Soo-Mi Park

An Adult Female With Resistance to Thyroid Hormone Mediated by Defective Thyroid Hormone Receptor α

CONTEXT The first human cases (female, age 6 y; father and daughter, ages 47 and 11 y, respectively) with growth retardation/short stature, skeletal dysplasia, constipation, and defective thyroid receptor α (TRα) have been recently described. OBJECTIVE A 45-year-old, short, overweight female with cognitive impairment, epilepsy, and constipation was investigated. DESIGN AND INTERVENTION Clinical, biochemical, and radiological assessment and THRA sequencing were undertaken. The patients thyroid status and her biochemical and physiological parameters were evaluated at baseline and after T4 therapy. RESULTS The patient exhibits disproportionate short stature, macrocephaly, low free T4/free T3 ratio and rT3 levels, together with subnormal heart and basal metabolic rate. She is heterozygous for a novel frameshift/premature stop (Ala382ProfsX7) THRA mutation, generating a mutant TRα with constitutive corepressor binding and negligible coactivator recruitment, which inhibits its wild-type counterpart in a dominant-negative manner-both in vitro and in mutation-containing patient blood mononuclear cells studied ex vivo. Her alertness and constipation responded to T4 therapy, which readily suppressed TSH levels, raised basal metabolic rate, and normalized elevated muscle creatine kinase, but cardiac parameters (heart rate, contractility) remained relatively refractory. The patient and a previous childhood case showed reduced red cell mass with macrocytosis unresponsive to T4 therapy. CONCLUSIONS Clinical (short stature, macrocephaly, constipation) and biochemical (low free T4/free T3 ratio, subnormal rT3) findings that are congruent with previous cases and newly recognized features (epilepsy) in this adult female with defective TRα define a shared phenotype in TRα-mediated resistance to thyroid hormone, with differential tissue responses to T4 treatment.

Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism

Background Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. Methods We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. Results Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as ‘NIPBL-like’. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. Conclusions Future diagnostic testing in ‘mutation-negative’ CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.

De novo loss-of-function mutations in SETD5, encoding a methyltransferase in a 3p25 microdeletion syndrome critical region, cause intellectual disability.

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations-four nonsense (c.1195A>T [p.Lys399(∗)], c.1333C>T [p.Arg445(∗)], c.1866C>G [p.Tyr622(∗)], and c.3001C>T [p.Arg1001(∗)]) and three frameshift (c.2177_2178del [p.Thr726Asnfs(∗)39], c.3771dup [p.Ser1258Glufs(∗)65], and c.3856del [p.Ser1286Leufs(∗)84])-were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.

X-linked cataract and Nance-Horan syndrome are allelic disorders

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication–triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.

Pathogenic Mitochondrial tRNA Point Mutations: Nine Novel Mutations Affirm Their Importance as a Cause of Mitochondrial Disease

Mutations in the mitochondrial genome, and in particular the mt‐tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies—outlined in a validated pathogenicity scoring system—is therefore invaluable in determining whether novel or rare mt‐tRNA variants are pathogenic. Here, we describe the identification of nine novel mt‐tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT‐TI; m.5541C>T, MT‐TW; m.5690A>G, MT‐TN; m.7451A>T, MT‐TS1; m.7554G>A, MT‐TD; m.8304G>A, MT‐TK; m.12206C>T, MT‐TH; m.12317T>C, MT‐TL2; m.16023G>A, MT‐TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as “definitely pathogenic” mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as ‘possibly pathogenic’ (m.4289T>C, m.7554G>A, and m.8304G>A).

The Clinical and Molecular Characterization of Patients With Dyshormonogenic Congenital Hypothyroidism Reveals Specific Diagnostic Clues for DUOX2 Defects

CONTEXT Mutations in the DUOX2 gene have been associated with transient or permanent congenital hypothyroidism due to a dyshormonogenic defect. OBJECTIVE This study aimed to verify the prevalence of DUOX2 mutations and the associated clinical features in children selected by criteria supporting a partial iodide organification defect (PIOD). PATIENTS AND METHODS Thirty children with PIOD-like criteria were enrolled and genotyped. A detailed clinical characterization was undertaken together with the functional analysis of the DUOX2 variations and the revision of the clinical and molecular data of the literature. RESULTS In this large selected series, the prevalence of the DUOX2 mutations was high (37%). We identified 12 missense variants, one splice site, and three frameshift DUOX2 mutations. Functional analyses showed significant impairment of H2O2 generation with five missense variants. Stop-codon mutants were shown to totally abolish DUOX2 activity by nonsense-mediated RNA decay, exon skipping, or protein truncation. DUOX2 mutations, either mono- or biallelic, were most frequently associated with permanent congenital hypothyroidism. Moreover, the present data suggested that, together with goiter and PIOD, the most significant features to select patients for the DUOX2 analysis are the low free T4 and the high TSH concentrations at the first postnatal serum sampling, despite borderline blood spot TSH. Interestingly, the analysis of previously described DUOX2 mutated cases confirmed the validity of these findings. CONCLUSIONS The defects in the peroxide generation system are common among congenital hypothyroidism patients with PIOD. The most robust clinical parameters for selecting patients for DUOX2 analysis have been identified, and several DUOX2 variants have been functionally characterized.

De novo, heterozygous, loss‐of‐function mutations in SYNGAP1 cause a syndromic form of intellectual disability

De novo mutations (DNM) in SYNGAP1, encoding Ras/Rap GTPase‐activating protein SynGAP, have been reported in individuals with nonsyndromic intellectual disability (ID). We identified 10 previously unreported individuals with SYNGAP1 DNM; seven via the Deciphering Developmental Disorders (DDD) Study, one through clinical analysis for copy number variation and the remaining two (monozygotic twins) via a research multi‐gene panel analysis. Seven of the nine heterozygous mutations are likely to result in loss‐of‐function (3 nonsense; 3 frameshift; 1 whole gene deletion). The remaining two mutations, one of which affected the monozygotic twins, were missense variants. Each individual carrying a DNM in SYNGAP1 had moderate‐to‐severe ID and 7/10 had epilepsy; typically myoclonic seizures, absences or drop attacks. 8/10 had hypotonia, 5/10 had significant constipation, 7/10 had wide‐based/unsteady gait, 3/10 had strabismus, and 2/10 had significant hip dysplasia. A proportion of the affected individuals had a similar, myopathic facial appearance, with broad nasal bridge, relatively long nose and full lower lip vermilion. A distinctive behavioral phenotype was also observed with aggressive/challenging behavior and significant sleep problems being common. 7/10 individuals had MR imaging of the brain each of which was reported as normal. The clinical features of the individuals reported here show significant overlap with those associated with 6p21.3 microdeletions, confirming that haploinsufficiency for SYNGAP1 is responsible for both disorders.

Production and application of polyclonal antibody to human thyroid transcription factor 2 reveals thyroid transcription factor 2 protein expression in adult thyroid and hair follicles and prepubertal testis.

Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.

SDHA related tumorigenesis: a new case series and literature review for variant interpretation and pathogenicity

To evaluate the role of germline SDHA mutation analysis by (1) comprehensive literature review, (2) description of novel germline SDHA mutations and (3) in silico structural prediction analysis of missense substitutions in SDHA.

Neuropathologic Characterization of Pontocerebellar Hypoplasia Type 6 Associated With Cardiomyopathy and Hydrops Fetalis and Severe Multisystem Respiratory Chain Deficiency due to Novel RARS2 Mutations

Abstract Autosomal recessive mutations in the RARS2 gene encoding the mitochondrial arginyl-transfer RNA synthetase cause infantile-onset myoencephalopathy pontocerebellar hypoplasia type 6 (PCH6). We describe 2 sisters with novel compound heterozygous RARS2 mutations who presented perinatally with neurologic features typical of PCH6 but with additional features including cardiomyopathy, hydrops, and pulmonary hypoplasia and who died at 1 day and 14 days of age. Magnetic resonance imaging findings included marked cerebellar hypoplasia, gyral immaturity, punctate lesions in cerebral white matter, and unfused deep cerebral grey matter. Enzyme histochemistry of postmortem tissues revealed a near-global cytochrome c oxidase-deficiency; assessment of respiratory chain enzyme activities confirmed severe deficiencies involving complexes I, III, and IV. Molecular genetic studies revealed 2 RARS2 gene mutations: a c.1A>G, p.? variant predicted to abolish the initiator methionine, and a deep intronic c.613-3927C>T variant causing skipping of exons 6–8 in the mature RARS2 transcript. Neuropathologic investigation included low brain weights, small brainstem and cerebellum, deep cerebral white matter pathology, pontine nucleus neuron loss (in 1 sibling), and peripheral nerve pathology. Mitochondrial respiratory chain immunohistochemistry in brain tissues confirmed an absence of complexes I and IV immunoreactivity with sparing of mitochondrial numbers. These cases expand the clinical spectrum of RARS2 mutations, including antenatal features and widespread mitochondrial respiratory chain deficiencies in postmortem brain tissues.