Viviana Varela

Identification of a mutation in the coding sequence of the human thyroid peroxidase gene causing congenital goiter.

Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones, and the TPO defects are believed to be the most prevalent causes of the inborn errors of thyroid metabolism. We investigated an adopted boy with iodide organification defect, who presented with florid hypothyroidism at the age of 4 mo, poorly complied with thyroxine treatment, and developed a compressive goiter necessitating partial resection at the age of 12 yr. Biochemical studies revealed the absence of TPO activity in the resected tissue. Genomic DNA studies identified a 4 base-pair insertion in the eighth exon of the TPO gene, and showed that the patient was homozygous for this frameshift mutation. The direct genetic diagnosis of this mutation can be made by digestion of polymerase chain reaction products with NaeI restriction enzyme. This will help assessing its prevalence among the heterogenous genetic group of TPO defects.

Two compound heterozygous mutations (c.215delA/c.2422T→C and c.387delC/c.1159G→A) in the thyroid peroxidase gene responsible for congenital goitre and iodide organification defect

Background  Iodide organification defects are frequently but not always associated with mutations in the thyroid peroxidase (TPO) gene and characterized by a positive perchlorate discharge test. These mutations phenotypically produce a congenital goitrous hypothyroidism, with an autosomal recessive mode of inheritance.

Qualitative and quantitative defects of thyroglobulin resulting in congenital goiter. Absence of gross gene deletion of coding sequences in the TG gene structure

Seven subjects belonging to three families (ME, MA, MO), with congenital goiter and various degrees of thyroid hypofunction, were investigated from the standpoints of clinical, biochemical, and molecular biology. In two of these families (ME, MA), 6 individuals had low serum levels of Tg-related antigens with a minor increase after bovine TSH (bTSH) stimulation. A large proportion of the tracer was incorporated into serum albumin, and Tg antigens in the thyroid extracts were barely detectable by RIA. (0.19 mg/g tissue; normal, 70–90 mg/g). Gel filtration (CL6B Sepharose gel) showed absence of a normal Tg peak, and SDS agarose gel electrophoresis indicated complete absence of Tg dimer and monomer. Immunoelectrophoresis confirmed the absence of Tg-related antigens. Thus, in these patients a quantitative defect of Tg gene expression was characterized. By contrast, in the MO family a high basal serum concentration of immunoreactive Tg was present, with an exaggerated response to bTSH. Thyroid extracts revealed elevated TPO activity and normal levels of Tg-related antigens. Tg was also eluted in the gel filtration columns with the same mobility as standard 19S Tg. Immunoelectrophoresis against rabbit and human Tg was abnormal, with two precipitin arcs being detected. The Tg molecule after hydrolysis yielded only DIT and MIT, with poor formation of iodothyronines. Microscopic studies revealed a pronounced lack of colloid in the follicular lumina, and overdistended endoplasmic reticulum cisternae. It was concluded that in this family the defective Tg gene expression is qualitative, with an abnormal Tg being formed and only partially transported to the follicular lumen. Southern blotting experiments to probe Tg gene structure indicated no significant differences in pattern between DNA samples from controls and those from goitrous patients. This suggests defective translation or abnormal routing of translation product through the membrane system of the cell. Further studies of the Tg mRNA and DNA structure will be needed to clarify the exact nature of both defects.

Differential levels of thyroid peroxidase and thyroglobulin messenger ribonucleic acids in congenital goiter with defective thyroglobulin synthesis

The biosynthesis of thyroid hormones requires iodide, thyroid peroxidase (TPO), thyroglobulin (Tg) and H2O2. We have studied two sisters with congenital large goiters and hypothyroidism. Perchlorate tests were negative. Serum T3 and T4 were decreased, TSH was increased and Tg was within the lower limit of normal. Biochemical and molecular studies were performed on goiter samples obtained after surgery. Tg content in both tissues was negligible. Paper chromatography of labeled iodocompounds showed a decrease in T4, and the presence of a pronase/pan-creatin-resistant iodoprotein. TPO activity was normal in the tissues. Sephacryl S-300 gel filtration demonstrated labeled iodoalbumin-like protein and the absence of a Tg peak. Salting out studies of soluble protein fraction gave an abnormal pattern. Agarose gel electrophoresis showed the presence of an iodoalbumin-like protein and the absence of Tg in the tissues. This last finding was confirmed by immunoelectrophoresis. The Tg and TPO mRNAs levels were also analyzed. Dot-blot hybridization studies with pM5 (TPO cDNA) and phTgM2 (Tg cDNA) probes showed increased and decreased signals, respectively. The increase in TPO mRNA can be explained as a compensatory mechanism vis a vis an increase in serum TSH caused by decreased serum T3 and T4 due to the impairment in Tg mRNA. The Tg mRNA of both patients was further studied with four different probes covering 5′ and 3′ regions (phTgM1, phTgB1, phTgB2 and phTgB3). Hybridization was observed with all four probes, thus excluding a dramatic deletion defect. Northern transfer showed a clear signal of hybridization with the phTgBI probe in the 8–9 Kb range. We may conclude that the biochemical and molecular abnormality of these patients is characterized by a decrease of Tg mRNA and of Tg translation.l higher (p < 0.01) than in normal men. This finding is probably related to the subnormal plasma levels of testosterone found in agonadal men during the replacement therapy; the analysis of data using a sampling interval of 10 min gave results similar to previous reports, confirming that the choice of sampling interval can markedly affect the evaluation of frequent LH pulsatile secretion.

Thyroid hormone modulation of VIP’s induced salivary secretion in the submaxillary glands of rats

The effect of changes in thyroid function upon vasoactive intestinal peptide (VIP) induced secretion of saliva were studied in male Wistar rats. Hyperthyroidism was induced by the sc administration every 12 h of 10 μg/100 g bwof l-triiodothyronine; hypothyroidism was induced by surgical thyroidectomy 2 weeks before the experiments. Preganglionar parasympathetic denervation was induced by sectioning the chorda tympani on the left side. The dose-response curves to increasing doses of VIP showed in the hypothyroid animals increased salivary secretion, while in the hyperthyroid ones the dose-response to the drug was reduced. This effect was seen on both sides, the denervated and the control ones. In the denervated glands there was a marked hypersensitivity to the administration of VIP producing greater responses with the same doses, in the 3 groups of animals. The negative modulation by thyroid hormones of the salivary response to VIP administration is compared with the positive modulation they induce in the salivary response to β-adrenergic and cholinergic drugs.

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.

Human thyroid tissue do not express thyroalbumin

Thyroid tissue total RNAs from multinodular goiter(G2) and from hereditary goiter with defective Tg synthesis (JNA) were hybridized with a 5’albumin cDNA probe (F-47), a 3’ albumin cDNA probe (B-44) and a thyroglobulin cDNA probe (phTgM3). JNA refers to tissue obtained from a patient with virtual absence of Tg in thyroid tissue and the presence of increased concentration of an albumin-like labeled protein in the thyroid. No hybridization signal was detected in both G2 and JNA with albumin probes at Northern Blot studies. Those results were confirmed by dot-blot analysis of total RNA where no hybridization signal was detected in G2 and JNA. To confirm that thyroid tissues do not express thyroalbumin total RNA from JNA and normal control thyroid tissue (C) were amplified by PCR using albumin and Tg primers. An expected fragment of 592 bp was observed in a human liver sample with the albumin primers. However JNA and C samples showed absence of an amplification product of the same size. We concluded that thyroid cells do not contain the albumin transcript. Albumin is probably taken up from circulation and iodinated by the thyroid follicular cell with subsequent release of iodoalbumin into the circulation.

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 β‐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 β‐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 β‐globins would affect the formation of the αβ dimers and their stability, which would further support the mechanism for the observed clinical features in both patients.

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 ‐α3.7 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.

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 [β6(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 [β6(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).