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Background: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. Methods: A genotype–phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. Results: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. Conclusion: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

Background: Pathogenic/likely pathogenic (P/LP) variants in the KCNQ1-encoded Kv7.1 potassium channel cause type 1 long QT syndrome (LQT1). Despite the revamped 2015 American College of Medical Genetics (ACMG) variant interpretation guidelines, the burden of KCNQ1 variants of uncertain significance (VUS) in patients with LQTS remains ∼30%. Objective: The purpose of this study was to determine whether a phenotype-enhanced (PE) variant classification approach could reduce the VUS burden in LQTS genetic testing. Methods: Retrospective analysis was performed on 79 KCNQ1 missense variants in 356 patients from Mayo Clinic and an independent cohort of 42 variants in 225 patients from Amsterdam University Medical Center (UMC). Each variant was classified initially using the ACMG guidelines and then readjudicated using a PE-ACMG framework that incorporated the LQTS clinical diagnostic Schwartz score plus 4 “LQT1-defining features”: broad-based/slow upstroke T waves, syncope/seizure during exertion, swimming-associated events, and a maladaptive LQT1 treadmill stress test. Results: According to the ACMG guidelines, Mayo Clinic variants were classified as follows: 17 of 79 P variants (22%), 34 of 79 LP variants (43%), and 28 of 79 VUS (35%). Similarly, for Amsterdam UMC, the variant distribution was 9 of 42 P variants (22%), 14 of 42 LP variants (33%), and 19 of 42 variants VUS (45%). After PE-ACMG readjudication, the total VUS burden decreased significantly from 28 (35%) to 13 (16%) (P = .0007) for Mayo Clinic and from 19 (45%) to 12 (29%) (P = .02) for Amsterdam UMC. Conclusion: Phenotype-guided variant adjudication decreased significantly the VUS burden of LQT1 case–derived KCNQ1 missense variants in 2 independent cohorts. This study demonstrates the value of incorporating LQT1-specific phenotype/clinical data to aid in the interpretation of KCNQ1 missense variants identified during genetic testing for LQTS.

ABCC8 and KCJN11 mutations cause the most severe diazoxide-resistant forms of congenital hyperinsulinism (CHI). Somatostatin analogues are considered as secondline treatment in diazoxide-unresponsive cases. Current treatment protocols include the first-generation somatostatin analogue octreotide, although pasireotide, a second-generation somatostatin analogue, might be more effective in reducing insulin secretion. Herein we report the first off-label use of pasireotide in a boy with a severe therapy-resistant form of CHI due to a homozygous ABCC8 mutation. After partial pancreatectomy, hyperinsulinism persisted; in an attempt to prevent further surgery, off-label treatment with pasireotide was initiated. Short-acting pasireotide treatment caused high blood glucose level shortly after injection. Long-acting pasireotide treatment resulted in more stable glycemic control. No side effects (e.g., central adrenal insufficiency) were noticed during a 2-month treatment period. Because of recurrent hypoglycemia despite a rather high carbohydrate intake, the boy underwent near-total pancreatectomy at the age of 11 months. In conclusion, pasireotide treatment slightly improved glycemic control without side effects in a boy with severe CHI. However, the effect of pasireotide was not sufficient to prevent near-total pancreatectomy in this case of severe CHI.

Background: Pituitary resistance to thyroid hormone (PRTH) is often seen in congenital hypothyroidism (CH), presenting as elevated thyrotropin (TSH) values despite (high-)normal thyroid hormone (TH) values achieved by levothyroxine treatment. In this study, we describe a girl with CH who was referred because of difficulties interpreting thyroid function tests. She was thought to have PRTH associated with CH, but genetic studies discovered a pathogenic variant in THRB, causing resistance to TH (RTH-β). Methods: Clinical, genetic, and biochemical data of the proband's family were collected. Results: The 3-year-old girl was diagnosed with CH due to a homozygous pathogenic c.470del p.(Asn157Thrfs*3) SLC5A5 variant in the neonatal period. She needed a notably high levothyroxine dose to normalize TSH, leading to high free thyroxine levels. There were no signs of hyperthyroidism. Sequencing identified a heterozygous pathogenic c.947G>A p.(Arg316His) THRB variant. Conclusions: To our knowledge, this is the first report of concomitant SLC5A5 and THRB variants causing CH and RTH-β.

Pathogenic variants in TSHB are known to cause severe isolated central congenital hypothyroidism (CH). In this study, we present the clinical, biochemical, and genetic features of the first patient with a mild central CH phenotype. We identified a novel homozygous variant in TSHB: (Chr1: NM_000549.5):c.290A>G p.(Tyr97Cys) in a newborn girl detected by neonatal CH screening, whose central CH was initially overlooked because of misinterpretation of her plasma-free thyroxine (fT4) concentration. This report adds to the phenotypic spectrum of TSHB variants and underlines the importance of using age-specific fT4 reference intervals to diagnose central CH.

Background and Aims: Adenosine triphosphate-binding cassette subfamily B member 4 (ABCB4) deficiency may lead to progressive familial intrahepatic cholestasis type 3 (PFIC3), biliary cirrhosis, low phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptive-induced cholestasis (CIC) or may remain asymptomatic. The long-term course, quality of life and histology were investigated in ABCB4 deficiency. Methods: Adult carriers of ABCB4 gene variants from two regional academic centres were analysed by history taking, electronic patient files, physical examination, blood analysis, abdominal ultrasound (US) and liver elastography. Patients completed a 36-Item Short Form Health Survey (SF-36) for quality of life and a Visual Analogue Scale (VAS) for pruritus. Available liver specimens were re-classified according to the Nakanuma scoring system, so far validated for primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) only. Quality of life data were compared to published data of patients with PBC, PSC and the general population. Results: Sixty-seven patients were identified, 64 (96%) were alive at the time of analysis and 62 (93%) were (at some time) treated with ursodeoxycholic acid (UDCA). Two patients died of cholangiocarcinoma (CCA), and one of decompensated biliary cirrhosis. Three additional deaths of CCA were reported in first-degree relatives. Transplant-free survival was 91% (median follow-up 14 years). Liver stiffness was normal (

Background Cardiotoxicity presenting as cardiomyopathy is a common side effect in cancer treatment especially with anthracyclines. The role of genetic predisposition is still being investigated. Case summary Four unrelated patients with a familial burden for cardiac disease, who developed cardiomyopathy after anthracycline treatment are presented. Case 1 received chemotherapy for breast cancer and developed a dilated left ventricle just after treatment. Her father had died unexpectedly while being screened for heart transplant. Case 2 was known with a family history of sudden cardiac death prior to her breast cancer diagnosis. She received anthracycline-containing chemotherapy treatment twice in 5 years due to recurrence of breast cancer. During that period, two brothers developed a cardiomyopathy. Eighteen years later, a genetic predisposition for cardiomyopathy was ascertained and at screening an asymptomatic non-ischaemic cardiomyopathy was established. Case 3 was diagnosed with a dilated cardiomyopathy 1 year after chemotherapy treatment for breast cancer. Her mother had developed a dilated cardiomyopathy several years before. Case 4 received chemotherapy treatment for Non-Hodgkin’s lymphoma and developed dilated cardiomyopathy 1 year later. His brother died from congestive heart failure which he developed after chemotherapy for Non-Hodgkin’s lymphoma and a grandmother had died suddenly during child delivery. In all four cases, genetic screening showed (likely) pathogenic variants in cardiomyopathy-associated genes. Discussion Current guidelines recommend cardiac evaluation in cancer patients receiving chemotherapy based on the presence of cardiovascular risk factors at the start of treatment. This series emphasizes the importance of including a thorough family history in this process.

Background: Next-generation sequencing gene panels are increasingly used for genetic diagnosis in inherited cardiac diseases. Besides pathogenic variants, multiple variants, variants of uncertain significance (VUS) and incidental findings can be detected. Such test results can be challenging for counselling and clinical decision making. Methods: We present patient cases to illustrate the challenges that can arise when unclear genetic test results are detected in cardiogenetic gene panels. Results: We identified three types of challenging gene panel results: 1) one or more VUS in combination with a pathogenic variant, 2) variants associated with another genetic heart disease, and 3) variants associated with a syndrome involving cardiac features. Conclusion: Large gene panels not only increase the detection rates of pathogenic variants but also of variants with uncertain pathogenicity, multiple variants and incidental findings. Gene panel results can be challenging for genetic counselling and require proper pre-test and post-test counselling. We advise evaluation of challenging cases by a multidisciplinary team.

Background Genetic heterogeneity is common in inherited cardiac diseases. Next-generation sequencing gene panels are therefore suitable for genetic diagnosis. We describe the results of implementation of cardiomyopathy and arrhythmia gene panels in clinical care. Methods We present detection rates for variants with unknown (class 3), likely (class 4), and certain (class 5) pathogenicity in cardiogenetic gene panels since their introduction into diagnostics. Results In 936 patients tested on the arrhythmia panel, likely pathogenic and pathogenic variants were detected in 8.8% (4.6% class 5; 4.2% class 4), and one or multiple class 3 variants in 34.8%. In 1970 patients tested on the cardiomyopathy panel, likely pathogenic and pathogenic variants were detected in 19.8% (12.0% class 5; 7.9% class 4), and one or multiple class 3 variants in 40.8%. Detection rates of all different classes of variants increased with the increasing number of genes on the cardiomyopathy gene panel. Multiple variants were detected in 11.7% and 28.5% of patients on the arrhythmia and cardiomyopathy panels respectively. In more recent larger versions of the cardiomyopathy gene panel the detection rate of likely pathogenic and pathogenic variants only slightly increased, but was associated with a large increase of class 3 variants. Conclusion Overall detection rates (class 3, 4, and 5 variants) in a diagnostic setting are 44% and 61% for the arrhythmia and cardiomyopathy gene panel respectively, with only a small minority of likely pathogenic and pathogenic variants (8.8% and 19.8% respectively). Larger gene panels can increase the detection rate of likely pathogenic and pathogenic variants, but mainly increase the frequency of variants of unknown pathogenicity. Electronic supplementary material The online version of this article (10.1007/s12471-019-1250-5) contains supplementary material, which is available to authorized users.

Aims Catecholaminergic polymorphic ventricular tachycardia (CPVT) and short QT syndrome (SQTS) are inherited arrhythmogenic disorders that can cause sudden death. Numerous genes have been reported to cause these conditions, but evidence supporting these gene–disease relationships varies considerably. To ensure appropriate utilization of genetic information for CPVT and SQTS patients, we applied an evidence-based reappraisal of previously reported genes. Methods and results Three teams independently curated all published evidence for 11 CPVT and 9 SQTS implicated genes using the ClinGen gene curation framework. The results were reviewed by a Channelopathy Expert Panel who provided the final classifications. Seven genes had definitive to moderate evidence for disease causation in CPVT, with either autosomal dominant (RYR2, CALM1, CALM2, CALM3) or autosomal recessive (CASQ2, TRDN, TECRL) inheritance. Three of the four disputed genes for CPVT (KCNJ2, PKP2, SCN5A) were deemed by the Expert Panel to be reported for phenotypes that were not representative of CPVT, while reported variants in a fourth gene (ANK2) were too common in the population to be disease-causing. For SQTS, only one gene (KCNH2) was classified as definitive, with three others (KCNQ1, KCNJ2, SLC4A3) having strong to moderate evidence. The majority of genetic evidence for SQTS genes was derived from very few variants (five in KCNJ2, two in KCNH2, one in KCNQ1/SLC4A3). Conclusions Seven CPVT and four SQTS genes have valid evidence for disease causation and should be included in genetic testing panels. Additional genes associated with conditions that may mimic clinical features of CPVT/SQTS have potential utility for differential diagnosis.

Two siblings presented with early lethal noncompaction cardiomyopathy (NCCM). Both carry compound heterozygous variants in the ryanodine receptor gene (RYR2). Evolving animal and human data have begun to implicate a role for RYR2 dysfunction in the development of NCCM. The identified RYR2 variants are therefore likely causative for this early lethal NCCM phenotype. Further research is needed to understand the role of RYR2 in the heart compaction process.

Supplemental Digital Content is available in the text., Background: Long QT syndrome (LQTS) is the first described and most common inherited arrhythmia. Over the last 25 years, multiple genes have been reported to cause this condition and are routinely tested in patients. Because of dramatic changes in our understanding of human genetic variation, reappraisal of reported genetic causes for LQTS is required. Methods: Utilizing an evidence-based framework, 3 gene curation teams blinded to each other’s work scored the level of evidence for 17 genes reported to cause LQTS. A Clinical Domain Channelopathy Working Group provided a final classification of these genes for causation of LQTS after assessment of the evidence scored by the independent curation teams. Results: Of 17 genes reported as being causative for LQTS, 9 (AKAP9, ANK2, CAV3, KCNE1, KCNE2, KCNJ2, KCNJ5, SCN4B, SNTA1) were classified as having limited or disputed evidence as LQTS-causative genes. Only 3 genes (KCNQ1, KCNH2, SCN5A) were curated as definitive genes for typical LQTS. Another 4 genes (CALM1, CALM2, CALM3, TRDN) were found to have strong or definitive evidence for causality in LQTS with atypical features, including neonatal atrioventricular block. The remaining gene (CACNA1C) had moderate level evidence for causing LQTS. Conclusions: More than half of the genes reported as causing LQTS have limited or disputed evidence to support their disease causation. Genetic variants in these genes should not be used for clinical decision-making, unless accompanied by new and sufficient genetic evidence. The findings of insufficient evidence to support gene-disease associations may extend to other disciplines of medicine and warrants a contemporary evidence-based evaluation for previously reported disease-causing genes to ensure their appropriate use in precision medicine.

BackgroundFour genetic causes of isolated congenital central hypothyroidism (CeH) have been identified, but many cases remain unexplained. We hypothesised the existence of other genetic causes of CeH with a Mendelian inheritance pattern.MethodsWe performed exome sequencing in two families with unexplained isolated CeH and subsequently Sanger sequenced unrelated idiopathic CeH cases. We performed clinical and biochemical characterisation of the probands and carriers identified by family screening. We investigated IRS4 mRNA expression in human hypothalamus and pituitary tissue, and measured serum thyroid hormones and Trh and Tshb mRNA expression in hypothalamus and pituitary tissue of Irs4 knockout mice.ResultsWe found mutations in the insulin receptor substrate 4 (IRS4) gene in two pairs of brothers with CeH (one nonsense, one frameshift). Sequencing of IRS4 in 12 unrelated CeH cases negative for variants in known genes yielded three frameshift mutations (two novel) in three patients and one male sibling. All male carriers (n=8) had CeH with plasma free thyroxine concentrations below the reference interval. MRI of the hypothalamus and pituitary showed no structural abnormalities (n=12). 24-hour thyroid-stimulating hormone (TSH) secretion profiles in two adult male patients showed decreased basal, pulsatile and total TSH secretion. IRS4 mRNA was expressed in human hypothalamic nuclei, including the paraventricular nucleus, and in the pituitary gland. Female knockout mice showed decreased pituitary Tshb mRNA levels but had unchanged serum thyroid hormone concentrations.ConclusionsMutations in IRS4 are associated with isolated CeH in male carriers. As IRS4 is involved in leptin signalling, the phenotype may be related to disrupted leptin signalling.

Background: Pathogenic RYR2 variants account for ≈60% of clinically definite cases of catecholaminergic polymorphic ventricular tachycardia. However, the rate of rare benign RYR2 variants identified in the general population remains a challenge for genetic test interpretation. Therefore, we examined the results of the RYR2 genetic test among patients referred for commercial genetic testing and examined factors impacting variant interpretability. Methods: Frequency and location comparisons were made for RYR2 variants identified among 1355 total patients of varying clinical certainty and 60 706 Exome Aggregation Consortium controls. The impact of the clinical phenotype on the yield of RYR2 variants was examined. Six in silico tools were assessed using patient- and control-derived variants. Results: A total of 18.2% (218/1200) of patients referred for commercial testing hosted rare RYR2 variants, statistically less than the 59% (46/78) yield among clinically definite cases, resulting in a much higher potential genetic false discovery rate among referrals considering the 3.2% background rate of rare, benign RYR2 variants. Exclusion of clearly putative pathogenic variants further complicates the interpretation of the next novel RYR2 variant. Exonic/topologic analyses revealed overrepresentation of patient variants in exons covering only one third of the protein. In silico tools largely failed to show evidence toward enhancement of variant interpretation. Conclusions: Current expert recommendations have resulted in increased use of RYR2 genetic testing in patients with questionable clinical phenotypes. Using the largest to date catecholaminergic polymorphic ventricular tachycardia patient versus control comparison, this study highlights important variables in the interpretation of variants to overcome the 3.2% background rate that confounds RYR2 variant interpretation.

Several patients with Beckwith-Wiedemann Syndrome (BWS) with multiple imprinting defects found by genetic analysis have been described. However, only two cases have been described with both genetic and clinical signs and symptoms of multiple diseases caused by imprinting defects. The girl in this case presented at the age of 6 months with morbid obesity (body mass index, +7.5 SDS) and a large umbilical hernia. Genetic analysis showed BWS (hypomethylation of the KCNQ1OT1 gene). Calcium homeostasis was normal, and she had no signs of Albright hereditary osteodystrophy. At the age of 10 years, she presented with fatigue, and laboratory analyses showed marked hypocalcemia with signs of PTH resistance, but without evidence for Albright hereditary osteodystrophy, thus suggesting pseudohypoparathyroidism type 1B. Consistent with this diagnosis, methylation analysis of the GNAS complex revealed hypomethylation (about 20%) of the GNAS exon 1A, NESPAS, and GNASXL loci and hypermethylation (100% methylation) of the NESP locus. Imprinting defects at several different loci can occur in some patients, thus causing multiple different diseases. Symptoms of pseudohypoparathyroidism type 1B may be absent at diagnosis of BWS, yet prolonged subclinical hypocalcemia and/or hyperphosphatemia can have negative consequences (eg, intracerebral calcifications, myocardial dysfunction). We therefore suggest that patients with an imprinting disorder should be monitored for elevations in PTH, and epigenetic analysis of the GNAS complex locus should be considered

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by myocardial atrophy, fibro-fatty replacement, and a high risk of ventricular arrhythmias that lead to sudden death. In 2009, genetic data from 57 publications were collected in the arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) Genetic Variants Database (freeware available at http://www.arvcdatabase.info), which comprised 481 variants in eight ACM-associated genes. In recent years, deep genetic sequencing has increased our knowledge of the genetics of ACM, revealing a large spectrum of nucleotide variations for which pathogenicity needs to be assessed. As of April 20, 2014, we have updated the ARVD/C database into the ARVD/C database to contain more than 1,400 variants in 12 ACM-related genes (PKP2, DSP, DSC2, DSG2, JUP, TGFB3, TMEM43, LMNA, DES, TTN, PLN, CTNNA3) as reported in more than 160 references. Of these, only 411 nucleotide variants have been reported as pathogenic, whereas the significance of the other approximately 1,000 variants is still unknown. This comprehensive collection of ACM genetic data represents a valuable source of information on the spectrum of ACM-associated genes and aims to facilitate the interpretation of genetic data and genetic counseling.

Tricho-rhino-phalangeal syndrome (TRPS) is characterized by craniofacial and skeletal abnormalities, and subdivided in TRPS I, caused by mutations in TRPS1, and TRPS II, caused by a contiguous gene deletion affecting (amongst others) TRPS1 and EXT1. We performed a collaborative international study to delineate phenotype, natural history, variability, and genotype-phenotype correlations in more detail.We gathered information on 103 cytogenetically or molecularly confirmed affected individuals. TRPS I was present in 85 individuals (22 missense mutations, 62 other mutations), TRPS II in 14, and in 5 it remained uncertain whether TRPS1 was partially or completely deleted.Main features defining the facial phenotype include fine and sparse hair, thick and broad eyebrows, especially the medial portion, a broad nasal ridge and tip, underdeveloped nasal alae, and a broad columella. The facial manifestations in patients with TRPS I and TRPS II do not show a significant difference. In the limbs the main findings are short hands and feet, hypermobility, and a tendency for isolated metacarpals and metatarsals to be shortened. Nails of fingers and toes are typically thin and dystrophic. The radiological hallmark are the cone-shaped epiphyses and in TRPS II multiple exostoses. Osteopenia is common in both, as is reduced linear growth, both prenatally and postnatally. Variability for all findings, also within a single family, can be marked.Morbidity mostly concerns joint problems, manifesting in increased or decreased mobility, pain and in a minority an increased fracture rate. The hips can be markedly affected at a (very) young age. Intellectual disability is uncommon in TRPS I and, if present, usually mild. In TRPS II intellectual disability is present in most but not all, and again typically mild to moderate in severity.Missense mutations are located exclusively in exon 6 and 7 of TRPS1. Other mutations are located anywhere in exons 4-7. Whole gene deletions are common but have variable breakpoints. Most of the phenotype in patients with TRPS II is explained by the deletion of TRPS1 and EXT1, but haploinsufficiency of RAD21 is also likely to contribute. Genotype-phenotype studies showed that mutations located in exon 6 may have somewhat more pronounced facial characteristics and more marked shortening of hands and feet compared to mutations located elsewhere in TRPS1, but numbers are too small to allow firm conclusions. (C) 2015 Elsevier Masson SAS. All rights reserved.

Background & Aims Hepatocellular secretory failure induced by drugs, toxins or transient biliary obstruction may sometimes persist for months after removal of the initiating factor and may then be fatal without liver transplantation. We characterized patients with severe persistent hepatocellular secretory failure (PHSF) and treated them with the pregnane X receptor (PXR) agonist, rifampicin. We also studied the effect of rifampicin on PXR-dependent expression of genes involved in biotransformation and secretion in vitro. Methods Thirteen patients (age 18–81 years, 6 male) with hepatocellular secretory failure that persisted after removal of the inducing factor (drugs/toxin: 9) or biliary obstruction (4) were identified over 6 years. Six of these patients were screened for ATP8B1 or ABCB11 mutations. All were treated with rifampicin (300 mg daily) for 1–10 weeks. Expression of genes involved in biotransformation and secretion was determined by rtPCR in human hepatocytes and intestinal cells incubated with rifampicin (10 μmol/L). Results Serum bilirubin of patients with PHSF ranged from 264 to 755 μmol/L. Normal γGT was found in 10/13 patients of whom 3/6 tested positive for ATP8B1/ABCB11 mutations. Serum bilirubin declined to

Background Conventional therapy with beta-blockers is incompletely effective in preventing arrhythmic events in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT). We have previously discovered that flecainide in addition to conventional drug therapy prevents ventricular arrhythmias in patients with genotype-positive CPVT. Objective To study the efficacy of flecainide in patients with genotype-negative CPVT. Methods We studied the efficacy of flecainide for reducing ventricular arrhythmias during exercise testing and preventing arrhythmia events during long-term follow-up. Results Twelve patients with genotype-negative CPVT were treated with flecainide. Conventional therapy failed to control ventricular arrhythmias in all patients. Flecainide was initiated because of significant ventricular arrhythmias (n = 8), syncope (n = 3), or cardiac arrest (n = 1). At the baseline exercise test before flecainide, 6 patients had ventricular tachycardia and 5 patients had bigeminal or frequent ventricular premature beats. Flecainide reduced ventricular arrhythmias at the exercise test in 8 patients compared to conventional therapy, similar to that in patients with genotype-positive CPVT in our previous report. Notably, flecainide completely prevented ventricular arrhythmias in 7 patients. Flecainide was continued in all patients except for one who had ventricular tachycardia at the exercise test on flecainide. During a follow-up of 48±94 months, arrhythmia events (sudden cardiac death and aborted cardiac arrest) associated with noncompliance occurred in 2 patients. Flecainide was not discontinued owing to side effects in any of the patients. Conclusions Flecainide was effective in patients with genotype-negative CPVT, suggesting that spontaneous Ca 2+ release from ryanodine channels plays a role in arrhythmia susceptibility, similar to that in patients with genotype-positive CPVT.

A 55-year-old woman was admitted to our intensive care department after intoxication with lithium. Her medical history was relevant for bipolar disorder for which she received medical treatment with lithium, haloperidol, and citalopram. In the week before admission, she had developed a clinical picture of gastroenteritis with diarrhea and vomiting, which resulted in dehydration and a marked deterioration in kidney function. During the last days before admission, she had become progressively lethargic and had developed dysarthria as well as postural tremors of the extremities. The tremors were coarse and irregular, most clearly present in the hands with a frequency of approximately 8 Hz. At admission, she opened her eyes spontaneously, localized pain, and showed normal verbal responses (E4M5V5). Physical examination showed severe dysarthria, ataxia, and no focal neurologic deficits. Reflexes were normal. Laboratory investigation revealed a de novo elevated creatinine level (220 μmol/L, normal 49–90) corresponding to an estimated glomerular filtration rate of 22 mL/min/1.73 m2 (normal >60) and a lithium level that was 5.8 mmol/L (target value 0.6–0.8 mmol/L). ECG at admission was normal except for a prolonged QTc interval of 533 milliseconds (normal

PathogenicFrequency and location comparisons were made forA total of 18.2% (218/1200) of patients referred for commercial testing hosted rareCurrent expert recommendations have resulted in increased use of

Background— Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene ( Ryr2 ) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease penetrance, expression, genotype-phenotype correlations, and arrhythmic event rates in relatives carrying the Ryr2 mutation is limited. Methods and Results— One-hundred sixteen relatives carrying the Ryr2 mutation from 15 families who were identified by cascade screening of the Ryr2 mutation causing CPVT in the proband were clinically characterized, including 61 relatives from 1 family. Fifty-four of 108 antiarrhythmic drug-free relatives (50%) had a CPVT phenotype at the first cardiological examination, including 27 (25%) with nonsustained ventricular tachycardia. Relatives carrying a Ryr2 mutation in the C-terminal channel-forming domain showed an increased odds of nonsustained ventricular tachycardia (odds ratio, 4.1; 95% CI, 1.5–11.5; P =0.007, compared with N-terminal domain) compared with N-terminal domain. Sinus bradycardia was observed in 19% of relatives, whereas other supraventricular dysrhythmias were present in 16%. Ninety-eight (most actively treated) relatives (84%) were followed up for a median of 4.7 years (range, 0.3–19.0 years). During follow-up, 2 asymptomatic relatives experienced exercise-induced syncope. One relative was not being treated, whereas the other was noncompliant. None of the 116 relatives died of CPVT during a 6.7-year follow-up (range, 1.4–20.9 years). Conclusions— Relatives carrying an Ryr2 mutation show a marked phenotypic diversity. The vast majority do not have signs of supraventricular disease manifestations. Mutation location may be associated with severity of the phenotype. The arrhythmic event rate during follow-up was low.

Objectives: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by fibrofatty replacement of cardiomyocytes. In around 50% of index patients, a genetic predisposition is demonstrated. The purpose of this study was to examine a plakophilin-2 (PKP2) splice site mutation, c.2489+4A>C, identified in 4 separately ascertained Dutch ARVD/C families. Methods: Genealogical studies and comprehensive screening of 5 desmosomal genes were undertaken. Reverse transcriptase PCR (RT-PCR) and subsequent sequencing was performed. Results: An A-to-C change (c.2489+4A>C) near the splice donor site of intervening sequence 12 of PKP2 was found in all 4 families. Based on pedigree data and haplotype sharing, a common ancestor should be situated more than 7 generations ago. RT-PCR demonstrated the presence of aberrant messenger RNA. Clinical manifestations ranged from severe disease to nonpenetrance in elderly mutation carriers. Conclusions: This founder mutation in PKP2 is predicted to lead to the presence of a dysfunctional PKP2 protein, whereas most truncating mutations are expected to lead to loss of protein. Mutation carriers displayed a wide range of disease severity, suggesting that PKP2 mutations alone are not sufficient to cause disease, which results in the variable expression and incomplete penetrance characteristic of ARVD/C mutations.

Identifying a mutation in a heterogeneous disease such as inherited cardiomyopathy is a challenge because classical methods, like linkage analysis, can often not be applied as there are too few meioses between affected individuals. However, if affected individuals share the same causal mutation, they will also share a genomic region surrounding it. High-density genotyping arrays are able to identify such regions shared among affected individuals. We hypothesize that the longest shared haplotype is most likely to contain the disease-causing mutation. We applied this method to two pedigrees: one with arrhythmogenic right ventricular cardiomyopathy (ARVC) and one with dilated cardiomyopathy (DCM), using high-density genome-wide SNP arrays. In the ARVC pedigree, the largest haplotype was on chromosome 12 and contained a causative PKP2 mutation. In the DCM pedigree, a causative MYH7 mutation was present on a large shared haplotype on chromosome 14. We calculated that a pedigree containing at least seven meioses has a high chance of correctly detecting the mutation-containing haplotype as the largest. Our data show that haplotype sharing analysis can assist in identifying causative genes in families with low penetrance Mendelian diseases, in which standard tools cannot be used due to lack of sufficient pedigree information.

Gallstones are very common. However, there is a small group of patients with low phospholipid-associated cholelithiasis (LPAC) that is characterized by symptomatic cholelithiasis at a young age ( A) in the ABCB4 gene.

Objectives This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2 -encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). Background Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by RYR2 has not been examined comprehensively in most patient cohorts. Methods Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 ± 15 years, mean QTc 428 ± 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc Results Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. Conclusions Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that ≈65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a hereditary cardiomyopathy characterized by fibrofatty replacement of cardiomyocytes, ventricular tachyarrhythmias and sudden death. ARVD/C is mainly caused by mutations in genes encoding desmosomal proteins. However, the pathogenicity of variants is not always clear. Therefore, we created an online database (www.arvcdatabase.info), providing information on variants in ARVD/C-associated genes. We searched the literature using ARVD/C and its underlying genes as search terms. From the selected papers and our unpublished data, we collected details on the type of mutation and information provided at the protein level. A "details page" contains clinical data and references. To aid the interpretation of missense mutations, we provide data from in silico prediction methods. In May 2009 the database contained 481 variants in eight genes. A total of 144 variants are considered pathogenic, 73 are unknown/unclassified, and 264 have no known pathogenicity. The database was converted into the Leiden Open Variation Database (LOVD) format, a gene-centered collection of DNA variations. The ARVD/C database will be useful for both researchers and clinicians. It can be searched to determine if variants have been published and whether they are considered pathogenic. External users are invited to add information to improve the quantity and quality of the data entered. Hum Mutat 30:1278-1283, 2009. (C) 2009 Wiley-Liss, Inc.

Objective: A low plasma high-density lipoprotein cholesterol (HDL-c) concentration is an important risk factor for the development of atherosclerotic cardiovascular disease. HDL-c levels are abnormally low in type I Gaucher disease (GD) patients. The aim Of this Study was to determine whether GD is associated with premature atherosclerosis. Methods: Lipid profiles, apolipoproteins, and carotid artery intima-media thickness (cIMT) were analyzed in 40 type I GD patients, 34 carriers and 41 control subjects. cIMT is a non-invasive validated biomarker for the Status of atherosclerosis and present and future cardiovascular disease risk. Results: Compared to control subjects, patients showed decreased HDL-c (1.1 +/- 0.3 mmol/L) as well as mildly decreased low-density lipoprotein cholesterol (LDL-c) levels (2.8 +/- 0.7 mmol/L), with an increased ApoB/ApoA1 ratio. In carriers, HDL-c levels were normal, but LDL-c levels were decreased (2.7 +/- 0.8 mmol/L). Mean cIMT measurements were not different in the three study groups (patients: 0.63 +/- 0.1 mm versus carriers: 0.64 +/- 0.1 mm versus control Subjects: 0.65 +/- 0.1 mm). Conclusion: In Gaucher disease low HDL-c levels do not lead to premature atherosclerosis as assessed by cIMT measurement. This indicates that the inverse relationship between levels of HDL-c and risk of cardiovascular disease in the general population may not be present in all conditions characterised by low HDL-c levels. (C) 2008 Elsevier Ireland Ltd. All rights reserved

Methylmalonic aciduria (MMA-uria) is an autosomal recessive inborn error of amino acid metabolism, involving valine, threonine, isoleucine, and methionine. This organic aciduria may present in the neonatal period with life-threatening metabolic acidosis, hyperammonemia, feeding difficulties, pancytopenia, and coma. Most affected patients have mutations in the methylmalonyl-coenzyme A (methylmalonyl-CoA) mutase gene. Mildly affected patients may present in childhood with failure to thrive and recurrent attacks of metabolic acidosis. Both a higher residual activity of methylmalonyl-CoA mutase as well as the vitamin B12–responsive defects (cblA and cblB) may form the basis of the mild disorder. A few patients with moderate MMA-uria are known in whom no defect could be identified. Here we present a 16-year-old female patient with persisting moderate MMA-uria (∼50 mmol/mol creatinine). She was born to consanguineous Caucasian parents. Her fibroblast mutase activity was normal and no effect of vitamin B12 supplementation could be established. Reduced incorporation of 14C-propionate into macromolecules suggested a defect in the propionate-to-succinate pathway. We found a homozygous nonsense mutation (c.139C>T) in the methylmalonyl-CoA epimerase gene (MCEE), resulting in an early terminating signal (p.R47X). Both parents were heterozygous for this mutation; they were found to excrete normal amounts of methylmalonic acid (MMA). This is the first report of methylmalonyl-CoA epimerase deficiency, thereby unequivocally demonstrating the biochemical role of this enzyme in human metabolism. Hum Mutat 27(7), 640–643, 2006. © 2006 Wiley-Liss, Inc.

Background Several genetic defects are associated with permanent congenital hypothyroidism. Immunologic, environmental, and iatrogenic (but not genetic) factors are known to induce transient congenital hypothyroidism, which spontaneously resolves within the first months of life. We hypothesized that molecular defects in the thyroid oxidase system, which is composed of at least two proteins, might be involved in the pathogenesis of permanent or transient congenital hypothyroidism in babies with defects in iodide organification, for which the oxidase system is required. Methods Nine patients were recruited who had idiopathic congenital hypothyroidism (one with permanent and eight With transient hypothyroidism) and an iodide-organification defect and who had been identified by the screening program for congenital hypothyroidism. The DNA of the patients and their relatives was analyzed for mutations in the genes for thyroid oxidase 1 (THOX1) and 2 (THOX2). Results The one patient with permanent and severe thyroid hormone deficiency and a complete iodide-organification defect had a homozygous nonsense mutation in the THOX2 gene that eliminates all functional domains of the protein. Three of the eight patients with mild transient congenital hypothyroidism and a partial iodide-organification defect had heterozygous mutations in the THOX2 gene that prematurely truncate the protein, thus abolishing its functional domains. Conclusions Biallelic inactivating mutations in the THOX2 gene result in complete disruption of thyroid-hormone synthesis and are associated with severe and permanent congenital hypothyroidism. Monoallelic mutations are associated with milder, transient hypothyroidism caused by insufficient thyroidal production of hydrogen peroxide, which prevents the synthesis of sufficient quantities of thyroid hormones to meet the large requirement for thyroid hormones at the beginning of life

BACKGROUND Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants.OBJECTIVE The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients.METHODS Nine variants in desmosomal (PKP2, JUP, DSG2, DSC2) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction.RESULTS An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression.CONCLUSION Six variants of uncertain clinical significance in the PKP2, JUP, and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiac disease with reduced penetrance and a highly variable expression. Mutations in the gene encoding the plakophilin-2 gene (PKP2) are detected in about 50% of ARVC/D patients. The p.Arg79X mutation in PKP2 has been identified in Europe and North America and has been functionally characterised. We evaluated the prevalence of the p.Arg79X mutation in PKP2 in the Dutch population.METHODS: Twelve index patients and 41 family members were evaluated in three university hospitals in the Netherlands. The diagnosis of ARVC/D was established according to the recently revised Task Force Criteria. Segregation of the p.Arg79X mutation was studied and haplotypes were reconstructed to determine whether the p.Arg79X mutation was a recurrent or a founder mutation.RESULTS: The p.Arg79X mutation in PKP2 was identified in 12 index patients. Haplotype analysis revealed a shared haplotype among Dutch p.Arg79X mutation carriers, indicating a common founder. Six index patients (50%) had a first- or second-degree relative who had died of sudden cardiac death below 40 years of age. At age 60, only 60% of the mutation carriers had experienced any symptoms. There was no significant difference in symptom-free survival and event-free survival between men and women.CONCLUSION: We have identified the largest series of patients with the same desmosome gene mutation in ARVC/D reported to date. This p.Arg79X mutation in PKP2 is a founder mutation in the Dutch population. The phenotypes of PKP2 p.Arg79X mutation carriers illustrate the clinical variability and reduced penetrance often seen in ARVC/D. (Neth Heart J 2010;18:583-91.).

Severe congenital hypothyroidism (CH) due to a total iodide organification defect (TIOD) is usually due to mutations in the thyroid peroxidase (TPO) gene located at chromosome 2p25. A homozygous deletion [DeltaT2512 (codon 808)] in exon 14 was identified in a patient with classical TIOD. The transmission pattern of the TPO gene in this family was anomalous; the mother was heterozygous for the deletion; and the mutation was absent in the father. Polymorphic short tandem repeat (STR) markers confirmed paternity and demonstrated on chromosome 2 that the propositus was homozygous for most markers on chromosome 2p and that these were identical to one of the maternal 2p homologs. A normal karyotype was found in the propositus, his parents and sister. We conclude that the homozygosity in the patient is due to partial maternal isodisomy of the short arm of chromosome 2, carrying a defective TPO gene. The patient, born small for gestational age, develops and grows well and appears healthy (while being treated with thyroxine) and has a normal phenotype except for a unilateral preauricular skin tag. This shows that partial maternal isodisomy for chromosome 2p (2pter - 2p12) is compatible with a minimal influence on normal development.

Presented is a cohort study to assess the nature and frequency of thyroid peroxidase (TPO) mutations in 45 patients (35 families) with congenital hypothyroidism due to a total iodide organification defect; incidence is 1:66,000 in The Netherlands. The presentation is consistently similar with a severe form of congenital hypothyroidism and also characterized by a complete and immediate release of accumulated radioiodide from the thyroid after sodium perchlorate administration. Sixteen different mutations were found, including eight novel mutations; the majority occurs in exons 8, 9, or 10. The GGCC insertion in exon 8 at nucleotide 1277, leading to an early termination signal in exon 9, is the most frequently occurring mutation. These mutations were detected in 29 families in both TPO alleles (13 homozygous and 16 compound heterozygous). In one family, partial maternal isodisomy of 2p was detected, in four families only one mutated TPO allele could be detected, and in one family no inactivating TPO mutation could be found. Because all patients clearly had the clinicopathologic features of a total iodide organification defect, we conclude that in these five families the mutations in the (other) alleles could be either located in the intronic sequences or in the promoter region. Mutations in the TPO gene result in total iodide organification defects.

OBJECTIVE To screen and subsequently sequence the TPO gene for mutations in patients with congenital goitre, hypothyroidism and evidence for an organification defect (positive perchlorate discharge test). PATIENTS We have studied seven hypothyroid and congenitally goitrous patients from three unrelated families. DESIGN AND MEASUREMENTS We have measured serum thyroid hormone levels, 131I uptake, serum TSH and serum Tg concentrations. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene. RESULTS DGGE identified the presence of two frameshift mutations: a GGCC duplication in exon 8 (homozygous in one family and heterozygous in the other family) and a heterozygous insertion of a single nucleotide (C) at position 2505-2511 in exon 14. In addition, we have detected an alteration in exon 11, not yet described in the literature, derived from a single nucleotide substitution of a C to G at position 2008, altering the well-conserved amino acid domain among the peroxidases superfamily. This mutation in exon 11 was present in two families that showed heterozygous mutation for exon 8 or for exon 14. CONCLUSIONS These results could support the hypothesis for a putative compound heterozygosity pattern in the affected patients. The altered phenotype (goitre and hypothyroidism since birth) seems justifiable in view of the possible inactivating character of this novel mutation in exon 11.

Recent advances have been made in the research on structure and function of thyroid peroxidase in the field of the reaction mechanism with respect to the involvement of compound I in iodination and thyroid hormonogenesis. New information has become available concerning the mechanism of thyroid peroxidase autoantibody formation and immunodominant domains on the thyroid peroxidase molecule. In certain types of hereditary congenital hypothyroidism, called total iodide organification defects, thyroid peroxidase is impaired owing to mutations in the thyroid peroxidase gene. Detailed investigations on the proteins expressed in vitro have been described.

Wild-type and mutant thyroid peroxidase (TPO) was expressed in a Semliki Forest Virus (SFV)-based transient expression system in Chinese hamster ovary-K1 cells. Twenty four hours after transfection proteins immunoreactive with TPO antibodies could be detected on a Western blot. Peroxidase activity was assayed using both the guaiacol and the I3- assay. Addition of hematin was necessary to obtain enzymatic active TPO. Thyroid peroxidase complementary DNA constructs containing mutations originally found in patients with hereditary congenital hypothyroidism caused by total iodide organification defects were analyzed using these techniques. In all cases TPO was expressed as shown by Western blotting and immunostaining. Enzymatic activity (measured by guaiacol and iodide oxidation assay) was below the detection level in four out of five mutants. The only mutant yielding TPO with enzymatic activity was G 1858 A (Gly 590 Ser). However, the mutation could affect splicing of TPO messenger RNA, leading to inactive TPO, because it is located at the exon 10/intron 10 border.

AIMS: We sought to determine the influence of genotype on clinical course and arrhythmic outcome among arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C)-associated mutation carriers.METHODS AND RESULTS: Pathogenic mutations in desmosomal and non-desmosomal genes were identified in 577 patients (241 families) from USA and Dutch ARVD/C cohorts. Patients with sudden cardiac death (SCD)/ventricular fibrillation (VF) at presentation (n = 36) were younger (median 23 vs. 36 years; P < 0.001) than those presenting with sustained monomorphic ventricular tachycardia (VT). Among 541 subjects presenting alive, over a mean follow-up of 6 ± 7 years, 12 (2%) patients died, 162 (30%) had sustained VT/VF, 78 (14%) manifested left ventricular dysfunction (EF < 55%), 28 (5%) experienced heart failure (HF), and 10 (2%) required cardiac transplantation. Patients (n = 22; 4%) with >1 mutation had significantly earlier occurrence of sustained VT/VF (mean age 28 ± 12 years), lower VT-/VF-free survival (P = 0.037), more frequent left ventricular dysfunction (29%), HF (19%) and cardiac transplantation (9%) when compared with those with only one mutation. Desmoplakin mutation carriers experienced more than four-fold occurrence of left ventricular dysfunction (40%) and HF (13%) than PKP2 carriers. Missense mutation carriers had similar death-/transplant-free survival and VT/VF penetrance (P = 0.137) when compared with those with truncating or splice site mutations. Men are more likely to be probands (P < 0.001), symptomatic (P < 0.001) and have earlier and more severe arrhythmic expression.CONCLUSIONS: Presentation with SCD/VF occurs at a significantly younger age when compared with sustained monomorphic VT. The genotype of ARVD/C mutation carriers impacts clinical course and disease expression. Male sex negatively modifies phenotypic expression.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is frequently associated with desmosomal mutations. However, nondesmosomal mutations may be involved. The aim of this study was to assess the contribution of a phospholamban (PLN) gene mutation to ARVD/C diagnosis according to the revised 2010 task force criteria (TFC). In 142 Dutch patients (106 men, mean age 51 ± 13 years) with proven ARVD/C (fulfillment of 2010 TFC for diagnosis), 5 known desmosomal genes (PKP2, DSP, DSC2, DSG2, and JUP) and the nondesmosomal PLN gene were screened. After genetic analysis, phenotypic characteristics of desmosomal versus PLN mutation carriers were compared. In 59 of 142 patients with ARVD/C (42%), no desmosomal mutation was found. In 19 of 142 patients (13%), the PLN founder mutation c.40_42delAGA (p.Arg14del) was identified. PLN mutation carriers more often had low-voltage electrocardiograms (p = 0.004), inverted T waves in leads V4 to V6 (p

Background: Although thyroid dysgenesis is the most common cause of congenital hypothyroidism (CH), its molecular basis remains largely elusive. Indeed, in only a minority of cases with thyroid dysgenesis (2%-3%) was it possible to identify an underlying genetic defect. The objective of this study was to screen the PAX8 gene and the PAX2 gene in a family with six cases of CH spanning three generations and presenting urogenital malformations. Herein, we report a case series and in vitro characterization of the PAX8 gene mutation. Methods: Investigations were conducted at a tertiary care referral center. The index case was diagnosed to have congenital hypothyroidism at 7 months of age when he presented with severe impairment of suckling, constipation, and poor development. Treatment with levothyroxine corrected the symptoms and was associated with catch-up growth. His progeny, including two sons, one daughter, and two granddaughters, were affected by CH, and three of them received the diagnosis at neonatal screening. Ultrasound demonstrated normally located thyroid glands with reduced volumes. Five of the six affected family members, including the index case, had urogenital malformations, including incomplete horseshoe kidney, undescended testicles, hydrocele, and ureterocele. Strabismus was found in three out of six affected patients. No other somatic malformations were found. Results: Direct sequencing of the PAX8 gene revealed a new heterozygous mutation (c.74C>G) in all affected individuals. This mutation leads to substitution of proline with arginine at codon 25 (P25R). Fluorescence microscopy showed that P25R is normally located in the nucleus. In transient transfection studies, this mutation causes reduced transcriptional activation ability when using a luciferase reporter construct under the control of a thyroglobulin promoter. This diminished transactivation ability is due to loss of DNA binding capability as shown in electrophoresis mobility shift assay. The sequencing analysis of the PAX2 gene was normal. Conclusions: We conclude that this novel PAX8 mutation is responsible for a severe form of dominantly inherited CH. The mutation seems to be associated with abnormalities of the urogenital tract

Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues., Graphical Abstract

Brain-lung-thyroid syndrome (BLTS) characterized by congenital hypothyroidism, respiratory distress syndrome, and benign hereditary chorea is caused by thyroid transcription factor 1 (NKX2-1/TTF1) mutations. We report the clinical and molecular characteristics of four cases presenting with primary hypothyroidism, respiratory distress, and neurological disorder. Two of the four patients presenting with the triad of BLTS had NKX2-1 mutations, and one of these NKX2-1 [c.890_896del (p.Ala327Glyfs(star)52)] is a novel variant. The third patient without any identified NKX2-1 mutations was a carrier of mitochondrial mutation; this raises the possibility of mitochondrial mutations contributing to thyroid dysgenesis. Although rare, the triad of congenital hypothyroidism, neurological, and respiratory signs is highly suggestive of NKX2-1 anomalies. Screening for NKX2-1 mutations in patients with thyroid, lung, and neurological abnormalities will enable a unifying diagnosis and genetic counseling for the affected families. In addition, identification of an NKX2-1 defect would be helpful in allaying the concerns about inadequate thyroxine supplementation as the cause of neurological defects observed in some children with congenital hypothyroidism

The molecular basis of a total iodide organification defect causing severe congenital hypothyroidism has been elucidated. The defect occurred in a family in which two of five siblings were affected. Thyroid tissue from one patient was available for investigation. The total thyroid peroxidase (TPO) messenger ribonucleic acid level was reduced and consisted mainly of the alternatively spliced form of TPO missing exon 10 (TPO-2). No TPO-1 (wild-type) protein was detected by Western blotting. The TPO-2 translation product of a slightly smaller mol wt was present in thyroid tissue of this patient. TPO activity was absent and thyroglobulin was not iodinated, showing that iodination in vivo did not occur. Denaturing gradient gel electrophoresis and subsequent sequencing revealed in both alleles of the patients a C--T transition of nucleotide 1708 of the TPO gene, involving a CpG dinucleotide. The mutation introduces a premature termination signal in exon 10 of the TPO gene, preventing the synthesis of enzymatic active peroxidase.