An in vivo functional assay to characterize human STAT5B genetic variants during zebrafish development.

Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.

Applying Bioinformatic Platforms, In Vitro, and In Vivo Functional Assays in the Characterization of Genetic Variants in the GH/IGF Pathway Affecting Growth and Development.

Heritability accounts for over 80% of adult human height, indicating that genetic variability is the main determinant of stature. The rapid technological development of Next-Generation Sequencing (NGS), particularly Whole Exome Sequencing (WES), has resulted in the characterization of several genetic conditions affecting growth and development. The greatest challenge of NGS remains the high number of candidate variants identified. In silico bioinformatic tools represent the first approach for classifying these variants. However, solving the complicated problem of variant interpretation requires the use of experimental approaches such as in vitro and, when needed, in vivo functional assays. In this review, we will discuss a rational approach to apply to the gene variants identified in children with growth and developmental defects including: (i) bioinformatic tools; (ii) in silico modeling tools; (iii) in vitro functional assays; and (iv) the development of in vivo models. While bioinformatic tools are useful for a preliminary selection of potentially pathogenic variants, in vitro-and sometimes also in vivo-functional assays are further required to unequivocally determine the pathogenicity of a novel genetic variant. This long, time-consuming, and expensive process is the only scientifically proven method to determine causality between a genetic variant and a human genetic disease.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.

Expression of acid-labile subunit (ALS) in developing and adult zebrafish and its role in dorso-ventral patterning during development.

Mammalian acid-labile subunit (ALS) is a serum protein that binds binary complexes between Insulin-like growth factors (IGFs) and Insulin-like growth factor-binding proteins (IGFBPs) extending their half-life and keeping them in the vasculature. Human ALS deficiency (ACLSD), due to homozygous or compound heterozygous mutations in IGFALS, leads to moderate short stature with reduced levels of IGF-I and IGFBP-3. There is only one corresponding zebrafish ortholog gene and it has not yet been studied. In this study we elucidate the role of igfals during zebrafish development. In zebrafish embryos igfals mRNA is expressed throughout development, mainly in the brain and subsequently also in the gut and swimbladder. To determine its role during development, we knocked down igfals gene product using morpholinos (MOs). Igfals morphant embryos displayed dorsalization in different degrees of severity, including a shortened trunk and loss of tail. Furthermore, co-injection of human IGFALS (hIGFALS) mRNA was able to rescue the MO-induced phenotype. Finally, overexpression of either hIGFALS or zebrafish igfals (zigfals) mRNA leads to ventralization of embryos including a reduced head and enlarged tail. These findings suggest that als plays an important role in dorso-ventral patterning during zebrafish development.

The role of acid-labile subunit (ALS) in the modulation of GH-IGF-I action.

Acid-labile subunit (ALS) deficiency (ACLSD) constitutes the first monogenic defect involving a member of the Insulin-like Growth Factor (IGF) binding protein system. The lack of ALS completely disrupts the circulating IGF system. Autocrine/paracrine action of local produced IGF-I could explain the mild effect on growth. In the present work we have revised the more relevant clinical and biochemical consequences of complete ACLSD in 61 reported subjects from 31 families. Low birth weight and/or length, reduced head circumference, height between -2 and -3 SD, pubertal delay and insulin resistance are commonly observed. Partial ACLSD could be present in children initially labeled as idiopathic short stature, presenting low IGF-I levels, suggesting that one functional IGFALS allele is insufficient to stabilize ternary complexes. Dysfunction of the GH-IGF axis observed in ACLSD may eventually result in increased risk for type-2 diabetes and tumor progression. Consequently, long term surveillance is recommended in these patients.

A novel heterozygous STAT5B variant in a patient with short stature and partial growth hormone insensitivity (GHI).

BACKGROUND: The most frequent monogenic causes of growth hormone insensitivity (GHI) include defects in genes encoding the GH receptor itself (GHR), the signal transducer and activator of transcription (STAT5B), the insulin like-growth factor type I (IGF1) and the acid-labile subunit (IGFALS). GHI is characterized by a continuum of mild to severe post-natal growth failure. OBJECTIVE: To characterize the molecular defect in a patient with short stature and partial GHI. PATIENT AND METHODS: The boy was born at term adequate for gestational age from non-consanguineous normal-stature parents. At 2.2 years, he presented proportionate short stature (height -2.77 SDS), wide forehead and normal mental development. Whole-exome analysis and functional characterization (site-directed mutagenesis, dual luciferase reporter assay, immunofluorescence and western immunoblot) were performed. RESULTS: Biochemical and endocrinological evaluation revealed partial GH insensitivity with normal stimulated GH peak (7.8 ng/mL), undetectable IGF1 and low IGFBP3 levels. Two heterozygous variants in the GH-signaling pathway were found: a novel heterozygous STAT5B variant (c.1896G>T, p.K632N) and a hypomorphic IGFALS variant (c.1642C>T, p.R548W). Functional in vitro characterization demonstrated that p.K632N-STAT5b is an inactivating variant that impairs STAT5b activity through abolished phosphorylation. Remarkably, the patient's immunological evaluation displayed only a mild hypogammaglobulinemia, while a major characteristic of STAT5b deficient patients is severe immunodeficiency. CONCLUSIONS: We reported a novel pathogenic inactivating STAT5b variant, which may be associated with partial GH insensitivity and can present without severe immunological complications in heterozygous state. Our results contribute to expand the spectrum of phenotypes associated to GHI.

A homozygous mutation in the highly conserved Tyr60 of the mature IGF1 peptide broadens the spectrum of IGF1 deficiency.

BACKGROUND: IGF1 is a key factor in fetal and postnatal growth. To date, only three homozygous IGF1 gene defects leading to complete or partial loss of IGF1 activity have been reported in three short patients born small for gestational age. We describe the fourth patient with severe short stature presenting a novel homozygous IGF1 gene mutation. RESULTS: We report a boy born from consanguineous parents at 40 weeks of gestational age with intrauterine growth restriction and severe postnatal growth failure. Physical examination revealed proportionate short stature, microcephaly, facial dysmorphism, bilateral sensorineural deafness and mild global developmental delay. Basal growth hormone (GH) fluctuated from 0.2 to 29 ng/mL, while IGF1 levels ranged from -1.15 to 2.95 SDS. IGFBP3 was normal-high. SNP array delimited chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located. IGF1 screening by HRM revealed a homozygous missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). The change of the highly conserved Tyr60 in the mature IGF1 peptide was consistently predicted as pathogenic by multiple bioinformatic tools. Tyr60 has been described to be critical for IGF1 interaction with type 1 IGF receptor (IGF1R). In vitro, HEK293T cells showed a marked reduction of IGF1R phosphorylation after stimulation with serum from the patient as compared to sera from age-matched controls. Mutant IGF1 was also less efficient in inducing cell growth. CONCLUSION: The present report broadens the spectrum of clinical and biochemical presentation of homozygous IGF1 defects and underscores the variability these patients may present depending on the IGF/IGF1R pathway activity.

Genetic Mutations in the GH/IGF Axis.

The GH/IGF axis plays an important role in the control of pre and postnatal growth. At least 48 monogenic defects have been described affecting the production, secretion, and action of GH and IGFs. Molecular defects of the GH/IGF axis resulting in short stature were arbitrarily classified into 4 groups: 1. Combined pituitary hormone deficiency (CPHD) (a. syndromic CPHD and b. non-syndromic CPHD), 2. Isolated GH deficiency (IGHD), 3. GH insensitivity, and 4. IGF-I insensitivity. Genetic diagnosis is obtained in about 30-40% of children with growth retardation, severe IGHD, CPHD, apparent GH or IGF-I insensitivity, and small for gestational age. Increased accessibility to next generation sequencing (NGS) techniques resulted in a significant number of likely pathogenic variants in genes previously associated with short stature as well as in completely novel genes. Functional in vitro assays and in vivo animal models are required to determine the real contribution of these findings.

Partial growth hormone insensitivity and dysregulatory immune disease associated with de novo germline activating STAT3 mutations.

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.

Characterization of four Latin American families confirms previous findings and reveals novel features of acid-labile subunit deficiency.

OBJECTIVE: Acid-labile subunit deficiency (ACLSD), caused by inactivating mutations in both IGFALS gene alleles, is characterized by marked reduction in IGF-I and IGFBP-3 levels associated with mild growth retardation. The aim of this study was to expand the known phenotype and genetic characteristics of ACLSD by reporting data from four index cases and their families. DESIGN: Auxological data, biochemical and genetic studies were performed in four children diagnosed with ACLSD and all available relatives. METHODS: Serum levels of IGF-I, IGFBP-3, acid-labile subunit (ALS), and in vitro ternary complex formation (ivTCF) were determined. After sequencing the IGFALS gene, pathogenicity of novel identified variants was evaluated by in vitro expression in transfected Chinese hamster ovarian (CHO) cells. ALS protein was detected in patients' sera and CHO cells conditioned media and lysates by Western immunoblot (WIB). RESULTS: Four index cases and four relatives were diagnosed with ACLSD. The following variants were found: p.Glu35Glyfs*17, p.Glu35Lysfs*87, p.Leu213Phe, p.Asn276Ser, p.Leu409Phe, p.Ala475Val and p.Ser490Trp. ACLSD patients presented low IGF-I and low or undetectable levels of IGFBP-3 and ALS. Seven out of 8 patients did not form ivTCF. CONCLUSIONS: This study confirms previous findings in ACLSD, such as the low IGF-I and a more severe reduction in IGFBP-3 levels, and a gene dosage effect observed in heterozygous carriers (HC). In addition, father-to-son transmission (father compound heterozygous and mother HC), preservation of male fertility, and marginal ALS expression with potential involvement in preserved responsiveness to rhGH treatment, are all novel aspects, not previously reported in this condition.

Assessment of pathogenicity of natural IGFALS gene variants by in silico bioinformatics tools and in vitro functional studies.

Acid-labile subunit (ALS) is essential for stabilization of IGF-I and IGFBP-3 in ternary complexes within the vascular system. ALS deficient (ALS-D) patients and a subset of children with idiopathic short stature (ISS), presenting IGFALS gene variants, show variable degree of growth retardation associated to IGF-I and IGFBP-3 deficiencies. The aim of this study was to evaluate the potential pathogenicity of eleven IGFALS variants identified in ALS-D and ISS children using in silico and in vitro approaches. We were able to classify seven of these variants as pathogenic since they present impaired synthesis (p.Glu35Lysfs*87, p.Glu35Glyfs*17, p.Asn276Ser, p.Leu409Phe, p.Ser490Trp and p.Cys540Arg), or partial impairment of synthesis and lack of secretion (p.Leu213Phe). We also observed significant reduction of secreted protein for variants p.Ala330Asp, Ala475Val and p.Arg548Trp, while still retaining their ability to form ternary complexes. These findings provide an approach to test the pathogenicity of IGFALS gene variants.

Enhancer turnover and conserved regulatory function in vertebrate evolution.

Mutations in regulatory regions including enhancers are an important source of variation and innovation during evolution. Enhancers can evolve by changes in the sequence, arrangement and repertoire of transcription factor binding sites, but whole enhancers can also be lost or gained in certain lineages in a process of turnover. The proopiomelanocortin gene (Pomc), which encodes a prohormone, is expressed in the pituitary and hypothalamus of all jawed vertebrates. We have previously described that hypothalamic Pomc expression in mammals is controlled by two enhancers-nPE1 and nPE2-that are derived from transposable elements and that presumably replaced the ancestral neuronal Pomc regulatory regions. Here, we show that nPE1 and nPE2, even though they are mammalian novelties with no homologous counterpart in other vertebrates, nevertheless can drive gene expression specifically to POMC neurons in the hypothalamus of larval and adult transgenic zebrafish. This indicates that when neuronal Pomc enhancers originated de novo during early mammalian evolution, the newly created cis- and trans-codes were similar to the ancestral ones. We also identify the neuronal regulatory region of zebrafish pomca and confirm that it is not homologous to the mammalian enhancers. Our work sheds light on the process of gene regulatory evolution by showing how a locus can undergo enhancer turnover and nevertheless maintain the ancestral transcriptional output.

Mutations in CDON, encoding a hedgehog receptor, result in holoprosencephaly and defective interactions with other hedgehog receptors.

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Screening of human LPHN3 for variants with a potential impact on ADHD susceptibility.

Attention deficit hyperactivity disorder (ADHD) is the most common behavioral disorder in childhood, and often has effects detectable into adulthood. Advances in genetic linkage and association analysis have begun to elucidate some of the genetic factors underlying this complex disorder. Recently, we identified LPHN3, a novel ADHD susceptibility gene harbored in 4q, and showed that a LPHN3 common haplotype confers susceptibility to ADHD and predicts effectiveness of stimulant medication. Here we present the mutational analysis of the entire coding region of LPHN3 in a cohort of 139 ADHD subjects and 52 controls from across the USA. We identified 21 variants, of which 14 have been reported and 7 are novel. These include 5 missense, 8 synonymous, and 8 intronic changes. Interestingly, neither susceptibility nor protective haplotype alleles are associated with obviously significant coding region changes, or canonical splice site alterations, suggesting that non-coding variations determining the quantity and/or quality of LPHN3 isoforms are the likely contributors to this common behavioral disorder.

Association of the TGrI29 microsatellite in thyroglobulin gene with autoimmune thyroiditis in a Argentinian population: a case-control study.

Autoimmune thyroid disease (AITD) is a multifactorial disorder that involves a putative association with thyroid autoantigen-specific and immune regulatory genes, as well as environmental factors. The thyroglobulin gene is the main identified thyroid autoantigen-specific gene associated to autoimmune thyroiditis. The aim of this work was to test for evidence of allelic association between autoimmune thyroiditis (AT) and thyroglobulin polymorphism markers in Argentinian patients. We studied six polymorphisms distributed throughout all the thyroglobulin gene: four microsatellites (Tgms1, Tgms2, TGrI29, and TGrI30), one insertion/deletion polymorphism (IndelTG-IVS18), and one exonic single nucleotide polymorphism (c.7589G>A) in 100 AT patients and 100 healthy control subjects. No differences in allele and genotype frequencies distribution were observed between autoimmune thyroiditis cases and controls for Tgms1, Tgms2, TGrI30, IndelTG-IVS18, and c.7589G>A. However, when we analyzed autoimmune thyroiditis patients with the TGrI29 microsatellite we found a significant association between the 197-bp allele and autoimmune thyroiditis (33.50% vs. 19.00% in control group) (P = 0.001). In addition, a significant major prevalence of the 197/201-bp genotype has been also seen in autoimmune thyroiditis subjects (59% vs. 24% in control group, P < 0.0001). In conclusion, our work showed the association between the thyroglobulin gene and autoimmune thyroiditis in Argentinian population and supports the described evidence of thyroglobulin as a thyroid-specific gene linked to AITD.

A novel SIX3 mutation segregates with holoprosencephaly in a large family.

Holoprosencephaly is the most common structural malformation of the forebrain in humans and has a complex etiology including chromosomal aberrations, single gene mutations and environmental components. Here we present the pertinent clinical findings among members of an unusually large kindred ascertained over 15 years ago following the evaluation and subsequent genetic work-up of a female infant with congenital anomalies. A genome-wide scan and linkage analysis showed only suggestive evidence of linkage to markers on chromosome 2 among the most likely of several pedigree interpretations. We now report that a novel missense mutation in the SIX3 holoprosencephaly gene is the likely cause in this family. Molecular genetic analysis and/or clinical characterization now show that at least 15 members of this family are presumed SIX3 mutation gene carriers, with clinical manifestations ranging from phenotypically normal adults (non-penetrance) to alobar holoprosencephaly incompatible with postnatal life. This particular family represents a seminal example of the variable manifestations of gene mutations in holoprosencephaly and difficulties encountered in their elucidation.

Mutations in the human SIX3 gene in holoprosencephaly are loss of function.

Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain; however, the genetics of this heterogeneous and etiologically complex malformation is incompletely understood. Heterozygous mutations in SIX3, a transcription factor gene expressed in the anterior forebrain and eyes during early vertebrate development, have been frequently detected in human HPE cases. However, only a few mutations have been investigated with limited functional studies that would confirm a role in HPE pathogenesis. Here, we report the development of a set of robust and sensitive assays of human SIX3 function in zebrafish and apply these to the analysis of a total of 46 distinct mutations (19 previously published and 27 novel) located throughout the entire SIX3 gene. We can now confirm that 89% of these putative deleterious mutations are significant loss-of-function alleles. Since disease-associated single point mutations in the Groucho-binding eh1-like motif decreases the function in all assays, we can also confirm that this interaction is essential for human SIX3 co-repressor activity; we infer, in turn, that this function is important in HPE causation. We also unexpectedly detected truncated versions with partial function, yet missing a SIX3-encoded homeodomain. Our data indicate that SIX3 is a frequent target in the pathogenesis of HPE and demonstrate how this can inform the genetic counseling of families.

Two distinct compound heterozygous constellations (R277X/IVS34-1G>C and R277X/R1511X) in the thyroglobulin (TG) gene in affected individuals of a Brazilian kindred with congenital goiter and defective TG synthesis.

In this study, we have extended our initial molecular studies of a nonconsanguineous family with two affected siblings and one of their nephews with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin synthesis. Genomic DNA sequencing revealed that the index patient (affected nephew) was heterozygous for a single base change of a cytosine to a thymine at nucleotide 886 in exon 7 (886C>T, mother's mutation) in one allele and for a novel guanine to cytosine transversion at position -1 of the splice acceptor site in intron 34 (IVS34-1G>C, father's mutation) in the other allele. The two affected siblings inherited the 886C>T mutation from their mother and a previously reported cytosine to thymine transition at nucleotide 4588 in exon 22 from their father (4588C>T). The 886C>T and 4588C>T substitutions resulted in premature stop codons at amino acids 277 (R277X) and 1511 (R1511X), respectively. In vitro transcription analysis showed that the exon 35 is skipped entirely when the IVS34-1G>C mutation is present, whereas the wild-type allele is correctly spliced. SSCP (exon 7 and 35) and restriction analysis (exon 22) using Taq I indicated that the two affected siblings, the affected nephew, his mother, and his unaffected brother were all heterozygous for the R277X mutation. The two affected siblings, their father, and three unaffected siblings were all heterozygous for the R1511X mutation, whereas the affected nephew and his father were heterozygous for the IVS34-1G>C mutation. Moreover, in this kindred, we have characterized polymorphisms (insertion/deletion, microsatellite, and single nucleotide polymorphism) located within introns 18 and 29 and exon 44 that are associated with the described mutations. Haplotype analysis with these polymorphic markers in two unrelated Brazilian families (present family studied and previously reported family) harboring the R277X mutation suggests a founder effect for the R277X mutation. In conclusion, the affected individuals of this family are either compound heterozygous for R277X/IVS34-1G>C or R277X/R1511X. This observation further supports that thyroglobulin gene mutations display significant intraallelic heterogeneity.

Five novel inactivating mutations in the thyroid peroxidase gene responsible for congenital goiter and iodide organification defect.

Thyroid peroxidase (TPO) defects, typically transmitted as autosomal recessive traits, result in hypothyroid goiters with failure to convert iodide into organic iodine. We analyzed the TPO gene in 14 unrelated patients with clinical evidence of iodide organification defects. Seven of the affected individuals harbored mutations in the TPO gene; one was compound heterozygous, the others were simply heterozygous for TPO mutations. Five novel mutations have been identified, one of which was found to be a single nucleotide deletion, while the other four were single nucleotide substitutions. A frameshift mutation c.387delC was detected in exon 5 which leads to an early termination signal in exon 7 (p.N129fsX208). Two missense mutations were identified in exon 8. The first, a c.920A>C transversion that results in a p.N307T substitution, was found in two patients. The second, a c.1297G>A transition, results in p.V433M. A c.1496C>T transition was detected in exon 9 that caused the substitution p.P499L. Finally, in exon 14 a c.2422T>C transition was identified, causing a p.C808R change. In addition, the previously reported GGCC duplication in exon 8 (c.1186_1187insGGCC; p.R396fsX472) was also detected in two affected individuals, one of whom was a compound heterozygous (p.R396fsX472/p.V433M).