Background:Heterozygous c.283+1G>A and c.283G>A variants in the THRB gene, encoding for thyroid hormone receptor (TR)β1 and β2, lead to autosomal dominant macular dystrophy (ADMD). We report the detailed clinical characterization of two first-degree relatives with ADMD, heterozygous for THRB c.283+1G>A, and an unrelated ADMD patient with a novel variant, c.283G>C. The genomic and molecular consequences of both variants were studied. Methods:gDNA and mRNA were obtained from leukocytes. Clinical characterization included biochemistry, bone density and body composition, ECG, echocardiography, ultrasound, audiometry and color-vision. In vitro assays investigated TR function and DNA binding. Results:The patients manifested no resistance to thyroid hormone beta (RTHβ) and had normal FT4 and TSH. Detailed studies in two patients showed no goiter, tachycardia, hypercholesterinemia or hepatic steatosis. Hearing was not impaired. Both had impaired color vision and reduced bone density. RT-PCR from all three patients revealed skipping of exon 4 exclusive to TRβ1, producing a deletion of 87 amino acids in the N-terminal domain (TRβ1ΔNTD). In vitro, DNA-binding affinity of TRβ1ΔNTD to DR4-TRE with or without RXRα was comparable to TRβ1WT. Surprisingly, TRβ1ΔNTD was transcriptionally twice more active than TRβ1WT with a similar EC50 for T3, demonstrating gain-of-function of TRβ1ΔNTD. THRA expression in leukocytes was increased by 3-fold compared to unrelated controls and different from RTHβ patients. Conclusion:These THRB splice site variants produce TRβ1 exon 4 skipping, resulting in a gain-of-function mutant, TRβ1ΔNTD. This explains the dominant ADMD phenotype devoid of RTHβ and suggests a TRβ1 gain-of-function syndrome.

A 19-year-old male presented with mild fatigue and chronic constipation and was found to have an elevated thyroid-stimulating hormone (TSH) level of 85 mIU/L, normal free thyroxine, and normal total triiodothyronine. This presentation was concerning for Macro-TSH, which is rare and may suggest hypothyroidism. This patient's diagnosis of Macro-TSH was confirmed by TSH antibody testing and gel filtration chromatography. We present this case to highlight the need to further evaluate TSH results when thyroid function tests are incompatible with the clinical presentation. Assessment for TSH antibodies, a commercially available test, or gold-standard gel filtration chromatography testing, which is not commercially available, can be used to diagnose Macro-TSH.

CONTEXT:Congenital hypothyroidism (CH) is a leading cause of preventable intellectual disability worldwide if left untreated. Thyroid peroxidase (TPO) is a key enzyme that uses hydrogen peroxide from the DUOX/DUOXA system to oxidize iodide for thyroid hormone synthesis. OBJECTIVE:This work aimed to identify the pathogenic TPO variants responsible for CH. METHODS:Variants identified by whole-exome sequencing were analyzed using in silico tools and structural modeling for pathogenicity. TPO function was assessed through in vitro studies on intracellular trafficking, enzymatic activity, and interaction with DUOX/DUOXA proteins. RESULTS:Six TPO variants were identified: p.G395D, p.V618M, p.M706V, and p.T725P in family 1, and p.R648G and p.G771R in families 2 and 3, respectively. Affected individuals in family 1 exhibited compound heterozygous or homozygous variants for the 4 variants. In silico analyses showed incomplete concordance in predicting pathogenicity. In vitro studies confirmed p.G395D as the primary pathogenic variant in family 1, and p.R648G and p.G771R in families 2 and 3. Notably, p.V618M, p.M706V, and p.T725P did not impair TPO function, either individually or in combination, suggesting that these regions are not critical for enzymatic activity. Further functional analyses revealed that p.G771R is essential for proper membrane insertion, whereas p.R648G is necessary for enzymatic activity. CONCLUSION:When multiple TPO variants occur within the same family, a combination of in silico and in vitro analyses can help identify the variant responsible for the phenotype. In silico methods, however, cannot predict the different mechanisms of impairment, such as enzyme activity vs cellular localization, where the protein's topology is essential for normal function.

Pendred syndrome (PDS) is an autosomal recessive disease caused by variants in SLC26A4 manifesting thyroid dyshormonogenesis. Patients typically present with goiter and sensorineural hearing loss (SNHL). The prevalence of PDS in non-African populations is estimated to be between 7.5 and 10 per 100,000, while its occurrence in African populations has not been reported with molecular analysis. This study, conducted at a university research center in Miami, USA and Khartoum, Sudan, to investigate PDS in Sudanese families with congenital hypothyroidism (CH). It involved 32 Sudanese families with children diagnosed with CH between 2016 and 2023. Patients underwent clinical evaluation, thyroid function tests, and genetic sequencing. Two disease-causing SLC26A4 variants were identified in two consanguineous families with first-cousin parents. One homozygous nonsense variant causing premature termination, p.Trp482*, previously reported as part of a compound heterozygous defect together with p.Gly102Arg, while the other homozygous defect was a previously reported missense variant, p.Thr410Met. In 32 families (72 individuals) whole exome sequencing data revealed 56.3 This report extends our understanding of the severity of the phenotypes caused by deleterious bi-allelic variants in SLC26A4. Recurrent SLC26A4 variants observed in our cohort likely reflect high consanguinity rather than a founder effect. SLC26A4 screening could be a part of the molecular testing for children presenting with congenital or early-onset SNHL in Sudan.

Objective: To evaluate the combined administration of propylthiouracil (PTU) and levothyroxine (LT4) in managing monocarboxylate transporter 8 (MCT8) deficiency and identify optimal therapeutic dosages. Methods: This multicenter case series involved 12 male patients with MCT8 deficiency whose parents/guardians consented to PTU and LT4 treatment. Data were collected from January 2008 to June 24, 2024. The study focused on treatment safety and outcomes, analyzing baseline and last encounter biochemical, metabolic, and anthropometric parameters. Statistical analyses included Wilcoxon signed ranks tests and generalized estimated equations to assess effects on thyroid and metabolic markers, and receiver operating characteristics curves to predict optimal dose. Results: Patients showed a significant reduction in serum total triiodothyronine (TT3) concentration and TT3/TT4 ratio, with increased serum TT4 and free T4 (fT4) concentrations. The use of PTU effectively reduced TT3 concentration by 25% at an average dose of 6.8 mg/kg/day, while LT4 increased fT4 concentration by 40% from baseline at an average dose of 4.3 µg/kg/day. Thyrotropin concentration was undetectable on treatment. No statistical differences were observed in metabolic and physical parameters between baseline and last encounter overall for the group, but six of eight patients for whom these data were available had an increase in weight (z-score). There were no adverse effects on liver function or granulocyte numbers noted throughout the period of observation. Conclusion: Combined treatment with PTU and LT4 normalized serum T3, fT4, and TT4 in patients with MCT8 deficiency. Individualized dose adjustments were crucial for achieving therapeutic goals, indicating the need for personalized treatment plans.

Resolving whether and how rare noncoding genetic variants cause Mendelian conditions remains challenging owing to the diverse mechanisms by which they cause disease. Here we demonstrate the utility of single-molecule chromatin fiber sequencing (Fiber-seq) for resolving the mechanistic basis of the Mendelian condition autosomal dominant resistance to thyrotropin (RTSH), which had previously been linked to noncoding variants within a short tandem repeat (STR) variant on chromosome 15.

Abstract Disclosure: H. Grasberger: None. A. Dumitrescu: None. X. Liao: None. E. Swanson: None. R.E. Weiss: None. P. Srichomkwun: None. T. Pappa: None. J. Chen: None. T. Yoshimura: None. P. Hoffmann: None. M. Franca: None. K. Onigata: None. S. Costagliola: None. J. Ranchalis: None. M.R. Vollger: None. A.B. Stergachis: None. J.X. Chong: None. M.J. Bamshad: None. G. Smits: None. G. Vassart: None. S. Refetoff: None. We previously identified a novel form of dominantly inherited resistance to TSH (RTSH), without TSH or TSH receptor mutations, that was linked to a locus on chromosome 15q (Grasberger et al., Hum. Genet. 2005). At birth, affected individuals present with nongoitrous euthyroid hyperthyrotropinemia that persists into adulthood and is frequently associated with high serum thyroglobulin levels not correlated with the degree of TSH elevation. Using whole genome sequencing we now report that noncoding mutations in an intergenic (TTTG)4 short tandem repeat (STR) underlie this form of RTSH in all 82 affected subjects from 12 unrelated families of different ethnic origins. Affected were heterozygous for the deletion of one repeat unit (10 families), or a single nucleotide change within the STR (two families). The STR maps to a primate-specific retrotransposon (AluSx1) with weak thyroid-specific enhancer-like chromatin signature. Paradoxically, some affected individuals appear prone to manifest proliferative thyroid disease since three of the affected subjects in our cohort required thyroidectomy for large nodular goiters in middle age. To investigate the consequence of these noncoding mutations we performed single-molecule chromatin fiber sequencing (Fiber-seq) and RNA-seq on thyroid specimens from subjects representing the two different mutations, and control thyroid tissues (healthy and nontoxic multinodular goiter) without STR mutation. Studies revealed that the mutant STRs stabilize transcription factor (TF) occupancy over the STR site, altering the spacing of adjacent FOXE1 binding elements. Increased TF occupancy at the mutated STR site actuates a complete enhancer cluster that selectively upregulates a bicistronic micro-RNA locus 35 kb downstream. The miRNA primary transcript is specifically overexpressed from the mutant STR allele leading to marked overexpression of the mature miRNA products, MIR7-5P and MIR1179. This was not observed in primary skin fibroblasts from RTSH subjects, consistent with thyroid specific regulation of the enhancer and lack of an extrathyroidal phenotype. An imbalance in signaling pathways targeted by MIR7-5P provides a working model for this novel cause of RTSH. These findings broaden our current knowledge of genetic defects altering pituitary-thyroid feedback regulation and highlight the function of this locus as primate-specific modulator of thyroid function. Presentation: 6/1/2024

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)(4) short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.

To evaluate the combined administration of propylthiouracil (PTU) and levothyroxine (LT4) in managing MCT8 deficiency and identify optimal therapeutic dosages.

The dynamic and continuously evolving field of ophthalmology necessitates rigorous regulatory oversight in the United States. This review outlines the multifaceted Food and Drug Administration's (FDA) approval process for ophthalmic products, detailing the classifications, pathways, and regulatory compliance for devices, drugs, biologics, and combination products. Particular emphasis is placed on distinct frameworks for Class I, II, and III devices, as well as regulations for drugs, biologics, and combination products. The organizational structure of the FDA is detailed, with highlights on specific Ophthalmology oversight divisions, historical regulatory evolution, and initiatives such as Patient-Focused Drug Development. An in-depth examination of the regulatory journey, ranging from initial research to post-marketing surveillance, includes practical guidance through stages such as Pre-Investigational New Drug/Pre-Submission consultations, clinical trials, new drug application/biologics license application/premarket approval submissions, and FDA advisory committee interactions. The article underscores the importance of early interactions with the health authorities, interdisciplinary team collaboration, adherence to current standards, and the anticipation of policy changes to ensure patient safety. It concludes with an analysis of 4 key FDA-approved ophthalmic products, including Eylea®, Luxturna®, Alphagan P®, and the Raindrop® Near Vision Inlay, detailing their contributions to ophthalmic care and offering valuable insights into their respective clinical trials, regulatory pathways, and potential implications. These case studies are included to illustrate both successful and failed ophthalmic product launches, thereby highlighting the importance of alignment with regulatory compliance.

Background: Congenital hypothyroidism due to defects in iodotyrosine deiodinase has variable phenotypes and can present as hypothyroid or with normal thyroid testing. Methods: Whole exome sequencing was performed in individuals from two families originating from different regions of Sudan. Mass spectrometry of urine and serum iodotyrosines was performed on subjects from both families. Results: A novel iodotyrosine deiodinase (IYD) mutation (c.835C>T; R279C) was identified in individuals from two Sudanese families inherited as autosomal recessive. The mutation was identified by multiple in silica analyses to likely be detrimental. Serum and urine monoiodotyrosine (MIT) and diiodotyrosine (DIT) were markedly elevated in the homozygous subjects. Conclusion: Measurement of serum and urine DIT and MIT was more sensitive than that of urine iodine or serum thyroid function tests to determine the effect of the IYD mutation.

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

A family with congenital hypothyroidism was identified with two novel deleterious compound heterozygous thyroid peroxidase (TPO) mutations (c.962C>A, and c.1577C>T). Serum thyroid tests showed higher-than-expected serum-free thyroxine (T4) relative to TT3, while reverse triiodothyronine (rT3) was also elevated. Two siblings manifested a more severe phenotype of developmental delay compared with another sibling and were found to harbor an additional novel heterozygous deleterious iodothyronine deiodinase 1 (DIO1) mutation (c.395G>A). In the context of L-T4 replacement, the decreased D1 activity results in abnormal thyroid hormone metabolism with decreased triiodothyronine (T3) generation from L-T4 and may result in decreased T3 bioavailability during critical stages of development.

As vaccines against COVID-19 became available for distribution, the University of Miami addressed several challenges to facilitate vaccine allocation to the highest risk employees, patients, and students. Advanced use of technology allowed for the automation of key processes in the mass vaccination effort, which expedited vaccine outreach and scheduling, while maintaining routine delivery of healthcare services. The University's employees were initially prioritized for vaccination; employees who opted in were stratified into 5 vaccine administration phases. A similar process was implemented for students. When the state of Florida mandated expansion of vaccine allocation to include individuals aged 65 and older, an algorithm for patients was designed, taking into account age, comorbidities, date of last visit, and presence of an activated patient portal account. Innovative use of technology allowed for 19 000 vaccines to be administered within the first 37 days, which comprised 100% vaccine allotment, without wasting a single vaccine dose.

For the first time in human history, obtaining a COVID-19 vaccine has become essential for the sustainability of our species. As an amazing product of collective ideation, remarkably safe and efficient vaccines have been invented, tested, distributed, and administered to the population on a voluntary basis. The fast-mutating individual behavior of the virus is probably guided by a similar goal of the sustainability of the species. With this commentary, we analyze and compare two means of sustainability through adaptability: collective ideation in the case of humans and individual mutations in the case of viruses — two very different species whose behaviors are driven by sustainability.

Background: Monocarboxylate transporter 8 (MCT8) deficiency is an X-chromosome-linked neurodevelopmental disorder resulting from impaired thyroid hormone transport across the cell membrane. The diagnosis of MCT8 deficiency is typically delayed owing to the late appearance of signs and symptoms as well as the inability of standard biomarkers of neonatal screening to provide early detection. In this study, we report, for the first time, the ability to detect MCT8 deficiency at birth using dried blood spot (DBS) samples. Methods: We retrospectively measured triiodothyronine (T3), thyroxine (T4), and reverse T3 (rT3) levels in DBS samples obtained at 4-5 days of life from 6 infants with genetically confirmed MCT8 deficiency and from 110 controls. The latter consisted of 58 healthy term neonates obtained at the same time, 16 were stored for more than 1 year before measurement to match samples from the MCT8-deficient infants. Ten DBS samples were collected at day 1 of life and 42 samples were from prematurely born neonates. Measurements were carried out in extract from eight millimeters diameter DBS using liquid chromatography-tandem mass spectrometry. Results: Contrary to characteristic iodothyronine abnormalities of MCT8 deficiency during later life, T3 and T4 values were not discriminatory from those of other study groups. In contrast, rT3 was significantly lower. The T3/rT3 ratio was higher in the DBS samples from the MCT8-deficient infants compared with all other groups with no overlap (p < 0.0001). Conclusions: rT3 and T3/rT3 ratio in DBS samples obtained from neonates can serve as biomarkers to detect MCT8 deficiency at birth.

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.

Resistance to thyroid hormone alpha (RTHα) is caused by mutations in thyroid hormone receptor α (THRA). Little is known about the natural history and treatment of RTHα, and diagnosis before the age of 1 year has not been previously reported. A de novo heterozygous THRA mutation (pC380SfsX9) was identified in a 10-month-old female investigated for developmental delay, hypotonia, macrocephaly, and severe constipation. Treatment with levothyroxine was accompanied by an appropriate rise in thyroxine (T4), triiodothyronine (T3), as well as decrease in thyrotropin levels and in the T3/T4 ratio with a trend toward normalization of peripheral markers of thyroid hormone action. THRA pC380SfsX9 results in extreme RTHα.

Background: Mutations of the thyroid hormone (TH)-specific cell membrane transporter, monocarboxylate transporter 8 (MCT8), produce an X-chromosome-linked syndrome of TH deficiency in the brain and excess in peripheral tissues. The clinical consequences include brain hypothyroidism causing severe psychoneuromotor abnormalities (no speech, truncal hypotonia, and spastic quadriplegia) and hypermetabolism (poor weight gain, tachycardia, and increased metabolism, associated with high serum levels of the active TH, T3). Treatment in infancy and childhood with TH analogues that reduce serum triiodothyronine (T3) corrects hypermetabolism, but has no effect on the psychoneuromotor deficits. Studies of brain from a 30-week-old MCT8-deficient embryo indicated that brain abnormalities were already present during fetal life. Methods: A carrier woman with an affected male child (MCT8 A252fs268*), pregnant with a second affected male embryo, elected to carry the pregnancy to term. We treated the fetus with weekly 500 μg intra-amniotic instillation of levothyroxine (LT4) from 18 weeks of gestation until birth at 35 weeks. Thyroxine (T4), T3, and thyrotropin (TSH) were measured in the amniotic fluid and maternal serum. Treatment after birth was continued with LT4 and propylthiouracil. Follow-up included brain magnetic resonance imaging (MRI) and neurodevelopmental evaluation, both compared with the untreated brother. Results: During intrauterine life, T4 and T3 in the amniotic fluid were maintained above threefold to twofold the baseline and TSH was suppressed by 80%, while maternal serum levels remained unchanged. At birth, the infant serum T4 was 14.5 μg/dL and TSH <0.01 mU/L compared with the average in untreated MCT8-deficient infants of 5.1 μg/ and >8 mU/L, respectively. MRI at six months of age showed near-normal brain myelination compared with much reduced in the untreated brother. Neurodevelopmental assessment showed developmental quotients in receptive language and problem-solving, and gross motor and fine motor function ranged from 12 to 25 at 31 months in the treated boy and from 1 to 7 at 58 months in the untreated brother. Conclusions: This is the first demonstration that prenatal treatment improved the neuromotor and neurocognitive function in MCT8 deficiency. Earlier treatment with TH analogues that concentrate in the fetus when given to the mother may further rescue the phenotype.