Proband is a 25-year-old male with phenotype of thalassemia intermedia. He received 4 units of blood transfusion so far. He is anemic with enlarged spleen, and ferritin was 630 ng/ml. Hematological indices are given in Table 1. Family genetic screening revealed that father was carrier for IVS1-5(G > C) while mother carried normal HBB gene alleles (Fig. 1). Sanger sequencing of the HBB gene further confirmed that proband was homozygous for IVS1-5(G > C) mutation, and only father was heterozygous for IVS1-5(G > C) mutation (Fig. 2). Parental identity was confirmed by VNTR D1S80 allele testing in the trio (Fig. 3). D1S80 is located in chromosome 1 and consists of 16 bp long tandem repeats. D1S80 allele varies from 224 to 656 bp in length. VNTR analysis proved that parents of case 1 were actually the biological parents of the proband. Thus, this IVS1-5(G > C) occurred de novo in the proband leading to thalassemia major disorder (Fig. 4).

Gel image of ARMS PCR for IVS1-5(G > C) mutation for Case 1 family is shown. Lane 1,2—proband; Lane 3,4—father; Lane 5,6—mother; N—normal allele ‘G’; and M—mutant allele ‘C.’ Proband is found to be IVS1-5CC, father is IVS1-5GC, and mother is IVS1-5GG. Arrow mark indicates position of 285 bp DNA band corresponding to IVS1-5(G > C) mutation. Similar result was obtained for Case 2 and Case 3 family

A: DNA sequence alignment in ClustalW for case 1 family is shown. B: Electrochromatogram of the case 1 family showing the IVS1-5G > C nucleotide change. Similar result was obtained for Case 2 and Case 3 family

Inheritance testing. D1S80 is a variable number tandem repeat (VNTR) located in chromosome 1 and consists of 16-bp long repeat unit. D1S80 allele varies from 224 to 656 bp in length. Lane 1,3,5—maternal DNA; Lane 2, 4, 6—proband DNA; Lane 7—paternal DNA (case1), and Lane M—1 kb DNA ladder. Common allele for D1S80 is present in both mother and child. Even paternity is proved as father, and child has a common allele

Haplotype comparison of proband with mother. Figure shows SNPs close to IVS1-5(G > C) mutation. Both mother and proband carry the same haplotype except for IVS1-5(G > C) mutation. Same was found in all the three cases. SNPs’ position and ‘rs’ numbers are labeled above the chromatograms. Figure shows data from Case 1 family, similar result was obtained for case 2 and case 3 family

Proband is a 17-year-old male, and there were no signs of pallor. He never received transfusions, ferritin was 182 ng/ml, and spleen was also within normal limits till the day of sample collection. Proband had high level of fetal hemoglobin (Table 1) and carried HPFH-3 (Fig. 5) mutation which may be responsible for HPFH phenotype but as the proband was homozygous for IVS1-5(G > C), his RBC was microcytic-like thalassemia major. Family screening revealed that father had beta-thalassemia trait but mother was absolutely normal. Further genetic testing revealed that proband was homozygous for IVS1-5(G > C) mutation but mother carried normal allele for the HBB gene by ARMS and Sanger sequencing (Fig. 2). As father had expired, his sample was not available for genetic testing, however, his hemoglobin analysis report was present which showed that father was beta-thalassemia trait. Proband inherited one mutant allele from his father but acquired de novo mutation in the other allele. Maternity was confirmed by VNTR D1S80 allele testing (Fig. 3).

Gel image for HPFH-3 mutation analysis. Lane 1—1 kb DNA ladder, Lane 2 and 3—family 2; Lane 4–5—family 3; and Lane 6,7,8—family 1. Mutant allele band 607 bp is present for both families 2 and 3 but absent for family 1. Normal allele band of 841 bp is present in all the three families. Thus, families 2 and 3 are heterozygous for HPFH-3 mutation

Proband is a 12-year-old female presentation with thalassemia major phenotype. She received more than 100 transfusions, and her spleen was removed. Her serum ferritin was 1131 ng/ml and received oral iron chelating drug deferasirox. Table 1 gives detail of hematological indices and hemoglobin analysis. Family screening showed that father was thalassemia trait but mother was normal. Father sample was not available for genetic testing. DNA analysis of proband and mother revealed that proband was homozygous for IVS1-5(G > C) mutation but mother carried normal allele. Both proband and mother had HPFH-3 mutation (Fig. 5). Sanger sequencing further confirmed that proband was homozygous for IVS1-5(G > C), and mother was normal. Maternity was confirmed by VNTR D1S80 allele, proband and mother shared a common allele for D1S80 VNTR (Fig. 3). Thus, proband acquired IVS1-5(G > C) homozygosity by de novo.