MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

An NK-like CAR T cell transition in CAR T cell dysfunction

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The effect of a pattern in palmar interdigital II on a-b ridge count in black and white Down syndrome cases and controls

An unconfirmed study by Fang (Ph.D. thesis, Univ. of London, 1950) in Britain showed that individuals with Down syndrome had lower total a-b ridge counts in palmar Interdigital area II (ID II) than a group of controls. This study compares 603 white Down syndrome cases and 93 black Down syndrome cases with 668 white and 402 black controls. Our results confirm those of Fang in that the Down syndrome cases in both racial groups had lower total a-b ridge counts than their respective controls. In addition, the black controls and Down syndrome cases had lower a-b ridge counts than their white counterparts. The mean a-b ridge count was significantly lower in individuals with a pattern in ID II compared to individuals without a pattern in ID II in both the Down syndrome and control groups. Some of the lower a-b ridge counts in the Down syndrome samples can be accounted for by the fact that there is an increased frequency of a pattern in ID II in Down syndrome cases. Both Down syndrome and normal individuals who had a pattern unilaterally had a lower than expected a-b ridge count on the contralateral hand that did not have a pattern. There was a tendency also for increased asymmetry in Down syndrome cases with a pattern in ID II.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Phenotype and Micro-array characterization of duplication 11q22.1-q25 and review of the literature

Partial duplication of 11q is related to several malformations like growth retardation, intellectual disability, hypoplasia of corpus callosum, short nose, palate defects, cardiac, urinary tract abnormalities and neural tube defects. We have studied the clinical and molecular characteristics of a patient with severe intellectual disabilities, dysmorphic features, congenital inguinal hernia and congenital cerebral malformation which is referred to as cytogenetic exploration. We have used FISH and array CGH analysis for a better understanding of the double chromosomic aberration involving a 7p microdeletion along with a partial duplication of 11q due to adjacent segregation of a paternal reciprocal translocation t(7;11)(p22;q21) revealed after banding analysis. The patient's karyotype formula was: 46,XY,der(7)t(7;11)(p22;q21)pat. FISH study confirmed these rearrangement and array CGH technique showed precisely the loss of at least 140 Kb on chromosome7p22.3pter and 33.4 Mb on chromosome11q22.1q25. Dysmorphic features, severe intellectual disability and brain malformations could result from the 11q22.1q25 trisomy. Our study provides an additional case for better understanding and delineating the partial duplication 11q.     

Diagnostic utility of HFE variants in Spanish patients: Association with HLA alleles and role in susceptibility to acute lymphoblastic leukemia

Two single nucleotide polymorphisms (SNPs) in the Human Hemochromatosis (HFE) gene, C282Y and H63D, are the major variants associated to altered iron status and it is well known that these mutations are in linkage disequilibrium with certain Human Leukocyte Antigen (HLA)-A alleles. In addition, the C282Y SNP has been previously suggested to confer susceptibility to acute lymphoblastic leukemia (ALL). We have aimed to assess the diagnosis utility of these polymorphisms in a population of Spanish subjects with suspicion of hereditary iron overload and to evaluate the effect of their associations with HLA-A alleles on the susceptibility to ALL. Both the 63DD [OR = 4.31 (1.7–11.2)] and 282YY (p for trend = 0.02) genotypes were more frequently found among subjects with suspicion of iron overload than among controls. 282YY carriers displayed significantly higher transferrin saturation index (TSI) values (p < 0.001) as well as serum iron (p = 0.01) and ferritin (p = 0.01) levels. In addition, transferrin levels were lower in these subjects (p = 0.01). Likewise, patients who were carriers of the compound heterozygous diplotype (282CY/63HD) showed significantly higher TSI and serum iron and ferritin concentrations. The H63D SNP did not significantly affect the analytical parameters measured. All 282YY carriers and 69.2% of compound heterozygotes showed an altered biochemical index. The frequencies of the HFE SNPs in ALL pediatric patients were lower than those found in controls, whereas the HLA-A*24 allele was significantly overrepresented in the patients group [OR = 3.76 (1.9–7.3)]. No HFE-HLA-A associations were found to modulate the ALL risk. These results suggest that it may be useful to test for both HFE H63D and C282Y polymorphisms in patients with iron overload, as opposed to just genotyping for the C282Y SNP, which is customary in some healthcare centers. These HFE variants and their associations with HLA-A alleles were not observed to be relevant for the susceptibility to ALL in our population.     

Prenatal diagnosis and molecular cytogenetic characterization of a de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14)

We present prenatal diagnosis of de novo proximal interstitial deletion of chromosome 4p (4p15.2→p14) and molecular cytogenetic characterization of the deletion using uncultured amniocytes. We review the phenotypic abnormalities of previously reported patients with similar proximal interstitial 4p deletions, and we discuss the functions of the genes of RBPJ, CCKAR, STIM2, PCDH7 and ARAP2 that are deleted within this region.     

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase—A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD⁺ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (k_cat), with less substantial effects on K_m. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.     

Characterization of seven novel mutations on the HEXB gene in French Sandhoff patients

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5′-end 16 kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.