Screening and Diagnosis of Rare Thalassemia Variants: Is Third-Generation Sequencing Enough?

CONTEXT.­: Rare thalassemia subtypes are often undiagnosed because conventional testing methods can only identify 23 common types of α- and ß-thalassemia. OBJECTIVE.­: To assess a comprehensive approach for the screening and diagnosis of rare thalassemia. DESIGN.­: The study cohort included 72 individuals with suspected rare thalassemia variants. Screening was conducted by next-generation sequencing (NGS) combined with third-generation sequencing (TGS) and chromosomal microarray analysis (CMA)/copy number variation sequencing. RESULTS.­: Of the 72 individuals with suspected rare thalassemia, 49 had rare α- or ß-gene variants. NGS combined with gap polymerase chain reaction detected a total of 42 cases, resulting in a positive detection rate of 58.3%. Additionally, 4 α-globin genetic deletions were identified by TGS, which increased the variant detection rate by 5.6%. Two samples with a microdeletion of chromosome 16 or 11 were detected by CMA, which increased the detection rate by 2.8%. For one sample, reanalysis of the NGS and TGS data confirmed the presence of the ß41-42/ßN and ßN/ßN mosaic. The HBB:c.315 + 2delT mutation was initially reported in Guangdong Province, China. Two HBB gene mutations (HBB:c.315 + 5G>C and HBB:c.295G>A) and 4 rare HBA gene deletions (-11.1, -α27.6, -α2.4, and -α21.9) were initially identified in the Zhonshan region. The hematologic phenotypes of all rare cases in this study were clarified. CONCLUSIONS.­: Rare thalassemia variants are more common than previously thought. Despite advancements in TGS, there is still no foolproof method for detection of all types of thalassemia. Thus, a comprehensive approach is necessary for accurate screening and diagnosis of rare thalassemia variants.

Fetal Hemoglobin H Hydrops Fetalis: Another Three Case Reports.

We report three cases of fetalis hydrops associated with nondeletional α-thalassemia. Two cases were caused by hemoglobin (Hb) H-Quong Sz disease, and one caused by homozygous Hb Constant Spring. Fetal hydrops occurred in the late second trimester in all three cases. Our study indicates that for pregnancies at risk for fetal nondeletional Hb H disease, strict ultrasound follow-up is particularly important. Even without techniques of intrauterine transfusion treatment, early prenatal diagnosis can enable parents to make timely decisions.

Can cell-free DNA testing be used in pregnancies with isolated fetal omphalocele? Preliminary evidence from cytogenetic results of prenatal cases.

Objective: To evaluate whether cell-free DNA (cfDNA) testing could replace an invasive procedure in pregnancies with isolated fetal omphalocele.Study design: This was a retrospective study of all pregnancies with sonographically detected fetal omphalocele at three tertiary referral centers between 2012 and 2016. Invasive diagnostic testing was performed for genetic investigations using conventional karyotyping or chromosomal microarray. cfDNA testing was assumed to be offered to patients with isolated fetal omphalocele for screening for common aneuploidies.Results: Invasive genetic testing was performed in a total of 107 pregnancies with a fetal omphalocele. Abnormal karyotype was found in 66% (31/47) of nonisolated omphalocele cases and in 1.7% (1/60) of isolated omphalocele cases. No pathogenic copy number variations (CNVs) were detected in 59 cases with isolated omphalocele and normal karyotype. If cfDNA screening was used in cases with isolated omphalocele, the affected fetus with trisomy 18 would be detected, and no rare chromosomal aberrations or submicroscopic pathogenic CNVs would be missed.Conclusions: cfDNA testing could be recommended for prenatal genetic evaluation in pregnancies with isolated fetal omphalocele after thorough pretest counseling.Key Message: A very low percentage of aneuploidies and rare chromosomal/subchromosomal abnormalities are found in prenatal cases of isolated omphalocele. It seems that for pregnancies with isolated omphalocele, cfDNA testing represents an alternative for patients who choose to continue the pregnancies and are reluctant to undertake invasive diagnostic testing.

Chromosomal microarray analysis in pregnancies at risk for a molecular disorder.

Objective: The aim of this study was to evaluate the utility of chromosomal microarray (CMA) in patients who were solely referred for molecular diagnosis.Methods: During a 2-year period, CMA was the patients' choice, whether to opt for it or not, for those at risk for fetal hemoglobin Bart's disease or ß-thalassemia major who were referred for invasive prenatal diagnosis and had a normal fetal genotype. CytoScan 750 K array (Affymetrix Inc., Santa Clara, CA) was used for CMA. The CMA testing results were collected.Results: There were 184 patients, who had a normal genotypic result, opting CMA testing without an obvious indication for fetal karyotyping. The median maternal age was 29 years (range, 17-34); the median gestational age was 13 weeks (range, 11-20). In two out of 184 (1.1%) cases unexpected de novo pathogenic microdeletions were found: a 3.2 Mb 22q11.21 microdeletion and a 0.8 Mb 16p11.2 microdeletion.Conclusions: Pregnancies at risk for thalassemia can also benefit from performing CMA. This information might be a part of the contents in comprehensive pretest counseling for those who are referred for diagnostic testing due to a molecular disorder.

δ-Thalassemia with Complete Absence of Hb A2 in a Chinese Family.

A Chinese family with δ-thalassemia (δ-thal) was found, in which the daughter is homozygous for δ-thal (HBD: c.-127T>C) with complete deficiency of Hb A2 and the mother is a heterozygote with low level of Hb A2. The father, however, is a heterozygote with a normal Hb A2 value due to coinheritance of a ß-thalassemia (ß-thal). Although no abnormal clinical or hematological findings were noted in the individuals with δ-thal, one should keep in mind that ß-thal can be missed during routine preliminary screening when ß-thal and δ-thal coexist in a subject.

Newborn screening for Hb H disease by determination of Hb Bart's using the Sebia capillary electrophoresis system in southern China.

Hb H (ß4) disease is an inherited hemoglobin (Hb) defect in which three of the four α-globin genes are deleted or dysfunctional. The clinical manifestations vary widely from mild asymptomatic anemia to a severely anemic state. Recent literature suggests that Hb H disease is not as benign a disorder as previously thought. Newborn screening for Hb H disease is especially appealing because the screening test is based on the detection of Hb Bart's (γ4) that is only possible within the newborn period. In a 2-year period of newborn screening, 18 babies were found to have Hb H disease in a total of 9490 newborns. The overall prevalence for Hb H disease among all newborns in southern China is approximately 1 in 500. The correct diagnosis would allow affected infants to be properly cared for and reduce mortality rate.

Codon 62 (GTG>GCG, Val→Ala) (α1) (HBA1: c.188T>C) causing nondeletional α-thalassemia in a Chinese family.

We report a novel α-globin gene point mutation detected during newborn screening for hemoglobinopathies. Sequence analyses identified a GTG>GCG substitution at codon 62 of the α1-globin gene. This mutation causes a silent α-thalassemia (α-thal).

Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] compromises neonatal screening for α-thalassemia with the Sebia Capillarys2 electrophoresis system.

We describe a Chinese newborn who was assumed to have α(0)-thalassemia (α(0)-thal) by determining the amount of Hb Bart's (γ4) in the cord blood, but was later shown to have only α(+)-thal. Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] was mistaken for Hb Bart's as both hemoglobin (Hb) variants have the same mobility.

A novel case of Hb Phnom Penh: codons 117/118 (+ATC) as a cause of α+ -thalassemia.

We report a novel case of Hb Phnom Penh [α117(GH5)Phe-Ile-α118(H1)Thr (α1)] detected through cord blood screening for hemoglobinopathies. Sequence analyses identified this in-frame mutation at codons 117/118 (+ATC) in exon 3 of the α1-globin gene. This mutation causes a silent α-thalassemia (α-thal).

Screening for common nondeletional α-thalassemias in Chinese newborns by determination of Hb Bart's using the Sebia Capillarys 2 electrophoresis system.

The interaction of the nondeletional α-thalassemia (α-thal) mutations with the Southeast Asian double α-globin gene deletion results in nondeletional Hb H (ß4) disease. Hb Constant Spring (Hb CS, α142, TAA>CAA at α2) and Hb Quong Sze [Hb QS, α125, CTG>CCG (α2)] are the most common nondeletional α-thalassemias in the Chinese population. These α-globin structural variants are unstable and undetectable by routine hemoglobin (Hb) electrophoresis. The amount of Hb Bart's (γ4) in the cord blood of newborns correlates with the number of α-globin genes that are deleted. We determined the quantity of Hb Bart's in cord blood at birth with the Sebia CapillaryS electrophoresis system. Using Hb Bart's levels at 0.1-2.5% as a cut-off range for nondeletional α-thal diagnosis, we detected 154 individuals in 6,525 newborns. Of the 154 samples, 12 were found to be Hb CS carriers, 10 Hb QS carriers, and one Hb Westmead [α122, CAC>CAG (α2)] carrier. We present the first report of the prevalence of Hb QS in our population.

A novel α-thalassemia frameshift mutation: codon 8 (-C).

We report a novel α-thalassemia (α-thal) point mutation detected during newborn screening for hemoglobinopathies. Sequence analyses identified a frameshift mutation at codon 8 (-C) in exon 1 of the α2-globin gene. This mutation causes an α(+)-thal phenotype.