Sequencing for LIPA mutations in patients with a clinical diagnosis of familial hypercholesterolemia.

BACKGROUND AND AIMS: We recently identified lysosomal acid lipase (LAL) deficiency, a recessive disease caused by mutations in LIPA, in 3 patients with a clinical diagnosis of familial hypercholesterolemia (FH). We aimed to determine the prevalence of LIPA mutations among individuals with a clinical FH diagnosis. METHODS: In 276 patients with phenotypic FH, in whom no genetic basis for their phenotype was found, LIPA was sequenced. All variants were assessed for pathogenicity using a literature search and in silico prediction models. RESULTS: We included 213 adults and 63 children with mean (±SD) LDL-C levels of 7.8 ± 1.3 and 4.4 ± 1.5 mmol/L, respectively. Twenty-one variants were identified. Six patients were heterozygous carrier of a (potentially) pathogenic mutation. No homozygous LIPA mutation carriers were identified. CONCLUSIONS: Our data show that LAL deficiency was not missed as diagnosis in our study population but the frequency of heterozygous LIPA mutations implies that the FH population might be relatively enriched with LIPA mutation carriers.

Genetic variation in APOB, PCSK9, and ANGPTL3 in carriers of pathogenic autosomal dominant hypercholesterolemic mutations with unexpected low LDL-Cl Levels.

Autosomal Dominant Hypercholesterolemia (ADH) is caused by LDLR and APOB mutations. However, genetically diagnosed ADH patients do not always exhibit the expected hypercholesterolemic phenotype. Of 4,669 genetically diagnosed ADH patients, identified through the national identification screening program for ADH, 75 patients (1.6%) had LDL-cholesterol (LDL-C) levels below the 50th percentile for age and gender prior to lipid-lowering therapy. The genes encoding APOB, PCSK9, and ANGPTL3 were sequenced in these subjects to address whether monogenic dominant loss-of-function mutations underlie this paradoxical phenotype. APOB mutations, resulting in truncated APOB, were found in five (6.7%) probands, reducing LDL-C by 56%. Rare variants in PCSK9, and ANGPTL3 completely correcting the hypercholesterolemic phenotype were not found. The common variants p.N902N, c.3842+82T>A, p.D2312D, and p.E4181K in APOB, and c.1863+94A>G in PCSK9 were significantly more prevalent in our cohort compared to the general European population. Interestingly, 40% of our probands carried at least one minor allele for all four common APOB variants compared to 1.5% in the general European population. While we found a low prevalence of rare variants in our cohort, our data suggest that regions in proximity of the analyzed loci, and linked to specific common haplotypes, might harbor additional variants that correct an ADH phenotype.

Initial characterization of C16orf89, a novel thyroid-specific gene.

BACKGROUND: Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. METHODS: We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. RESULTS: C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. CONCLUSIONS: We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.

Molecular characterization of iodotyrosine dehalogenase deficiency in patients with hypothyroidism.

CONTEXT: The recent cloning of the human iodotyrosine deiodinase (IYD) gene enables the investigation of iodotyrosine dehalogenase deficiency, a form a primary hypothyroidism resulting from iodine wasting, at the molecular level. OBJECTIVE: In the current study, we identify the genetic basis of dehalogenase deficiency in a consanguineous family. RESULTS: Using HPLC tandem mass spectrometry, we developed a rapid, selective, and sensitive assay to detect 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine in urine and cell culture medium. Two subjects from a presumed dehalogenase-deficient family showed elevated urinary 3-monoiodo-l-tyrosine and 3,5-diodo-l-tyrosine levels compared with 57 normal subjects without thyroid disease. Subsequent analysis of IYD revealed a homozygous missense mutation in exon 4 (c.658G>A p.Ala220Thr) that co-segregates with the clinical phenotype in the family. Functional characterization of the mutant iodotyrosine dehalogenase protein showed that the mutation completely abolishes dehalogenase enzymatic activity. One of the heterozygous carriers for the inactivating mutation recently presented with overt hypothyroidism indicating dominant inheritance with incomplete penetration. Screening of 100 control alleles identified one allele positive for this mutation, suggesting that the c.658G>A nucleotide substitution might be a functional single nucleotide polymorphism. CONCLUSIONS: This study describes a functional mutation within IYD, demonstrating the molecular basis of the iodine wasting form of congenital hypothyroidism. This familial genetic defect shows a dominant pattern of inheritance with incomplete penetration.

The quantity of thyroid hormone in human milk is too low to influence plasma thyroid hormone levels in the very preterm infant.

BACKGROUND: Thyroid hormone is crucial for brain development during foetal and neonatal life. In very preterm infants, transient low levels of plasma T4 and T3 are commonly found, a phenomenon referred to as transient hypothyroxinaemia of prematurity. We investigated whether breast milk is a substantial resource of thyroid hormone for very preterm neonates and can alleviate transient hypothyroxinaemia. Both the influence of breast feeding on plasma thyroid hormone levels and the thyroid hormone concentration in preterm human milk were studied. METHODS: Two groups were formed from the placebo group of a randomized thyroxine supplementation trial in infants born at < 30 weeks' gestational age on the basis of the mean breast milk intake during the third, fourth and fifth weeks of life. One group received more than 50% breast milk (mean breast milk intake 84%, n = 32) and the other group less than 25% breast milk (mean breast milk intake 3.3%, n = 25). Plasma thyroid hormone concentrations were compared between the two groups. Breast milk was collected from mothers of infants participating in the same trial and the thyroxine concentration in breast milk was measured with RIA after extraction. RESULTS: No significant differences were found between both groups in plasma concentrations of T4, free T4, T3, TSH, rT3 and thyroxine-binding globulin (TBG), which were measured once a week. Thyroxine concentration in breast milk ranged between 0.17 microg/l and 1.83 microg/l (mean 0.83, SD 0.3 microg/l) resulting in a maximum T4 supply of 0.3 microg/kg via ingested breast milk. In formula milk, the T4 concentration was equally low. Protease treatment did not influence the measured T4 concentrations. CONCLUSIONS: No differences in plasma thyroid hormone between breast milk-fed and formula-fed infants were found. The amount of T4 present in human milk and formula milk is too low to alter the hypothyroxinaemic state of preterm infants.