The Masked Thalassemia: A Rare Case of a Patient with Normal HbA2 Levels, β-Thalassemia Pathogenic Variant (CD39 C>T), and a Novel δ-Globin Gene Deletion

Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF level. On the other hand carriers of severe alpha-thalassemia also have similar CBC parameters to that of β-thalassemia with normal HbA2 level. Co-presence of mutations in the β-globin and delta-globin genes (point mutations or deletions) usually give normal HbA2 and elevated HbF level. We report a β-thal carrier with normal level of HbA2 and increased level of HbF who had a point mutation in CD39 on the beta-globin gene and a point mutation in CD27 on the δ-globin gene named Hb-Yialousa. Materials & Methods: An individual with low hematological indices, normal HbA2 and elevated HbF was referred to our center as routine premarital screening program. Mutations in the β-globin and δ-globin genes were screened using ARMS and sequencing methods. Results: The mutation in β- and δ-globin genes were identified as CD39 and CD27 (HbYialousa) respectively. No point mutation or deletion in α-globin gene was identified. Discussion: We showed that normal HBA2 with elevated HbF level is due to co-inheritance of delta-globin gene mutation with mutation in the β-globin gene. When screening for β-thalassemia, one has to either rule out presence of α-globin gene mutation of mutation in the delta-globin gene.

Genetic insights into the clinical diversity of beta thalassaemia.

Beta thalassaemia is one of the most common inherited haemoglobinopathies, characterised by reduced or absent production of the beta globin chain. In India, the carrier frequency of thalassaemia is estimated to be 3-4%. The prevention of Beta thalassaemia is the best strategy, and this can be achieved through carrier screening and prenatal diagnosis. Carriers of beta thalassaemia can be easily identified using haematological parameters such as complete blood count and High Performance Liquid Chromatography (HPLC) for haemoglobin analysis. The characteristic findings observed in thalassaemia carriers include microcytosis, hypochromia, with a Mean Corpuscular Volume (MCV) of less than 80 fL and Mean Corpuscular Haemoglobin (MCH) of less than 28 pg. They also present with elevated levels of HBA2 (α2δ2) ≥3.5%. Carrier screening for beta thalassaemia primarily relies on the observation of elevated HbA2 levels. However, in rare cases, some carriers can have normal HbA2 levels, leading to false-negative screening results. In a case involving a married couple who underwent routine preconceptional screening by complete blood count and HPLC for thalassaemia screening, the male partner had elevated HbA2 levels (5.2%), while the female partner had normal HbA2 levels (1.6%). Molecular testing revealed that the male partner was heterozygous for the Intervening Sequence (IVS) 1-5 (G>C) mutation, while the female partner was found to be heterozygous for the CD41-42 (-CTTT) mutation. It is important to consider molecular testing of the HBB gene in couples, even if one partner is a carrier and the other partner has normal or borderline HbA2 levels.

BackgroundAlport syndrome is an inherited renal disease caused by mutations in COL4A3, COL4A4, or COL4A5 genes. Coexisting mutations in either two of the three genes in Alport patients have been reported recently. However, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in X‐linked Alport syndrome (XLAS) patients is unclear.MethodsUsing targeted next‐generation sequencing, six unrelated Chinese children were identified to have a combination of a pathogenic variant in COL4A5 and a heterozygous mutation in COL4A3 or COL4A4. They were three males and three females. Another three XLAS males each with only one pathogenic variant in COL4A5 were included. The clinical data were analyzed and compared between the males in two groups (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5).ResultsPatients with XLAS who also had heterozygous pathogenic COL4A3 or COL4A4 variants accounted for 1% of Alport syndrome. In this study, three children showed coexisting pathogenic variants in COL4A5 and COL4A3. Two children showed pathogenic variants in COL4A5 and COL4A4. One child had pathogenic variants in the three COL4A3‐5 genes, in which the pathogenic variant in COL4A5 was de novo and the pathogenic variants in COL4A4 and COL4A3 were inherited independently (in trans). The site and type of mutations in COL4A5 were similar between the two groups. It was revealed that males in group 1 presented more severe proteinuria than males in group 2 (p < 0.05).ConclusionThe present study provides further evidence for complicated genotype in Alport syndrome. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found that heterozygous pathogenic COL4A3 or COL4A4 variants are likely to make XLAS disease more serious.

Identification of a Point Mutation in Position – 83 (G>A) of the β Globin Gene Promoter and the Influence of Globin Genes Mutation on the HbA2 Level

The molecular basis of β-thalassemia (β-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous β-thal may cause misdiagnosis, especially in countries with a high prevalence of β-thal where prevention programs have been implemented. The coinheritance of β- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of β-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C>T) linked to a polyadenylation (polyA) site [AATAAA>A(–AATAA)] 5 bp deletional β-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during β-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a β-thal major (β-TM) child. This has important implications in prenatal diagnosis.

Although δ-globin gene (HBD MIM#142000) mutations have no immediate physical consequence, it can interfere with the diagnosis of β-thalassemia (β-thal), which can be severe. In the present study, of 40,863 samples referred for thalassemia trait screening, 167 samples with lower than expected Hb A2 levels, in the presence or absence of a second Hb A2 fraction, were selected for our analysis and 152 samples (0.4%) were positive for δ-globin gene mutations. Twenty-one different mutations were detected, and of these 12 have not been previously described. We found that −77 (T>C) was the most common mutation in Chinese followed by −30 (T>C), together accounting for almost 82.3% of the total number of δ-globin gene defects. Since compound heterozygotes for β-thal and a δ-globin gene mutation may have low mean cell volume (MCV) and normal Hb A2 levels, and therefore be overlooked as β-thal heterozygotes, a detailed molecular analysis for both α- and β-thal is necessary, especially when one partner has been identified to have β-thal trait.

Heterozygous pathogenic variants in POMC are not responsible for monogenic obesity: Implication for MC4R agonist use

We report the case of a father and daughter who are heterozygous for a duplication of 65 bp within exon 2 of the β-globin gene, resulting in an altered and truncated β-globin chain that is predicted to be non functional. The β-globin gene mutation is in cis with the common Hb A2 ′ missense mutation of the δ-globin gene (HBD: c.49G>C), resulting in β-thalassemia (β-thal) trait with normal levels of Hb A2. This is the second report of this β0-thal mutation, and both families were associated with the Hb A2 ′ variant and normal levels of Hb A2. Laboratories should be aware of the rare occurrence of β-thal trait with normal levels of Hb A2.

Genetic predisposition is has been identified as a cause of cancer, yet little is known about the role of adult cancer predisposition syndromes in childhood cancer. We examined the extent to which heterozygous pathogenic germline variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, MSH2, MSH6, MLH1, and PMS2 contribute to cancer risk in children and adolescents. We conducted a meta-analysis of 11 studies that incorporated comprehensive germline testing for children and adolescents with cancer. ClinVar pathogenic or likely pathogenic variants (PVs) in genes of interest were compared with 2 control groups. Results were validated in a cohort of mainly European patients and controls. We employed the Proxy External Controls Association Test to account for different pipelines. Among 3975 children and adolescents with cancer, statistically significant associations with cancer risk were observed for PVs in BRCA1 and 2 (26 PVs vs 63 PVs among 27 501 controls, odds ratio = 2.78, 95% confidence interval = 1.69 to 4.45; P < .001) and mismatch repair genes (19 PVs vs 14 PVs among 27 501 controls, odds ratio = 7.33, 95% confidence interval = 3.64 to 14.82; P<.001). Associations were seen in brain and other solid tumors but not in hematologic neoplasms. We confirmed similar findings in 1664 pediatric cancer patients primarily of European descent. These data suggest that heterozygous PVs in BRCA1 and 2 and mismatch repair genes contribute with reduced penetrance to cancer risk in children and adolescents. No changes to predictive genetic testing and surveillance recommendations are required.

HBD: c.442T>C is a new mutation at the stop codon (TGA>CGA) of the δ-globin gene, which produces a new codon for arginine. This substitution causes a 51 nucleotides longer open reading frame determining the synthesis of a potential larger δ subunit, which is a probable target of mechanisms for the degradation of aberrant proteins as well as the defective synthesized mRNA molecules, and may also be rapidly degraded by a variety of RNA surveillance pathways. We identified this molecular defect in four patients: three women with a reduced HbA2 level and a 37-year-old male showing the typical phenotype of an α-thalassemia (α-thal) carrier with reduced values of red cell indices and normal HbA2 level (2.5%). The mutation on the δ-globin gene was found to have been coinherited with a β-globin gene defect leading to a normalized HbA2 level. These data support the necessity of investigating these cases at a molecular level, particularly if the partner is also a β-thalassemia (β-thal) carrier. The present data emphasizes the importance of a careful evaluation of correlation between genotypes resulting from DNA analysis and phenotypes, especially in cases of atypical hematological parameters, in order to carry out an adequate diagnostic process finalized to appropriate genetic counseling.

Context: Neonatal severe hyperparathyroidism (NSHPT) is a rare and life-threatening emergency. It includes generalized hyperparathyroid bone disease and respiratory distress from combinations among a narrowed thorax, rib fractures, hypotonia, and biochemical disturbances. Successful therapy is compatible with long life and a healthy prognosis. However, neuromotor retardation may persist after otherwise successful therapy. The time and amplitude of hypercalcemia likely correlate with irreversible neuromotor retardation; thus, early intervention seems critical in many cases. NSHPT is usually caused by homozygous or heterozygous pathogenic variant(s) of the CASR; a heterozygous variant of this gene is also the usual cause of familial hypocalciuric hypercalcemia (FHH or FHH1). Homozygotes and heterozygotes with NSHPT are often not distinguished in the current literature. In theory, their management should differ. Optimum treatment in homozygotes is early total parathyroidectomy with induction of postoperative hypoparathyroidism. Optimal management of heterozygotes is more complex. It consists in temporizing measures and varies from careful observation without surgery, to bisphosphonates and/or calcimimetics, and to subtotal parathyroidectomy. The heterozygotes can then develop into healthy babies with asymptomatic FHH1.Evidence Acquisition: Each case met strict criteria for “severe” and neonatal disease. We analyzed the core biochemical parameters of the maximal serum calcium and maximal PTH. To compare different immunoassays, PTH was analyzed as a ratio (thus without units) of the raw data divided by the upper limit of its normal range. Each case also required information about the allelic dosage of the pathogenic or likely pathogenic CASR variant(s).Evidence Synthesis: There were 36 cases with homozygous pathogenic CASR variants and 21 cases with heterozygous pathogenic or likely pathogenic variants. Maximal serum calcium was far higher in homozygotes 5.8 +/- 1.5 versus 3.2 +/- 0.2 (P<.001) (normal 2.2–2.6 mM). Maximal serum PTH as a ratio was also far higher in homozygotes 17 +/- 12 versus 8 +/- 9 (P=.003) (normal ratio 0.3–1.0). We defined extreme hypercalcemia empirically as any calcium value above 4.5 mM. No heterozygote had a maximal calcium above 3.7 mM; however, 30 of 36 (81%) of the homozygotes had maximal calcium above 4.5 mM. Whenever tested early, this extreme hypercalcemia was usually recognized in the first week of life.Conclusions: Extreme hypercalcemia greater than 4.5 mM was newly identified as a conservative cutoff that was 100% predictive of homozygosity for pathogenic CASR variants. Extreme hypercalcemia in NSHPT is an early, facile, rapid, and inexpensive determinant of homozygosity for pathogenic variants of the CASR. It should be sought and used to promote early and definitive total parathyroidectomy, whenever it is identified in NSHPT.

Background: FOXN1 belongs to the Foxhead-box (FOX) family of transcription factors and is fundamental for the successful development of T-cells in the thymus. Homozygous pathogenic variants in the FOXN1 gene are associated with a severe combined immunodeficiency (SCID) phenotype. In contrast, individuals carrying a heterozygous single pathogenic variant in FOXN1 demonstrate a less severe phenotype. Patients described thus far can present with frequent mild respiratory infections and associated T-cell lymphopenia that has been shown to improve with age. We present three patients with heterozygous variants in FOXN1 presenting with a mild clinical phenotype. Methods: Informed consent was obtained from patients and families. Case data was compiled retrospectively from the patients’ medical charts. Genetic testing used an augmented exome slice of 251 genes implicated in inborn errors of immunity, with Sanger backfill of select genes. Case Presentation: Probands P1 and P2 are two unrelated pediatric patients identified up by a positive newborn screen for SCID. P3 is the father of P2. All were found to have heterozygous pathogenic variants in FOXN1. In P1 and P2, laboratory investigations revealed T-cell lymphopenia on initial bloodwork. Lymphopenia improved with age. A surveillance approach was taken to manage both Pediatric patients, based on local experience with similar presentations, and both have done well thus far. Aside from a typical number of minor infections including common upper respiratory tract illnesses and otitis media, neither child had infections requiring hospitalizations or IV antibiotics. Additionally, P1 and P2 tolerated live viral vaccines. P3 had a history of one severe dental infection but had otherwise been well prior to diagnosis, and since. Conclusion: With the increasing implementation of newborn screening, patients with heterozygous pathogenic variants in FOXN1 are increasingly identified. No clear care pathway has been established. Our cases add to the growing body of literatures in presenting a cohort of patients with heterozygous pathogenic FOXN1 variants whose phenotype indicates mild, self-resolving infections despite T-lymphopenia. It will be important to identify and follow these patients longitudinally to consider recommendation of a surveillance approach if appropriate, and to provide guidance to affected families regarding the trajectory of this condition.

Should We Diagnose Autosomal Dominant Alport Syndrome When There Is a Pathogenic Heterozygous COL4A3 or COL4A4 Variant?

Variability In Hb A2 levels among Individuals with Beta-Thalassemia Trait: Is Iron Deficiency Associated with Abnormally Low Hb A2?