA from AAUAAA to AAUAAG, resulting in inefficient mRNA processing, and in some instances, the use of an alternate cryptic downstream polyadenylation signal [10], as shown in Figure 1A.This variation thus has significant impact on the levels of the mature mRNA and translated gene producthemoglobin α protein-produced from HBA2.Of note, HBA2 encodes 2-3 times more protein than HBA1 [14], which explains the greater impact of HBA2 on alpha-thalassemia.
The phenotypes and genetic studies were all necessary for assessing the fetal risk of 'hemoglobin H disease' or 'hemoglobin Bart's hydrops fetalis syndrome' in the studied couple.The results showed the predicted genotypes of the offspring were either (α T α/α T α) for the father or (−α 3.7 /α T α) for the mother.As both were healthy and their anemia was clinically insignificant, they proceeded with the marriage.The determined genotypes are also helpful for other family members [15][16][17].

Figure 1 .
Figure 1.(A) Human HBA2 gene located on chromosome 16 (NCBI RefSeq Accession: NC_000016.10)between 172876-173710.The three exons of HBA2 are shown as green blocks.Light green in exon 1 represents 5 UTR, and in exon 3, it represents the 3 UTR.The polyadenylation signal (AATAAA) is located between 173689 and 173694.In the α T variant (HBA2:c.*94A>G;rs63751269), this sequence is altered to AATAAG.(B) Human HBA2 and HBA1 genes are located on human chromosome 16 (NCBI RefSeq Accession: NC_000016.10)between 172876-173710 and 176680-177522, respectively.The three exons of HBA2 are shown as orange blocks, while the three exons of HBA1 are shown as yellows blocks.The −α 3.7 variants involves a deletion of 3.7 kilobases as indicated by the red dashed box.(C) Genotypes of the studied couple.

Table 1 .
Characteristics of α-thalassemia in the studied couple.