Curating the gnomAD database: Report of novel variants in the globin-coding genes and bioinformatics analysis.

Massive parallel sequencing technologies are facilitating the faster identification of sequence variants with the consequent capability of untangling the molecular bases of many human genetic syndromes. However, it is not always easy to understand the impact of novel variants, especially for missense changes, which can lead to a spectrum of phenotypes. This study presents a custom-designed multistep methodology to evaluate the impact of novel variants aggregated in the genome aggregation database for the HBB, HBA2, and HBA1 genes, by testing and improving its performance with a dataset of previously described alterations affecting those same genes. This approach scored high sensitivity and specificity values and showed an overall better performance than sequence-derived predictors, highlighting the importance of protein conformation and interaction specific analyses in curating variant databases. This study also describes the strengths and limitations of these structural studies and allows identifying residues in the globin chains more prone to tolerate substitutions.

Two novel unstable hemoglobin variants due to in-frame deletions of key amino acids in the ß-globin chain.

Hemoglobinopathies are the most common autosomal recessive disorders and are mostly inherited in a recessive manner. However, certain mutations can affect the globin chain stability, leading to dominant forms of thalassemia. The aim of this work was the molecular and structural characterization of two heterozygous in-frame deletions, leading to ß-globin variants in pediatric patients in Argentina. The HBB gene of the probands and their parents was sequenced, and other markers of globin chain imbalance were analyzed. Several structural analyses were performed, and the effect of the mutations on the globin chain stability was analyzed. In Hb JC-Paz, HBB:c.29_37delCTGCCGTTA (p.Ala10_Thr12del), detected in an Argentinean boy, one α-helix turn is expected to be lost. In Hb Tavapy, HBB:c.182_187delTGAAGG (p.Val60_Lys61del), the deleted residues are close to distal histidine (His63) in the heme pocket. Both mutations are predicted to have a destabilizing effect. The development of computational structural models and bioinformatics algorithms is expected to become a useful tool to understand the impact of the mutations leading to dominant thalassemia.

The Chaperones Involved in Hemoglobin Synthesis Take the Spotlight: Analysis of AHSP in the Argentinean Population and Review of the Literature.

Hemoglobin (Hb) synthesis is a complex, well-coordinated process that requires molecular chaperones. These intervene in different steps: regulating epigenetic mechanisms necessary for the adequate expression of the α- and ß-globin clusters, binding the nascent peptides and helping them acquire their native structure, preventing oxidative damage by free globin chains and preventing the cleavage of essential erythroid transcription factors. This study analyzed the distribution of the single nucleotide polymorphism (SNP) rs4296276 in intron 1 of the α-globin chaperone α Hb-stabilizing protein (AHSP) in the Argentinean population. The risk allele was found in thalassemia patients who exhibited more severe phenotypes than expected. Future studies may help establish the role of these chaperones as modifiers in pathological states with globin chain imbalance, such as thalassemia.

Multiple copy number variants in a pediatric patient with Hb H disease and intellectual disability.

Two distinct syndromes that link α-thalassemia and intellectual disability (ID) have been described: ATR-X, due to mutations in the ATRX gene, and ATR-16, a contiguous gene deletion syndrome in the telomeric region of the short arm of chromosome 16. A critical region where the candidate genes for the ID map has been established. In a pediatric patient with Hemoglobin H disease, dysmorphic features and ID, 4 novel and clinically relevant Copy Number Variants were identified. PCR-GAP, MLPA and FISH analyses established the cause of the α-thalassemia. SNP-array analysis revealed the presence of 4 altered loci: 3 deletions (arr[hg19]Chr16(16p13.3; 88,165-1,507,988) x1; arr[hg19]Chr6(6p21.1; 44,798,701-45,334,537) x1 and arr[hg19]Chr17(17q25.3; 80,544,855-81,057,996) x1) and a terminal duplication (arr[hg19]Chr7(7p22.3-p22.2; 4,935-4,139,785) x3). The -α(3.7) mutation and the ∼1.51 Mb in 16p13.3 are involved in the alpha-thalassemic phenotype. However, the critical region for ATR-16 cannot be narrowed down. The deletion affecting 6p21.1 removes the first 2 exons and part of intron 2 of the RUNX2 gene. Although heterozygous loss of function mutations affecting this gene have been associated with cleidocranial dysplasia, the patient does not exhibit pathognomonic signs of this syndrome, possibly due to the fact that the isoform d of the transcription factor remains unaffected. This work highlights the importance of searching for cryptic deletions in patients with ID and reiterates the need of the molecular analysis when it is associated to microcytic hypochromic anemia with normal iron status.

[Regulation of the ß-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies]. / Regulación de expresión de genes de la familia de ß-globina humana, útil en la búsqueda de nuevos blancos terapéuticos para tratamiento de hemoglobinopatías.

Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and ß-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the ß-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2). In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the ß-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

Hb Wilde and Hb Patagonia: two novel elongated beta-globin variants causing dominant beta-thalassemia.

We describe here the molecular and hematological characteristics of novel frameshift mutations in exon 2 of the HBB gene (in heterozygous state) found in two Argentinean pediatric patients with dominant ß-thalassemia-like features. In Hb Wilde, HBB:c.270_273delTGAG(p.Glu90Cysfs*67), we detected the deletion of the third base of the codon 89 (T) and the codon 90 (GAG), whereas in Hb Patagonia, HBB:c.296_297dupGT(p.Asp99Trpfs*59), the frameshift mutation was due to a duplication of a 'GT' dinucleotide after the second base of codon 98 (GTG). The Hb Patagonia and Hb Wilde mutations would result in elongated ß-globin chains with modified C-terminal sequences and a total of 155 and 157 amino acids residues, respectively. Based on bioinformatics and structural analysis, as well as protein modeling, we predict that the elongated ß-globins would affect the formation of the αß dimers and their stability, which would further support the mechanism for the observed clinical features in both patients.

[Molecular bases of α-thalassemia in Argentina]. / Bases moleculares de alfa-talasemia en la Argentina.

The α-thalassemia is one of the most common hereditary disorders worldwide. Currently, molecular diagnostics is the only available tool to achieve an accurate diagnosis. The purpose of this study was to characterize the molecular bases of these syndromes in our environment and to establish genotype-phenotype associations. Through a combination of different molecular techniques and fluorescent in situ hybridization (FISH),we were able to find α-thalassemic mutations in 145 of the 184 patients (78.8%) studied with hematological parameters compatible with α-thalassemia. Deletions of the α-globin genes resulted the major molecular cause of the disease, and the most frequent mutation was -α(3.7), found in homozygous and heterozygous genotypes. In patients with α° phenotypes, other prevalent mutations were( _MED) and (_CAL/CAMP). The description of a sub-telomeric deletion in a patient with α-thalassemia and mental retardation was also achieved. ß-thalassemic mutations in heterozygous state were found in 7.6% of the patients, who presented α-thalassemic clinical features (microcytosis and Hb A2levels below 3.5%). Hematologic profiles for the α+ and α° genotypes were established for adult and pediatric patients. Hopefully, this work will provide guidelines for the detection of possible α-thalassemic carriers. It also highlights the collaborative work of hematologists, the biochemical and molecular biology laboratory and genetists, in order to provide appropriate genetic counseling.

Coinheritance of a novel mutation on the HBA1 gene: c.187delG (p.W62fsX66) [codon 62 (-G) (α1)] with the α212 patchwork allele and Hb S [ß6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T].

We describe a novel frameshift mutation on the HBA1 gene (c.187delG), causative of α-thalassemia (α-thal) in a Black Cuban family with multiple sequence variants in the HBA genes and the Hb S [ß6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T] mutation. The deletion of the first base of codon 62 resulted in a frameshift at amino acid 62 with a putative premature termination codon (PTC) at amino acid 66 on the same exon (p.W62fsX66), which most likely triggers nonsense mediated decay of the resulting mRNA. This study also presents the first report of the α212 patchwork allele in Latin America and the description of two new sequence variants in the HBA2 region (c.-614G>A in the promoter region and c.95+39 C>T on the first intron).

The p.Cys1281Tyr variant in the hinge module/flap region of thyroglobulin causes intracellular transport disorder and congenital hypothyroidism.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) variants causes very low serum TG levels with normal or enlarged thyroid glands, depending on the severity of the defect, and with autosomal recessive inheritance. The purpose of this study was to functionally characterize p.Cys1281Tyr variant in the TG gene in order to increase our knowledge of the molecular mechanisms associated with CH. In order to find evidence that support the hypothesis that the p.Cys1281Tyr variant would affect the TG folding were performed amino acid prediction, 3D modeling and transient expression analysis in HEK293T cells. 18 of the 21″in silico" algorithms predict a deleterious effect of the p.Cys1281Tyr variant. The full-length 3D model p.Cys1281Tyr TG showed disulfide bond cleavage between the cysteines at positions 1249 and 1281 and rearrangement of the TG structure, while transient expression analysis indicated that p.Cys1281Tyr causes retention of the protein inside the cell. Consequently, these results show that this pathogenic variant makes it impossible for TG to fulfill its function in the biosynthesis process of thyroid hormones, causing CH. In conclusion, our results confirm the pathophysiological importance of misfolding of TG as a consequence of p.Cys1281Tyr variant located in the hinge module/flap region of TG.

Identification of a new HBA1 gene mutation (HBA1:c.301-2A>T) in cis with Hb Riccarton (HBA1:c.154G>A) [α51(CE9)Gly→Ser].

We report two point mutations found in a heterozygous state on the HBA1 gene of an 88-year-old Argentinean patient with an α(+)-thalassemia (α(+)-thal) phenotype: Hb Riccarton HBA1:c.154G>A) [α51(CE9)Gly→Ser] and a novel mutation, HBA1:c.301-2A>T that affects the splicing acceptor site of the second intron and leads to a non functional α-globin chain. Cloning of the HBA1 PCR (polymerase chain reaction) product and direct sequencing of the clones revealed that both mutations were in cis.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review.

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

Curating the gnomAD database: Report of novel variants in the thyrogobulin gene using in silico bioinformatics algorithms.

Thyroglobulin (TG) is a large glycosylated protein of 2767 amino acids, secreted by the thyrocytes into the follicular lumen. It plays an essential role in the process of thyroid hormone synthesis. TG gene variants lead to permanent congenital hypothyroidism. In the present work, we report a detailed population and bioinformatic prediction analyses of the TG variants indexed in the Genome Aggregation Database (gnomAD). The results showed a clear predominance of nonsense variants in the European (Finnish), European (Non-Finnish) and Ashkenazi Jewish ethnic groups, whereas the splice site variants predominate in South Asian and African/African-American populations. In total, 282 novel TG variants were described (47 missense involving the wild-type cysteine residues, 177 missense located in the ChEL domain and 58 splice site variants) which were not reported in the literature and that would have deleterious effects in prediction programs. In the gnomAD population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:320. In conclusion, we provide an updated and curated reference source for the diagnosis of thyroid disease, mainly to congenital hypothyroidism due to TG deficiency. The identification and characterization of TG variants is undoubtedly a valuable approach to study the TG structure/function relations and an important tool for clinical diagnosis and genetic counseling.

Defects in protein folding in congenital hypothyroidism.

Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the most common preventable causes of both cognitive and motor deficits. CH is a heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis, hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1, DUOXA2, IYD or TG genes, which usually originate goiter. About 800 genetic mutations have been reported to cause CH in patients so far, including missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for proteasomal degradation by the ER-associated degradation (ERAD) machinery. Despite of all these remarkable advances in the field of the CH pathogenesis, several points on the development of this disease remain to be elucidated. The continuous study of thyroid gene mutations with the application of new technologies will be useful for the understanding of the intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge on the disorders in the protein folding caused by thyroid genes mutations.

p.L571P in the linker domain of rat thyroglobulin causes intracellular retention.

Thyroglobulin (TG), a large glycosylated protein secreted by thyrocytes into the thyroid follicular lumen, plays an essential role in thyroid hormone biosynthesis. Rattus norvegicus TG (rTG) is encoded by a large single copy gene, 186-kb long, located on chromosome 7 composed of 48 exons encoding a 8461-kb mRNA. Although the TG gene displays sequence variability, many missense mutations do not impose any adverse effect on the TG protein, whereas other nucleotide substitutions may affect its TG stability and/or TG intracellular trafficking. In order to gain a further understanding of the protein domains regulating its intracellular fate, we cloned a full-length cDNA from rTG into the pcDNA6/V5-His B expression vector. However, transient expression of the cDNA in HEK293T cells showed that the encoded protein was not a wild-type molecule, as it was unable to be secreted in the culture supernatant. Sequencing analyses revealed three random mutations, which accidentally emerged during the course of cloning: c.1712T>C [p.L571P] in the linker domain (amino acid positions 360 to 604), c.2027A>G [p.Q676R] in TG type 1-6 repeat and c.2720A>G [p.Q907R] in the TG type 1-7 repeat. Expression of cDNAs encoding a combination of two mutations [p.Q676R-p.Q907R], [p.L571P-p.Q907R] or [p.L571P-p.Q676R] indicated that any TG bearing the p.L571P substitution was trapped intracellularly. Indeed, we expressed the single point mutant p.L571P and confirmed that this point mutation was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Endo H analysis showed that the p.L571P mutant is completely sensitive to the enzyme, whereas the will-type TG acquires full N-glycan modifications in Golgi apparatus. This data suggest that the p.L571P mutant contains the mannose-type N-glycan, that was added at the first stage of glycosylation. Complex-type N-glycan formation in the Golgi apparatus does not occur, consistent with defective endoplasmic reticulum exit of the mutant TG. Moreover, predictive analysis of the 3D linker domain showed that the p.L571P mutation would result in a significant protein conformational change. In conclusion, our studies identified a novel amino acid residue within the linker domain of TG associated with its conformational maturation and intracellular trafficking.

Regulación de expresión de genes de la familia de ß-globina humana, útil en la búsqueda de nuevos blancos terapéuticos para tratamiento de hemoglobinopatías / Regulation of the ß-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies

Durante la etapa embrionaria, el desarrollo fetal y la vida posnatal se expresan isoformas funcionalmente distintas de hemoglobina, producto de la combinación de cadenas polipeptídicas sintetizadas a partir de los distintos genes que componen las familias de α- y β-globina. En función de que la presencia de altos niveles de hemoglobina fetal (Hb F) es beneficiosa en síndromes falciformes y talasémicos graves, se plantea revisar las bases de la regulación de la expresión de los genes de la familia de β-globina, en particular los genes que codifican las cadenas de γ-globina (HBG1 y HBG2). En este trabajo se revisan los conocimientos sobre factores de transcripción y reguladores epigenéticos que gobiernan los eventos de encendido y apagado de los genes de la familia de β-globina. Se espera que la consolidación de estos conocimientos permita hallar nuevos blancos terapéuticos para el tratamiento de hemoglobinopatías.

Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β- globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the γ-globin chains (HBG1 and HBG2). In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

Bases moleculares de alfa-talasemia en la Argentina

La α-talasemia, es uno de los desórdenes hereditarios más frecuentes mundialmente. Al presente, el diagnóstico molecular es la única herramienta que permite el diagnóstico certero. El propósito de este trabajo fue caracterizar las bases moleculares de estos síndromes en nuestro medio, y establecer relaciones genotipo-fenotipo. Mediante la complementación de distintas técnicas de biología molecular e hibridación fluorescente in situ (FISH), se logró poner en evidencia la presencia de mutaciones α-talasémicas en 145 de 184 (78.8%) pacientes estudiados con perfil hematológico compatible con α-talasemia. Dentro de este grupo, las deleciones correspondieron al defecto genético más frecuente, prevaleciendo la mutación -α3.7 en genotipos heterocigotas y homocigotas. Asimismo, en pacientes con fenotipo α0 las deleciones prevalentes fueron -MED y -CAL/CAMP. Este estudio permitió también describir una deleción de la región sub-telomérica en un paciente con α-talasemia y retraso mental. En el 7.6% de los pacientes caracterizados clínicamente como posibles α-talasémicos (microcitosis con valores de Hb A2 inferiores al 3.5%), se hallaron mutaciones β-talasémicas en estado heterocigota. Se lograron establecer perfiles hematológicos asociados a los genotipos α+ y α0 para pacientes adultos y niños. Esperamos que este trabajo pueda servir como guía para reconocer posibles portadores α-talasémicos. También permite destacar el trabajo en conjunto de médicos hematólogos, el laboratorio (bioquímico y de biología molecular) y de los médicos genetistas, con el fin de proporcionar adecuado consejo genético.

The α-thalassemia is one of the most common hereditary disorders worldwide. Currently, molecular diagnostics is the only available tool to achieve an accurate diagnosis. The purpose of this study was to characterize the molecular bases of these syndromes in our environment and to establish genotype-phenotype associations. Through a combination of different molecular techniques and fluorescent in situ hybridization (FISH),we were able to find α-thalassemic mutations in 145 of the 184 patients (78.8%) studied with hematological parameters compatible with α-thalassemia. Deletions of the a-globin genes resulted the major molecular cause of the disease, and the most frequent mutation was -α3.7, found in homozygous and heterozygous genotypes. In patients with α0 phenotypes, other prevalent mutations were -MED and -CAL/CAMP. The description of a sub-telomeric deletion in a patient with α-thalassemia and mental retardation was also achieved. β-thalassemic mutations in heterozygous state were found in 7.6% of the patients, who presented α-thalassemic clinical features (microcytosis and Hb A2 levels below 3.5%). Hematologic profiles for the α+ and α0 genotypes were established for adult and pediatric patients. Hopefully, this work will provide guidelines for the detection of possible α-thalassemic carriers. It also highlights the collaborative work of hematologists, the biochemical and molecular biology laboratory and genetists, in order to provide appropriate genetic counseling.