Increased Prevalence of TG and TPO Mutations in Sudanese Children With Congenital Hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) is due to dyshormonogenesis in 10% to 15% of subjects worldwide but accounts for 60% of CH cases in the Sudan. OBJECTIVE: To investigate the molecular basis of CH in Sudanese families. DESIGN: Clinical phenotype reporting and serum thyroid hormone measurements. Deoxyribonucelic acid extraction for whole-exome sequencing and Sanger sequencing. SETTING: University research center. PATIENTS: Twenty-six Sudanese families with CH. INTERVENTION: Clinical evaluation, thyroid function tests, genetic sequencing, and analysis. Our samples and information regarding samples from the literature were used to compare TG (thyroglobulin) and TPO (thyroid peroxidase) mutation rates in the Sudanese population with all populations. RESULTS: Mutations were found in dual-oxidase 1 (DUOX1), dual-oxidase 2 (DUOX2), iodotyrosine deiodinase (IYD), solute-carrier (SLC) 26A4, SLC26A7, SLC5A5, TG, and TPO genes. The molecular basis of the CH in 7 families remains unknown. TG mutations were significantly higher on average in the Sudanese population compared with the average number of TG mutations in other populations (P < 0.05). CONCLUSIONS: All described mutations occur in domains important for protein structure and function, predicting the CH phenotype. Genotype prediction based on phenotype includes low or undetectable thyroglobulin levels for TG gene mutations and markedly higher thyroglobulin levels for TPO mutations. The reasons for higher incidence of TG gene mutations include gene length and possible positive genetic selection due to endemic iodine deficiency.

Central Congenital Hypothyroidism Caused by a Novel Mutation, C47W, in the Cysteine Knot Region of TSHß.

BACKGROUND: Isolated central congenital hypothyroidism (ICCH) is a rare form (1:50,000 newborns) of congenital hypothyroidism, which can present with growth and neuropsychological retardation. Unlike the more common primary CH (1:1,500-1:4,000), which presents on newborn screening with elevated serum thyroid-stimulating hormone (TSH) and low thyroxine (T4) and triiodothyronine (T3), ICCH presents with low TSH and low thyroid hormone levels. ICCH, therefore, may be missed in most newborn screens that are based only on elevated TSH. Most cases of ICCH have been associated with mutations in the TSHß gene. PATIENT: We present a consanguineous Sudanese family where the proband was diagnosed with "atypical" CH (serum TSH was low, not high). INTERVENTION AND OUTCOME: The propositus underwent whole-exome sequencing, and the C47W TSHß mutation was identified. Sanger sequencing confirmed the proband to be homozygous for C47W, and both parents were heterozygous for the same mutation. The mutation was predicted by several in silico methods to have a deleterious effect (SIFT 0.0, Damaging; Polyphen2_HDIV 0.973, probably damaging; MutationTaster 1, disease causing; and CADD 3.17, 16.62). C47W affects the first cysteine of the cysteine knot of the TSHß subunit. The cysteine knot region of TSHß is highly conserved across species and is critical for binding to the TSH receptor. Only two other mutations were previously reported along the cysteine knot and showed consistently low or undetectable serum TSH and low T4 and T3 levels. Other TSHß gene mutations causing ICCH have been reported in the "seatbelt" region, necessary for TSHß dimerization with the alpha subunit. CONCLUSIONS: Identification of a mutation in the TSHß gene reinforces the importance of identifying ICCH that can occur in the absence of elevated serum TSH and demonstrates the functional significance of the TSHß cysteine knot.

A NOVEL MUTATION CAUSING COMPLETE THYROID BINDING GLOBULIN DEFICIENCY (TBG-CD MIA) IN A MALE WITH COEXISTING GRAVES DISEASE.

OBJECTIVE: An asymptomatic male was found on screening to have a low serum TSH and total T4. The diagnosis of Graves' disease was made with positive thyroid stimulating immunoglobulin (TSI) and elevated free T4 in the presence of complete TBG deficiency (TBG-CD). Genetic testing of the patient and family members revealed a novel frameshift mutation in the TBG (SERPINA7) gene resulting in a complete deficiency of the protein. METHODS: The laboratory testing included total T4, free T4 by analog method and direct dialysis and TBG measurements. Sequencing of genomic DNA was performed from peripheral blood. RESULTS: A 35-year-old East Indian male was referred to endocrinology because of abnormal thyroid function tests (TFTs): TSH 0.01 mIU/L (0.4-3.6), total T4 3.0 µg/dl (5.5-10.5) done as part of a "routine office visit". Upon further testing, the serum free T4 2.0 ng/dl (0.8-1.8) and TSI 355% (<140% baseline) were elevated and the diagnosis of Graves' disease was made. TBG deficiency was suspected because the total T4 concentration was inconsistent with hyperthyroidism and further testing confirmed TBG was undetectable. Sequencing of the TBG gene revealed a novel hemizygous frameshift mutation: p.Ala64ProfsTer106, TBG-CD Mia (numbering excludes 20 a.a. signal peptide) associated with the complete deficiency of TBG in a patient with Graves' disease. CONCLUSION: Patients with Graves' disease harboring a TBG mutation have conflicting TFTs. If a clinically hyperthyroid patient presents with normal or low total T4, serum TBG should be measured to identify an abnormality and prevent unnecessary testing.

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy.

Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.