A truncating TPO mutation (Y55X) in patients with hypothyroidism and total iodide organification defect.

UNLABELLED: Absract Purpose: Mutations in the TPO gene have been reported to cause congenital hypothyroidism (CH), and our aim in this study was to determine the genetic basis of congenital hypothyroidism in two affected children coming from a consanguineous family. METHODS: Since CH is usually inherited in autosomal recessive manner in consanguineous/multi case-families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in linked-gene by Sanger sequencing. RESULTS: The family showed potential linkage to the TPO gene and we detected a non-sense mutation (Y55X) in both cases that had total iodode organification defect (TIOD). The mutation segregated with disease status in the family. Y55X is the only truncating mutation in the exon 2 of the TPO gene reported in the literature and results in the earliest stop codon known in the gene to date. CONCLUSIONS: This study confirms the pathogenicity of Y55X mutation and demonstrates that a nonsense mutation in the amino-terminal coding region of the TPO gene could totally abolish the function of the TPO enzyme leading to TIOD. Thus it helps to establish a strong genotype/phenotype correlation associated with this mutation. It also highlights the importance of molecular genetic studies in the definitive diagnosis and accurate classification of CH.

PLA2G6, encoding a phospholipase A2, is mutated in neurodegenerative disorders with high brain iron.

Neurodegenerative disorders with high brain iron include Parkinson disease, Alzheimer disease and several childhood genetic disorders categorized as neuroaxonal dystrophies. We mapped a locus for infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation (NBIA) to chromosome 22q12-q13 and identified mutations in PLA2G6, encoding a calcium-independent group VI phospholipase A2, in NBIA, INAD and the related Karak syndrome. This discovery implicates phospholipases in the pathogenesis of neurodegenerative disorders with iron dyshomeostasis.

Thyroid dyshormonogenesis is mainly caused by TPO mutations in consanguineous community.

OBJECTIVE: In this study, we aimed to investigate the genetic background of thyroid dyshormonogenesis (TDH). CONTEXT: Thyroid dyshormonogenesis comprises 10-15% of all cases of congenital hypothyroidism (CH), which is the most common neonatal endocrine disorder, and might result from disruptions at any stage of thyroid hormone biosynthesis. Currently seven genes (NIS, TPO, PDS, TG, IYD, DUOX2 and DUOXA2) have been implicated in the aetiology of the disease. DESIGN: As TDH is mostly inherited in an autosomal recessive manner, we planned to conduct the study in consanguineous/multi-case families. PATIENTS: One hundred and four patients with congenital TDH all coming from consanguineous and/or multi-case families. MEASUREMENTS: Initially, we performed potential linkage analysis of cases to all seven causative-TDH loci as well as direct sequencing of the TPO gene in cases we could not exclude linkage to this locus. In addition, in silico analyses of novel missense mutations were carried out. RESULTS: TPO had the highest potential for linkage and we identified 21 TPO mutations in 28 TDH cases showing potential linkage to this locus. Four of 10 distinct TPO mutations detected in this study were novel (A5T, Y55X, E596X, D633N). CONCLUSIONS: This study underlines the importance of molecular genetic studies in diagnosis, classification and prognosis of CH and proposes a comprehensive mutation screening by new sequencing technology in all newly diagnosed primary CH cases.

Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease.

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.

Jervell and Lange-Nielsen syndrome: homozygous missense mutation of KCNQ1 in a Turkish family.

Long QT syndrome is one of the most common cardiac ion channel diseases, but its morbidity and mortality rate can be lessened with an early diagnosis and proper treatment. This cardiac ventricular repolarization abnormality is characterized by a prolonged QT interval and a propensity for ventricular tachycardia (VT) of the torsades de pointes type. The long QT syndrome represents a high risk for presyncope, syncope, cardiac arrest, and sudden death. Jervell and Lange-Nielsen syndrome (JLNS) is a recessively inherited form of long QT syndrome characterized by profound sensorineural deafness and prolongation of the QT interval. Findings have shown that JLNS occurs due to homozygous and compound heterozygous pathogenic variants in KCNQ1 or KCNE1. A 3.5-year-old girl presented to the hospital with recurrent syncope, seizures, and congenital sensorineural deafness. Her electrocardiogram showed a markedly prolonged QT interval, and she had a diagnosis of JLNS. The sequence analysis of the proband showed the presence of a pathogenic homozygous missense variant (c.728G>A, p.Arg243His). Heterozygous mutations of KCNQ1 were identified in her mother, father, and sister, demonstrating true homozygosity. Even with high-dose beta-blocker therapy, the patient had two VT attacks, so an implantable cardioverter defibrillator was fitted. The authors suggest early genetic diagnosis for proper management of the disease in the proband and genetic counseling for both the proband and the girl's extended family.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome.

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotype-phenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR.

Mild and severe congenital primary hypothyroidism in two patients by thyrotropin receptor (TSHR) gene mutation.

Congenital hypothyroidism (CH) is the most commonly encountered endocrinological birth defect, with an incidence of approximately 1 in 3000-4000 live births. It could be sporadic or familial as well as goitrous or non-goitrous. Inactivating mutations of TSHR , which is one of the genes responsible for non-goitrogenic congenital hypothyroidism, are mostly inherited autosomal recessively and result in a wide clinical spectrum owing to the extent of receptor function loss. Here, we report detailed clinical features of two CH cases with TSHR mutations. The first case was diagnosed before the initiation of the national screening program and had a severe clinical phenotype associated with a homozygous inactivating TSHR mutation (P556R), whereas the second case was diagnosed after the introduction of the national screening program and showed a mild clinical presentation and carried another homozygous missense mutation (P162A) in the TSHR gene. We compared the clinical features of our cases with those of previously reported patients with TSHR mutations to enhance the genotype/phenotype correlations between these mutations and corresponding clinical phenotypes.

TSHR is the main causative locus in autosomal recessively inherited thyroid dysgenesis.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and results in mental retardation if untreated. Eighty-five percent of CH cases are due to disruptions in thyroid organogenesis and are mostly sporadic, but about 2% of thyroid dysgenesis is familial, indicating the involvement of genetic factors in the aetiology of the disease. In this study, we aimed to investigate the Mendelian (single-gene) causes of non-syndromic and non-goitrous congenital hypothyroidism (CHNG) in consanguineous or multi-case families. Here we report the results of the second part (n=105) of our large cohort (n=244), representing the largest such cohort in the literature, and interpret the overall results of the whole cohort. Additionally, 50 sporadic cases with thyroid dysgenesis and 400 unaffected control subjects were included in the study. In familial cases, first, we performed potential linkage analysis of four known genes causing CHNG (TSHR, PAX8, TSHB, and NKX2-5) using microsatellite markers and then examined the presence of mutations in these genes by direct sequencing. In addition, in silico analyses of the predicted structural effects of TSHR mutations were performed and related to the mutation specific disease phenotype. We detected eight new TSHR mutations and a PAX8 mutation but no mutations in TSHB and NKX2-5. None of the biallelic TSHR mutations detected in familial cases were present in the cohort of 50 sporadic cases. Genotype/phenotype relationships were established between TSHR mutations and resulting clinical presentations. Here we conclude that TSHR mutations are the main detectable cause of autosomal recessively inherited thyroid dysgenesis. We also outline a new genetic testing strategy for the investigation of suspected autosomal recessive non-goitrous CH.

Computational analysis of missense filamin-A variants, including the novel p.Arg484Gln variant of two brothers with periventricular nodular heterotopia.

BACKGROUND: Periventricular nodular heterotopia (PNH) is a cell migration disorder associated with mutations in Filamin-A (FLNA) gene on chromosome X. Majority of the individuals with PNH-associated FLNA mutations are female whereas liveborn males with FLNA mutations are very rare. Fetal viability of the males seems to depend on the severity of the variant. Splicing or severe truncations presumed loss of function of the protein product, lead to male lethality and only partial-loss-of-function variants are reported in surviving males. Those variants mostly manifest milder clinical phenotypes in females and thus avoid detection of the disease in females. METHODS: We describe a novel p.Arg484Gln variant in the FLNA gene by performing whole exome analysis on the index case, his one affected brother and his healthy non-consanguineous parents. The transmission of PNH from a clinically asymptomatic mother to two sons is reported in a fully penetrant classical X-linked dominant mode. The variant was verified via Sanger sequencing. Additionally, we investigated the impact of missense mutations reported in affected males on the FLNa protein structure, dynamics and interactions by performing molecular dynamics (MD) simulations to examine the disease etiology and possible compensative mechanisms allowing survival of the males. RESULTS: We observed that p.Arg484Gln disrupts the FLNa by altering its structural and dynamical properties including the flexibility of certain regions, interactions within the protein, and conformational landscape of FLNa. However, these impacts existed for only a part the MD trajectories and highly similar patterns observed in the other 12 mutations reported in the liveborn males validated this mechanism. CONCLUSION: It is concluded that the variants seen in the liveborn males result in transient pathogenic effects, rather than persistent impairments. By this way, the protein could retain its function occasionally and results in the survival of the males besides causing the disease.

Novel TSHR mutations in consanguineous families with congenital nongoitrous hypothyroidism.

OBJECTIVE: Nonsyndromic autosomal recessively inherited nongoitrous congenital hypothyroidism (CHNG) can be caused by mutations in TSHR, PAX8, TSHB and NKX2-5. We aimed to investigate mutational frequencies of these genes and genotype/phenotype correlations in consanguineous families with CHNG. DESIGN: Because consanguinity in individuals with a presumptive genetic condition is often an indicator of an autosomal recessive inheritance and allows firmer correlations to be established between genotype and phenotype, we planned to execute our study in consanguineous families. PATIENTS: Hundred and thirty-nine children with CHNG phenotype born to consanguineous families. MEASUREMENTS: First, we investigated cases for evidence of linkage to the four known CHNG genes by microsatellite marker analysis. Mutation analysis by direct sequencing was then performed in those cases in whom linkage to the relevant candidate gene could not be excluded. In addition, in silico analysis of the predicted structural effects of TSHR mutations was performed and related to the mutation-specific disease phenotype. RESULTS: Homozygous germline TSHR mutations were detected in six families (5%), but no mutations were detected in PAX8, TSHB and NKX2-5. Four of TSHR mutations had not previously been described. Genotype-phenotype correlations were established and found to be related to the predicted structural effects of the mutations. CONCLUSIONS: Known causative genes account for the development of CHNG only in a minority of cases, and our cohort should provide a powerful resource to identify novel causative genes and to delineate the extent of locus heterogeneity in autosomal recessively inherited CHNG.

Pantothenate kinase-associated neurodegeneration (PKAN): molecular confirmation of a Turkish patient with a rare frameshift mutation in the coding region of the PANK2 gene.

Here we report the clinical, neuroimaging, and molecular findings of a classic pantothenate kinase-associated neurodegeneration (PKAN) patient of Turkish origin. Our patient is the first reported case of PKAN in Turkey with molecular genetic confirmation of the diagnosis. The frameshift mutation c.821_822delCT of the PANK2 gene detected in our patient has only been described in such classic patients to date, and our case provides further evidence of the association of this mutation with the classic PKAN phenotype. Since this mutation is a rare disease-causing mutation in other populations, further studies of more Turkish PKAN patients will show if it is the result of a founder effect in this population. In our case, molecular diagnosis allowed accurate prenatal genetic testing and counseling for this family. This case report highlights the importance of magnetic resonance imaging and molecular investigation in children who have progressive neurodegenerative symptoms of parkinsonism, dystonia, pyramidal features, and dementia.

Association of urokinase gene 3'-UTR T/C polymorphism with calcium oxalate urolithiasis in children.

INTRODUCTION AND OBJECTIVES: Urokinase is synthesized by various cells such as kidney, pneumocytes, and phagocytes. It cleaves plasminogen to plasmin and hence stimulates fibrinolysis. Urokinase breaks down the matrix protein within the stone and thus prevents stone formation and growth. Urokinase concentrations are lower and urokinase gene 3'-UTR T/C polymorphism is higher in patients with recurrent stones. Our aim was to investigate the role of urokinase gene 3'-UTR T/C polymorphism in childhood recurrent stone disease. MATERIAL AND METHODS: A control group of 40 healthy children having no history of stone formation (group 1) (mean age 10.5+/-4.2 years), 40 children (mean age 10.5+/-4.33 years) who had calcium oxalate stones for the first time (group 2), and 40 patients (mean age 11.2+/-3.8 years) with recurrent calcium oxalate stone disease (group 3) were included in the study. The groups were compared with respect to age, gender and urokinase gene 3'-UTR T/C polymorphism. Polymerase chain reaction-based restriction analysis was used to identify C/T polymorphism of the urokinase gene. RESULTS: No significant difference was observed between the three groups with respect to age and gender, while urokinase gene 3'-UTR T/C gene polymorphism was observed in four patients (10%) from group 3. In groups 1 and 2 there was no patient with T/C polymorphism. CONCLUSIONS: Urokinase 3'-UTR T/C gene polymorphism seems to appear more commonly in children with recurrent calcium oxalate stone disease than in healthy children and in those with stones for the first time. These results suggest that the urokinase gene might play a role in childhood recurrent calcium oxalate stone disease.

Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism.

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.

Digenic DUOX1 and DUOX2 Mutations in Cases With Congenital Hypothyroidism.

Context: The DUOX2 enzyme generates hydrogen peroxide (H2O2), a crucial electron acceptor for the thyroid peroxidase-catalyzed iodination and coupling reactions mediating thyroid hormone biosynthesis. DUOX2 mutations result in dyshormonogenetic congenital hypothyroidism (CH) that may be phenotypically heterogeneous, leading to the hypothesis that CH severity may be influenced by environmental factors (e.g., dietary iodine) and oligogenic modifiers (e.g., variants in the homologous reduced form of NAD phosphate-oxidase DUOX1). However, loss-of-function mutations in DUOX1 have not hitherto been described, and its role in thyroid biology remains undefined. Case Description: We previously described a Proband and her brother (P1, P2) with unusually severe CH associated with a DUOX2 homozygous nonsense mutation (p.R434*); P1, P2: thyrotropin >100 µU/mL [reference range (RR) 0.5 to 6.3]; and P1: free T4 (FT4) <0.09 ng/dL (RR 0.9 to 2.3). Subsequent studies have revealed a homozygous DUOX1 mutation (c.1823-1G>C) resulting in aberrant splicing and a protein truncation (p.Val607Aspfs*43), which segregates with CH in this kindred. Conclusion: This is a report of digenic mutations in DUOX1 and DUOX2 in association with CH, and we hypothesize that the inability of DUOX1 to compensate for DUOX2 deficiency in this kindred may underlie the severe CH phenotype. Our studies provide evidence for a digenic basis for CH and support the notion that oligogenicity as well as environmental modulators may underlie phenotypic variability in genetically ascertained CH.

Comprehensive Screening of Eight Known Causative Genes in Congenital Hypothyroidism With Gland-in-Situ.

CONTEXT: Lower TSH screening cutoffs have doubled the ascertainment of congenital hypothyroidism (CH), particularly cases with a eutopically located gland-in-situ (GIS). Although mutations in known dyshormonogenesis genes or TSHR underlie some cases of CH with GIS, systematic screening of these eight genes has not previously been undertaken. OBJECTIVE: Our objective was to evaluate the contribution and molecular spectrum of mutations in eight known causative genes (TG, TPO, DUOX2, DUOXA2, SLC5A5, SLC26A4, IYD, and TSHR) in CH cases with GIS. Patients, Design, and Setting: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. PATIENTS, DESIGN, AND SETTING: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. RESULTS: Twenty-nine cases harbored likely disease-causing mutations. Monogenic defects (19 cases) most commonly involved TG (12), TPO (four), DUOX2 (two), and TSHR (one). Ten cases harbored triallelic (digenic) mutations: TG and TPO (one); SLC26A4 and TPO (three), and DUOX2 and TG (six cases). Novel variants overall included 15 TG, six TPO, and three DUOX2 mutations. Genetic basis was not ascertained in 20 patients, including 14 familial cases. CONCLUSIONS: The etiology of CH with GIS remains elusive, with only 59% attributable to mutations in TSHR or known dyshormonogenesis-associated genes in a cohort enriched for familial cases. Biallelic TG or TPO mutations most commonly underlie severe CH. Triallelic defects are frequent, mandating future segregation studies in larger kindreds to assess their contribution to variable phenotype. A high proportion (∼41%) of unsolved or ambiguous cases suggests novel genetic etiologies that remain to be elucidated.

A Homozygous Nonsense Thyroid Peroxidase Mutation (R540X) Consistently Causes Congenital Hypothyroidism in Two Siblings Born to a Consanguineous Family.

OBJECTIVE: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder, and mutations in the thyroid peroxidase (TPO) gene have been reported to cause the disease. Our aim in this study was to determine the genetic basis of CH in two affected children coming from a consanguineous family. METHODS: First, we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in the linked gene by Sanger sequencing. By using next-generation sequencing, we also checked if any other mutation was present in the remaining 10 causative CH genes. RESULTS: The family showed potential linkage to the TPO gene, and we detected a homozygous nonsense mutation (R540X) in both cases. The two patients had total iodide organification defect (TIOD). Both the microsatellite marker haplotypes and the mutation segregated with the disease status in the family, i.e. all healthy subjects were either heterozygous carriers or homozygous wild-type, confirming the pathogenic nature of the mutation. Neither was the mutation present in any of the 400 control chromosomes nor were there any other mutations in the remaining causative CH genes. CONCLUSION: This study proves the pathogenicity of R540X mutation and demonstrates the strong genotype/phenotype correlation associated with this mutation. It also highlights the power of working with familial cases in revealing the molecular basis of CH and in establishing accurate genotype/phenotype relationships associated with disease causing mutations.

A Homozygous TPO Gene Duplication (c.1184_1187dup4) Causes Congenital Hypothyroidism in Three Siblings Born to a Consanguineous Family.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disease, and germ-line mutations in the TPO gene cause the inherited form of the disease. Our aim in this study was to determine the genetic basis of congenital hypothyroidism in three affected children coming from a consanguineous Turkish family. Because CH is usually inherited in autosomal recessive manner in consanguineous/multicase families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First, we investigated the potential genetic linkage of the family to any known CH locus, using microsatellite markers, and then screened for mutations in linked-gene by conventional sequencing. The family showed potential linkage to the TPO gene and we detected a homozygous duplication (c.1184_1187dup4) in all cases. The mutation segregated with disease status in the family. This study confirms the pathogenicity of the c.1184_1187dup4 mutation in the TPO gene and helps establish a genotype/phenotype correlation associated with this mutation. It also highlights the importance of molecular genetic studies in the definitive diagnosis and accurate classification of CH.

The Missense Alteration A5T of the Thyroid Peroxidase Gene is Pathogenic and Associated with Mild Congenital Hypothyroidism.

Congenital hypothyroidism (CH) occurs with a prevalence of approximately 1:4000 live births. Defects of thyroid hormone synthesis account for 15-20% of these cases. Thyroid peroxidase (TPO) gene is the most common cause for dyshormonogenesis. So far, more than 60 mutations in the TPO gene have been described, resulting in a variable decrease in TPO bioactivity. We present an 8-day-old male with mild CH who was identified to have a G to A transition in the fifth codon of the TPO gene (c.13G>A; p.Ala5Thr). The unaffected family members were heterozygous carriers of the mutation, whereas 400 healthy individuals of the same ethnic background did not have the mutation. Mutation analysis of 11 known causative CH genes and 4 of our own strong candidate genes with next-generation sequencing revealed no mutations in the patient nor in any other family members. The results of in silico functional analyses indicated partial loss-of-function (LOF) in the resulting enzyme molecule due to mutation. The patient's clinical finding s were consistent with the effect of this partial LOF of the mutation. In conclusion, we strongly believe that A5T alteration in the TPO gene is actually pathogenic and suggest that it should be classified as a mutation.