Functional and molecular studies in primary carnitine deficiency.

Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported (n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen-2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency.

Correlation assessment among clinical phenotypes, expression analysis and molecular modeling of 14 novel variations in the human galactose-1-phosphate uridylyltransferase gene.

Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by deleterious variations in the GALT gene can cause a potentially lethal disease called classic galactosemia. In this study, we selected 14 novel nucleotide sequence changes in the GALT genes found in galactosemic patients for expression analysis and molecular modeling. Several variants showed decreased levels of expression and decreased abundance in the soluble fraction of the Escherichia coli cell extracts, suggesting altered stability and solubility. Only six variant GALT enzymes had detectable enzymatic activities. Kinetic studies showed that their V(max) decreased significantly. To further characterize the variants at molecular level, we performed static and dynamic molecular modeling studies. Effects of variations on local and/or global structural features of the enzyme were anticipated for the majority of variants. In-depth studies with molecular dynamic simulations on selected variants predicted the alteration of the protein structure even though static models apparently did not highlight any perturbation. Overall, these studies offered new insights on the molecular properties of GALT enzyme, with the aim of correlating them with the clinical outcome. Hum Mutat 33:1107-1115, 2012. © 2012 Wiley Periodicals, Inc.

Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2-associated RET sequence variation and reference for genotype/phenotype correlations.

Multiple endocrine neoplasia type 2 (MEN2) is an inherited, autosomal-dominant disorder caused by deleterious mutations within the RET protooncogene. MEN2 RET mutations are mainly heterozygous, missense sequence changes found in RET exons 10, 11, and 13-16. Our group has developed the publicly available, searchable MEN2 RET database to aid in genotype/phenotype correlations, using Human Genome Variation Society recommendations for sequence variation nomenclature and database content. The MEN2 RET database catalogs all RET sequence variation relevant to the MEN2 syndromes, with associated clinical information. Each database entry lists a RET sequence variation's location within the RET gene, genotype, pathogenicity classification, MEN2 phenotype, first literature reference, and comments (which may contain information on other clinical features, complex genotypes, and additional literature references). The MEN2 phenotype definitions were derived from the International RET Mutation Consortium guidelines for classification of MEN2 disease phenotypes. Although nearly all of the 132 RET sequence variation entries initially cataloged in the database were from literature reports, novel sequence variation and updated phenotypic information for any existing database entry can be submitted electronically on the database website. The database website also contains links to selected MEN2 literature reviews, gene and protein information, and RET reference sequences. The MEN2 RET database (www.arup.utah.edu/database/MEN2/MEN2_welcome.php) will serve as a repository for MEN2-associated RET sequence variation and reference for RET genotype/MEN2 phenotype correlations.

Genotypes and serum concentrations of human alpha-1-antitrypsin "P" protein variants in a clinical population.

BACKGROUND: Alpha-1-antitrypsin (AAT) deficiency is a relatively common genetic disorder that can lead to the development of pulmonary disorders. Diagnosis of AAT deficiency is typically performed by isoelectric focusing (IEF) protein phenotyping in concert with determination of AAT serum concentration levels. The "P" phenotypic variant is associated with several known genetic variants that are found at unknown relative frequencies. AIMS: To investigate the genetic variation of "P" alleles in patient samples. METHODS: A DNA sequencing protocol for the full AAT coding region from serum was developed. Additionally, a retrospective evaluation of AAT concentrations in serum samples containing "P" allele IEF phenotype variants was undertaken. RESULTS: "P" phenotypic variants are observed in approximately 1 of every 900 samples received in the reference laboratory. Heterozygous "MP" allele samples exhibited a wide range of serum protein concentrations. Genotyping revealed the presence of the deleterious P lowell variant in six heterozygous MP samples, two heterozygous PZ samples, and one homozygous PP sample. A non-deleterious P st albans variant was observed in a single MP sample. A novel heterozygous AAT M"P" variant, P salt lake was identified, that did not exhibit a reduced AAT serum concentration. CONCLUSIONS: Genetic heterogeneity is present in clinical "P" phenotype variants identified by IEF, and the deleterious P lowell variant appears to be relatively common. Sequencing of "P" phenotype variants can provide useful clinical information, especially when the "P" phenotype variant is paired with a deficiency phenotype allele.

Mutation database for the galactose-1-phosphate uridyltransferase (GALT) gene.

Classical galactosemia is an autosomal recessive disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. Our group developed a disease-specific database containing all of the reported sequence variants in GALT (Available at: http://arup.utah.edu/database/galactosemia/GALT_welcome.php; Last accessed: 13 April 2007). Currently the database contains a total of 229 sequence variants, of which 196 are mutations (including nine novel mutations identified in our laboratory), 31 polymorphisms in both introns and exons, and two variants of unknown or uncertain significance. All sequence variants have been verified for their position within the GALT gene and named following standard nomenclature. Sequence variants are reported with accompanying information on protein effect, classification of mutation vs. polymorphism, mutation type (when applicable) based on how each was first described in the literature, and accompanying link to pertinent publication. Unpublished variants are described with relevant clinical information that supports their classification as causative of the disease vs. polymorphisms. Other features of this database include disease information, relevant links for galactosemia and literature, reference sequences, ability to query by various criteria, and submit of novel variations to the database. This free online scientific resource was developed with the clinical laboratory in mind to serve as a reference and repository for novel findings that are periodically collected, verified, and updated into the database.

Clinical and analytical sensitivities in hereditary hemorrhagic telangiectasia testing and a report of de novo mutations.

Hereditary hemorrhagic telangiectasia is a vascular dysplasia with variable onset and expression. Through identification of a mutation in a proband, mutation testing can be offered to family members. Mutation carriers can receive medical surveillance and treatment before potentially fatal complications arise. In this study, we assessed the significance of clinical evaluations as part of hereditary hemorrhagic telangiectasia diagnostic testing to determine the clinical sensitivity of molecular testing and to report novel mutations. Based on reported clinical symptoms, we classified 142 consecutive cases as affected, suspected, or unlikely affected. We performed temperature gradient capillary electrophoresis and full gene sequencing of both ACVRL1 and ENG genes. We then compared the mutation detection rates between these groups, categorizing sequence variants as mutations, variants of uncertain significance (VUS), or known polymorphisms. Our mutation and VUS detection rate in affected individuals was 74% and 16% in the suspected/unlikely affected group. Sixty-one percent of the mutations and all VUS were novel. The mutation detection rate for temperature gradient capillary electrophoresis was 97%. Our results suggest that a careful clinical evaluation increases the mutation detection rate. We have confirmed the occurrence of de novo mutations in three patients. Our results also show that temperature gradient capillary electrophoresis is an efficient mutation screening method.

A fourth locus for hereditary hemorrhagic telangiectasia maps to chromosome 7.

Hereditary hemorrhagic telangiectasia (HHT) is a genetically and clinically heterogeneous multisystem vascular dysplasia. Mutations of the endoglin and ACVRL1 genes are known to cause HHT. However, existence of HHT families in which linkage to these genes has been excluded has suggested that other gene(s) can cause HHT in some families. Recently, a family was reported to be linked to chromosome 5q, the HHT3 locus. Here we report on linkage results on a family with classic features of HHT, albeit a less severe phenotype with regards to epistaxis and telangiectases, in which linkage to HHT1, HHT2, and HHT3 is ruled out. Whole genome linkage analysis and fine mapping results suggested a 7 Mb region on the short arm of chromosome 7 (7p14) between STR markers D7S2252 and D7S510. We obtained a maximum two point LOD score of 3.60 with the STR marker D7S817. This region was further confirmed by haplotype analysis. These findings suggest the presence of another gene causing HHT (HHT4). The features in this family that strongly suggest the presence of a hereditary, multisystem vascular dysplasia would be easily missed during the typical evaluation and management of a patient with an AVM. This family helps emphasize the need to obtain a very detailed, targeted medical and family history for even mild, infrequent but recurring nosebleed, subtle telangiectases. Further studies of the candidate region and the identification of the gene responsible for the vascular anomalies in this family will add to our understanding of vascular morphogenesis and related disorders.

A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders.

Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia (PD-G) have been epidemiologically linked to an environment severely deficient in calcium (Ca2+) and magnesium (Mg2+). Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein containing both channel and kinase domains that has been proposed to be involved in the homeostatic regulation of intracellular Ca2+, Mg2+, and trace metal ion concentration. There is evidence that TRPM7 is constitutively active and that the number of available channels is dependent on intracellular free Mg2+ levels. We found a TRPM7 variant in a subset of ALS-G and PD-G patients that produces a protein with a missense mutation, T1482I. Recombinant T1482I TRPM7 exhibits the same kinase catalytic activity as WT TRPM7. However, heterologously expressed T1482I TRPM7 produces functional channels that show an increased sensitivity to inhibition by intracellular Mg2+. Because the incidence of ALS-G and PD-G has been associated with prolonged exposure to an environment severely deficient in Ca2+ and Mg2+, we propose that this variant TRPM7 allele confers a susceptibility genotype in such an environment. This study represents an initial attempt to address the important issue of gene-environment interactions in the etiology of these diseases.