Diagnostic and predictive value of ultrasound and isotope thyroid scanning, alone and in combination, in infants referred with thyroid-stimulating hormone elevation on newborn screening.

OBJECTIVE: To determine the diagnostic and predictive value of ultrasound and radioisotope scans of the thyroid, alone and in combination, during a single visit after initial referral by the screening laboratory with thyroid-stimulating hormone (TSH) elevation. STUDY DESIGN: Retrospective blind review of ultrasound and radioisotope images followed by final diagnosis based on clinical features, biochemistry, imaging, and molecular genetic study. RESULTS: Infants (n = 97; 61 female) with median birthweight 3.38 kg (range 2.04-4.86) and gestation 40 weeks (range 33-42), underwent successful dual thyroid ultrasound and technetium-99m pertechnetate radioisotope scan in a single center. Combined scanning at the initial visit resulted in a correct final diagnosis in 79 of 97 (81%) cases. One patient was misdiagnosed initially as having athyreosis as the result of delayed radioisotope scan and the diagnosis of ectopia made later on diagnostic challenge. The specificity/sensitivity for radioisotope scan and for ultrasound was as follows: 100%/97% and 100%/55% for ectopia (n = 39); 81%/100% and 54%/100% for athyreosis (n = 18); and 89%/90% and 80%/95% for dyshormonogenesis (n = 20). Neither modality, alone or in combination, predicted final diagnosis in eutopic glands due to hypoplasia (n = 4), transient TSH elevation (n = 12), and status still uncertain (n = 4). CONCLUSION: More than 80% of newborn infants with TSH elevation can be diagnosed correctly on initial imaging with combined radioisotope scan and ultrasound. Ultrasound cannot reliably detect thyroid ectopia. Radioisotope scan, especially if performed late, may show no uptake despite the presence of a eutopic gland.

A clinically euthyroid child with a large goiter due to a thyroglobulin gene defect: clinical features and genetic studies.

A 10-year old child born to consanguineous parents presented with an extremely large goiter, a low free T4 level and free T4 index, and normal TSH concentration. The findings of undetectable thyroglobulin (TG) and low free T4, and an elevated free T3/free T4 ratio suggested the possibility of a defect in TG synthesis. Noteworthy aspects of this case were the extremely elevated thyroidal radioiodide uptake despite a normal TSH concentration and the fact that the reduction in the size of her goiter only occurred when her TSH was suppressed below the normal range. Gene sequencing revealed that the patient was homozygous for a donor splice site mutation in intron 30 (IVS30+1G>C). Isolation of RNA obtained from the thyroid gland by fine needle aspiration and sequencing of the TG cDNA confirmed the prediction that exon 30 was skipped, resulting in an in-frame loss of 46 amino acids.

Novel heterozygous thyrotropin receptor mutation presenting with neonatal hyperthyrotropinaemia, mild thyroid hypoplasia and absent uptake on radioisotope scan.

Hyperthyrotropinaemia [mildly elevated thyrotropin (TSH) with normal thyroxine (T4) levels] demands a full assessment, including clinical examination, thyroid imaging and, where indicated, molecular genetic investigations. A male infant, both of whose parents were on T4 treatment, was referred at age 57 days with mild but persistent TSH elevation (12.7 mU/L) and normal free T4 (19.6 pmol/L), following notification by the screening laboratory of a capillary TSH of 10.7 mU/L (reference range, 1.7-9.1 mU/L) on day 8. Assessment showed a venous free T4 level of 15 pmol/L, venous TSH of 20.9 mU/L, serum thyroglobulin of 63 µg/L (reference range, <50 µg/L), and negative thyroglobulin and thyroid peroxidase antibodies. Thyroid ultrasound showed a eutopic, slightly small gland with heterogeneous texture; however, there was no uptake on radioisotope scan. Molecular genetic studies demonstrated a novel missense heterozygous mutation in the TSH receptor (TSHR) gene (c.1169G>T;p.Cys390Phe) in the child, mother and maternal grandmother, but not in the father. The infant was treated with T4 but this was discontinued at age 3 years when repeat testing showed a free T4 of 16.7 pmol/L (reference range, 9-23 pmol/L) and TSH of 8.5 mU/L (reference range, 0.3-5.5 mU/L). A heterozygous TSHR mutation should be considered in the context of hyperthyrotropinaemia and reduced/absent uptake on radioisotope scan. Detection of this mutation has allowed our patient to discontinue T4 treatment for the moment, with a view to staying off treatment in the long-term.

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and humero-spinal dysostosis.

Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.

TBX15 mutations cause craniofacial dysmorphism, hypoplasia of scapula and pelvis, and short stature in Cousin syndrome.

Members of the evolutionarily conserved T-box family of transcription factors are important players in developmental processes that include mesoderm formation and patterning and organogenesis both in vertebrates and invertebrates. The importance of T-box genes for human development is illustrated by the association between mutations in several of the 17 human family members and congenital errors of morphogenesis that include cardiac, craniofacial, and limb malformations. We identified two unrelated individuals with a complex cranial, cervical, auricular, and skeletal malformation syndrome with scapular and pelvic hypoplasia (Cousin syndrome) that recapitulates the dysmorphic phenotype seen in the Tbx15-deficient mice, droopy ear. Both affected individuals were homozygous for genomic TBX15 mutations that resulted in truncation of the protein and addition of a stretch of missense amino acids. Although the mutant proteins had an intact T-box and were able to bind to their target DNA sequence in vitro, the missense amino acid sequence directed them to early degradation, and cellular levels were markedly reduced. We conclude that Cousin syndrome is caused by TBX15 insufficiency and is thus the human counterpart of the droopy ear mouse.

A new heterozygous mutation (D196N) in the Gs alpha gene as a cause for pseudohypoparathyroidism type IA in a boy who had gallstones.

BACKGROUND: Pseudohypoparathyroidism (PHP) is characterized by hypocalcemia and hyperphosphatemia in association with an increased secretion of parathyroid hormone (PTH) due to decreased target tissue responsiveness to PTH. Patients with PHP type Ia are not only resistant to PTH, but also to other hormones that bind to receptors coupled to stimulatory G protein (Gsalpha). PHP Ia and Albright hereditary osteodystrophy (AHO) are caused by a reduced activity of the Gsalpha protein. Heterozygous inactivating Gs alpha (GNAS) gene mutations have been identified in these patients. METHODS: We studied a boy with PHP Ia. During follow-up the patient developed elevated liver enzyme serum levels and abdominal discomfort. Gsalpha activity was measured in erythrocyte membranes from the patient and the GNAS coding region of Gsalpha sequenced. RESULTS: Gsalpha activity was reduced (62%) and molecular analysis revealed a new heterozygous GNAS gene mutation (D196N). Gallstones were diagnosed and cholecystectomy was performed. Biochemical analysis revealed cholesterol stones, a condition that was not reported before in PHP Ia. CONCLUSIONS: Cholesterol gallstones may rarely be associated with PHP Ia and should be taken into account.

Non-immune goiter and hypothyroidism in a 19-week fetus: a plea for conservative treatment.

Hypothyroidism was documented by cordocentesis at 19 weeks in a fetus with non-immune goiter. Intra-amniotic thyroxine was injected at 25 weeks when amniotic fluid volume increased. Psychomotor outcome was normal. We argue that intra-amniotic thyroxine should not be used to treat the hypothyroidism but only to correct the development of polyhydramnios.

A Novel Homozygous Mutation in the Solute Carrier Family 26 Member 7 Gene Causes Thyroid Dyshormonogenesis in a Girl with Congenital Hypothyroidism.

We investigated the genetic cause of thyroid dyshormonogenesis in a girl with congenital hypothyroidism. Genetic analysis showed that she was homozygous for a hitherto not described mutation (c.1432_1433delGT, p.V478KfsX11) in the solute carrier family 26 member 7 (SLC26A7) gene. SLC26A7 is proposed to be an anion transporter in the thyroid gland. The mutation leads to a frameshift and a premature stop codon. The predicted protein is truncated and very likely to be nonfunctional if it was expressed at all. In addition, in silico studies predict the mutation to be pathogenic.

Determination of thyroid volume in infants with suspected congenital hypothyroidism-the limitations of both subjective and objective evaluation.

OBJECTIVE: To compare two methods of assessing gland size on thyroid ultrasound in newborn infants with suspected congenital hypothyroidism (CH). METHODS: Images from infants with eutopic glands referred between 2007 and 2013 were evaluated blind by two sets of observers. Subjective gland size was categorised as small, borderline-small, normal, borderline-large and large. Objective gland volume, calculated as the sum of each lobe using the prolate ellipsoid formula (length x width x depth x π/6), was put into corresponding categories: <0.8, 0.81-1.0, 1.1- <2.2, 2.2-2.4 and >2.4 ml, derived from normative Scottish data. RESULTS: Of 36 infants, permanent CH was present in 17, transient CH in 17, status uncertain in 2. Mean (SD) intraobserver error for thyroid volume measurement was 0.11 (0.23) ml [8.3%]. Subjective assessment by two observers was discordant in only four (10.8%) infants. However, subjective vs objective evaluation was discordant in 14 (39%). Eight (three permanent, five transient CH) had large glands subjectively but normal glands objectively; and six (four transient CH) had normal glands subjectively but small glands objectively. The former infants all showed a single flattened curve to the anterior thyroid margin, giving an impression of bulkiness. Gland shape was normal in the latter infants. CONCLUSION: Neither subjective nor objective evaluation predicts permanent vs transient CH. Altered gland shape may confound both methods, and undermine use of the conventional formula for measuring lobe volume. ADVANCES IN KNOWLEDGE: Until more refined methods are available for assessing thyroid size, both subjective and objective evaluation are recommended in CH.

Identification of Transient Receptor Potential Channel 4-Associated Protein as a Novel Candidate Gene Causing Congenital Primary Hypothyroidism.

BACKGROUND: Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. METHODS: To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (TRPC4AP), was sequenced in 179 further patients with TD. Expression of TRPC4AP in human thyroid was investigated using RT-PCR. Trpc4ap- functional analysis was performed in Xenopus laevis using Morpholino (MO) antisense oligomers. RESULTS: WES identified a likely damaging mutation in TRPC4AP leading to a de novo stop codon p.Q552*. Targeted sequencing of TRPC4AP demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that TRPC4AP is expressed in human thyroid gland tissue. Using Xenopus laevis, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in Trpc4ap MO knockdowns compared to controls and by 41% in "Clustered Regularly Interspaced Short Palindromic Repeats"/Cas9-mediated gene knockout experiments. DISCUSSION: A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by IKBKG gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. CONCLUSION: TRPC4AP was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.

Brachy-syndactyly caused by loss of Sfrp2 function.

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.

Novel Mutations in the NKX2.1 gene and the PAX8 gene in a Boy with Brain-Lung-Thyroid Syndrome.

OBJECTIVE: To elucidate the molecular mechanism which causes thyroid dysgenesis (TD) in a boy with brain-lung-thyroid syndrome. DESIGN, PATIENTS, MEASUREMENTS: We describe a patient with TD, respiratory disease and cerebral palsy who is heterozygous for mutations in two different genes, the PAX8 (p.E234K) and the NKX2.1 (p.A329GfsX108). In vitro studies were performed to functionally characterize these mutations. Congenital hypothyroidism (CH) was identified by neonatal screening associated with a hypoplastic thyroid gland. Postpartum he developed a brain-lung-thyroid syndrome with severe respiratory failure, symptomatic epilepsy and a considerable psychomotor retardation. The DNA-binding capability and the transcriptional activity of the two mutated transcription factors were investigated in vitro. RESULTS: The NKX2.1 mutation did not show any transcriptional activity and had almost no DNA-binding. The PAX8 mutation was normally located to the nucleus and showed a normal transactivation and a normal binding to the known downstream targets. CONCLUSIONS: The molecular defect explaining the phenotype of brain-lung-thyroid syndrome was identified. To what extent the PAX8 mutation contributes to the phenotype needs to be further investigated. We recommend to screen patients with CH and TD for mutations in all known TD candidate genes.

A further case of brain-lung-thyroid syndrome with deletion proximal to NKX2-1.

Brain-lung-thyroid syndrome (OMIM #610978) is associated with mutations in the NK2 homeobox 1 (NKX2-1) gene, a transcription factor important in development. 50% of patients are affected by the full triad, comprising congenital hypothyroidism, benign hereditary chorea and infant respiratory distress syndrome. Four cases have previously been reported where a patient has features consistent with brain-lung-thyroid syndrome and a chromosome 14q13 deletion adjacent to, but not disrupting, NKX2-1. We present a patient who has a phenotype consistent with brain-lung-thyroid syndrome, featuring congenital hypothyroidism and choreoathetoid movements with gross motor delay. Thyroid ultrasound showed a small-normal gland and spontaneous resolution of hypothyroidism. Array CGH revealed a de novo 14q13.2-3 deletion adjacent to but not directly involving NKX2-1. Sequencing of NKX2-1 was normal. This report highlights a further case of chromosomal deletion adjacent to NXK2-1 in a patient with a phenotype consistent with brain-lung-thyroid syndrome, and confirms that array-CGH is a useful test in the investigation of congenital hypothyroidism. Deletion of the adjacent gene MBIP in most reported cases so far may be relevant to the pathogenesis of brain-lung-thyroid syndrome. Deletion of nearby promoter or enhancer elements acting on NKX2-1 could also be an important factor. However, further work is needed to elucidate the pathogenesis of the brain-lung-thyroid phenotype in such cases.

Familial Central Hypothyroidism Caused by a Novel IGSF1 Gene Mutation.

BACKGROUND: Congenital hypothyroidism of central origin (CH-C) is a rare disease in which thyroid hormone deficiency is caused by insufficient thyrotropin stimulation of a normal thyroid gland. A recently described syndrome of isolated CH-C and macroorchidism was attributed to loss-of-function mutations of the immunoglobulin superfamily, member 1 gene (IGSF1). PATIENTS AND METHODS: CH-C was diagnosed in three siblings. The TRH, TRHR, and TSHB genes were sequenced followed by whole-exome sequencing in the proband. A mutation identified in IGSF1 was analyzed by direct PCR sequencing in family members. The effects of the mutation were assessed by in vitro studies in HEK293 cells. RESULTS: The index case was negative for mutations in TRH, TRHR, and TSHB. Whole-exome sequencing revealed a novel insertion mutation in IGSF1, c.2284_2285insA, p.R762QfsX7, which was confirmed by direct PCR sequencing and was identified in six additional family members. The mutation introduces a frame-shift and premature stop codon in the seventh Ig loop, thereby truncating IGSF1. In vitro studies revealed that the mutated IGSF1-R762QfsX7 migrates as a doublet at ∼28 kDa, which is far smaller than the wild type protein (130-140 kDa). Both bands were endonuclease H sensitive, indicating immature glycosylation and failure of the protein to traffic out of the endoplasmic reticulum to the plasma membrane. Further phenotypic findings in the family included macroorchidism and infertility in the uncle and mild neurological phenotypes in the affected males, such as hypotonia, delayed psychomotor development, clumsy behavior, and attention deficit disorder. CONCLUSIONS: We identified a novel insertion mutation in the IGSF1 gene and further delineated the phenotype of the IGSF1-deficiency syndrome. Our findings indicate a possible association between an IGSF1 mutation and neurological phenotypes.

Expression profiling of human fetal growth plate cartilage by EST sequencing.

The differentiation of mesenchymal stem cells into hypertrophic chondrocytes is an integral and multistep process important in pattern formation, endochondral ossification, and postnatal growth of the skeleton. In recent years, novel genes involved in these processes have been identified, but still only little is known about the large-scale gene expression profile during skeletal development. We initiated an expressed sequence tag (EST) project aiming at the identification of genes and pathways involved in this complex process. Candidate genes are expected to be of value for diagnosis and treatment of monogenic and multigenic heritable disorders of the skeleton. Here, we describe the sequences of 4,748 clones from a human growth plate cartilage cDNA library generated from 20 weeks prenatal-2 years postnatal specimens. In silico analysis of these sequences revealed 1,688 individual transcription units, corresponding to known (1,274) and to novel, yet uncharacterised potential genes (414). The tissue specificity of the library was reflected by its corresponding EST profile representing a total of approximately 10% proteins already shown to be involved in cartilage/bone development or homeostasis. The EST profile also reflects the developmental stage of the tissue with significant differences in the expression of matrix proteins compared to corresponding EST profiles from 8-12 and 12-20 week human fetal cartilage. Calculation of the relative frequency of transcripts in our cDNA library, as compared to their abundance in other EST datasets, revealed a set of approximately 200 genes, including 81 novel, yet uncharacterised genes, showing increased expression. These genes represent candidates for the large number of osteochondrodysplasias for which the causative gene defects have not yet been identified.

Identification of four novel COL10A1 missense mutations in schmid metaphyseal chondrodysplasia: further evidence that collagen X NC1 mutations impair trimer assembly.

Schmid metaphyseal chondrodysplasia (SMCD) is an autosomal dominant disorder affecting the growth plate cartilage of long bones caused by heterozygous mutations in the gene for collagen X (COL10A1), a short-chain collagen expressed by hypertrophic chondrocytes of growth plate cartilage. In this paper we analyzed six unrelated patients clinically determined as affected by SMCD, and characterized four missense mutations, c.52G>A (p.G18R), c.1744T>G (p.Y582D), c.1792T>G (p.Y598D) and c.1958A>C (p.Q653P). These mutations were clustered in the two regions of the collagen X protein shown to contain all previous SMCD mutations; the signal sequence cleavage site (p.G18R), or the C-terminal NC1 trimerization domain (p.Y582D, p.Y598D and p.Q653P). To determine the functional effect of the mutations we produced engineered p.Y582D, p.Y598D and p.Q653P cDNA and expressed these in vitro. Our data showed that while the wild-type collagen X assembled in vitro into trimers that were stable to SDS-PAGE analysis, p.Y582D (the most N-terminal of the SMCD NC1 mutations described), p.Q653P, and the previously analyzed p.Y598D impair collagen X trimerization. However, in two patients no mutations were detected despite complete sequence analysis of the COL10A1 coding region, the exon-intron splice consensus sequences and the 500bp gene promoter region. Heterozygosity for known polymorphisms ruled out major COL10A1 gene deletions and Southern analysis excluded major rearrangements. The data suggest that in these two patients, SMCD results from mutations at another gene locus. No mutations were detected in RMRP, the gene for cartilage-hair hypoplasia that has phenotypic overlap with SMCD.

Cytochrome P4501B1 mutations cause only part of primary congenital glaucoma in Ecuador.

PURPOSE: To determine the role of cytochrome P450IBI (CYP1B1) mutations in causing primary congenital glaucoma (PCG) in a cohort of Native Americans from Quito, Ecuador. MATERIALS AND METHODS: Seventeen patients with PCG from 15 Native American families were recruited from the Ophthalmology Clinic at Hospital Metropolitano, Quito, Ecuador. Experienced ophthalmologists examined all affected study subjects. Purified DNA was prepared from peripheral blood samples and CYP1B1 coding exons (exons 2 and 3) were amplified and sequenced. Southern blot was performed only on those affected patients who showed no mutations in the CYP1B1 coding exons. RESULTS: The molecular basis of PCG in two families was determined: two novel mutations (a deletion and a point mutation) and one novel polymorphism in CYP1B1 were identified in addition to a previously described single amino acid substitution. Southern blot analyses on whole genomic DNA from affected individuals in whom no mutations were identified by the direct PCR/sequencing approach did not detect any large rearrangements or mutations outside the coding region. CONCLUSION: These findings suggest that mutations in CYPIBI are not a major cause of PCG in this population and that at least one additional locus for this condition is responsible for most cases. Further, the PCG phenotype did not correlate readily with the molecular basis of the disorder, suggesting that careful clinical analysis of the phenotype cannot predict the molecular basis of the disease with accuracy.

A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey.

BACKGROUND: Isolated central congenital hypothyroidism (ICCH) is rare but important. Most ICCH patients are diagnosed later, which results in severe growth failure and intellectual disability. OBJECTIVE: We describe a boy with ICCH due to a large homozygous TSHß gene deletion. RESULTS: A 51-day-old male Turkish infant, whose parents were first cousins, was admitted for evaluation of prolonged jaundice. His clinical appearance was compatible with hypothyroidism. Venous thyrotropin (TSH) was undetectably low, with a subsequent low free T4 and a low free T3, suggestive of central hypothyroidism. Using different PCR protocols, we could not amplify both coding exons of the boy's TSHß gene, which suggested a deletion. An array comparative genomic hybridization (aCGH) using specific probes around the TSHß gene locus showed him to be homozygous for a 6-kb deletion spanning all exons and parts of the 5' untranslated region of the gene. CONCLUSIONS: Infants who are clinically suspected of having hypothyroidism should be evaluated thoroughly, even if their TSH-based screening result is normal. In cases with ICCH and undetectably low TSH serum concentrations, a TSHß gene deletion should be considered; aCGH should be performed when gene deletions are suspected. In such cases, PCR-based sequencing techniques give negative results.

A new mutation in the promoter region of the PAX8 gene causes true congenital hypothyroidism with thyroid hypoplasia in a girl with Down's syndrome.

BACKGROUND: Thyroid dysfunction is common in newborn infants with Down's syndrome (DS), but defects causing classic thyroid dysgenesis (TD) with permanent congenital hypothyroidism (CH) have not been described. OBJECTIVE: We studied a girl with DS and CH who had a mutation in the promoter sequence of the PAX8 gene. RESULTS: A female infant was found to have trisomy 21 and CH, with a venous thyrotropin (TSH) of >150 mU/L and a free thyroxine (fT4) of 15.1 pmol/L (day 12). Thyroid peroxidase antibodies and thyroglobulin antibodies were elevated. Scintigraphy showed normal uptake, but ultrasound identified a small gland with heterogenous echotexture and cystic changes. Sequence analysis of the PAX8 gene revealed a new heterozygous maternally inherited mutation (-3C>T) close to the transcription initiation site. Electromobility shift assay studies of the wild type and the mutant PAX8 sequence incubated with nuclear extracts from PCCL3 cells exhibited that the sequence at position -3 is not involved in specific protein binding. However, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro tested in HEK, PCCL3, as well as in HeLa cells, indicating that this mutation is very likely to lead to reduced PAX8 expression. CONCLUSIONS: The persistent CH in this patient with DS is likely to be attributable to the diminished PAX8 expression due to a new heterozygous mutation in the PAX8 promoter sequence. Our case shows that true CH may occur in DS, as in the general population. Furthermore, it is possible that the trisomy 21 itself may have resulted in a more severe phenotypic expression of the PAX8 mutation in the child than the mother.