Medical sequencing at the extremes of human body mass

Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96-Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese population compared with the lean population. In addition, computational analysis predicted a greater fraction of deleterious variants within the obese cohort. Together, these data suggest that multiple rare alleles contribute to obesity in the population and provide a medical sequencing-based approach to detect them.     

The 2-methoxy group of ubiquinone is essential for function of the acceptor quinones in reaction centers from Rba. sphaeroides

The orientation of a methoxy substituent is known to substantially influence the electron affinity and vibrational spectroscopy of benzoquinones, and has been suggested to be important in determining the function of ubiquinone as a redox cofactor in bioenergetics. Ubiquinone functions as both the primary (Q(A)) and secondary (Q(B)) quinone in the reaction centers of many purple photosynthetic bacteria, and is almost unique in its ability to establish the necessary redox free energy gap for 1-electron transfer between them. The role of the methoxy substitution in this requirement was examined using monomethoxy analogues of ubiquinone-4 - 2-methoxy-3,5-dimethyl-6-isoprenyl-1,4-benzoquinone (2-MeO-Q) and 3-methoxy-2,5-dimethyl-6-isoprenyl-1,4-benzoquinone (3-MeO-Q). Only 2-MeO-Q was able to simultaneously act as Q(A) and Q(B) and the necessary redox potential tuning was shown to occur in the Q(B) site. In the absence of active Q(B), the IR spectrum of the monomethoxy quinones was examined in vitro and in the Q(A) site, and a novel distinction between the two methoxy groups was tentatively identified, consistent with the unique role of the 2-methoxy group in distinguishing Q(A) and Q(B) functionality.     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Risk of obesity and metabolic syndrome associated with FTO gene variants discloses clinically relevant gender difference among Turks

Gene variations in the fat mass- and obesity-associated gene (FTO) have shown controversial associations with obesity and metabolic syndrome (MetS) in several populations. We explored the association of FTO gene with obesity, MetS, and insulin-related parameters separately in men and women. Two SNPs in the FTO, gene rs9939609 and rs1421085, were genotyped by the Taqman System in 1967 adults (mean age of the whole group 50.1 ± 12.0; 48.4 % male). A random sample of the Turkish Adult Risk Factor cohort was cross-sectionally analyzed. Both SNPs exhibited strong linkage disequilibrium (r² = 0.85) and minor alleles were associated with risk of obesity in women and of MetS in men. Carriers of the rs1421085 C-allele exhibited higher body mass index (BMI) in each gender. Adjusted fasting insulin and HOMA index were significantly higher in C-allele carriers in men alone. Logistic regression analysis demonstrated significantly increased likelihood for obesity in female C-risk allele carriers (OR 1.61; 95 % CI 1.19–2.18), after adjustment for age, smoking status, alcohol usage, physical activity grade and presence of diabetes mellitus. Male C-allele carriers were at increased risk for MetS (OR 1.44; 95 % CI 1.07–1.95), adjusted for age, smoking status, alcohol consumption, and physical activity. Further adjustment for BMI attenuated the MetS risk, indicating interaction between C-allele, gender and BMI. The FTO gene in Turkish adults contributes independently to obesity in women and—by interacting with BMI—to MetS and insulin resistance in men.     

Diabetes Alters Aromatase Enzyme Levels in Sciatic Nerve and Hippocampus Tissues of Rats

Diabetes mellitus (DM) is associated with increased risk of impaired cognitive function. Diabetic neuropathy is one of the most common and important complications of DM. Estrogens prevent neuronal loss in experimental models of neurodegeneration and accelerate nerve regeneration. Aromatase catalyzes the conversion of androgens to estrogens and expressed in a variety of tissues including neurons. Although insulin is known to regulate the activity of aromatase there is no study about the effects of diabetes on this enzyme. Present study was designed to investigate the effects of experimental diabetes on aromatase expression in nervous system. Gender-based differences were also investigated. Rats were injected with streptozotocin to induce diabetes. At the end of 4 and 12 weeks sciatic nerve and hippocampus homogenates were prepared and evaluated for aromatase proteins. Aromatase expressions in sciatic nerves of both genders were decreased in 4 weeks of diabetes, but in 12 weeks the enzyme levels were increased in females and reached to control levels in male animals. Aromatase levels were not altered in hippocampus at 4 weeks but increased at 12 weeks in female diabetic rats. No significant differences were observed at enzyme levels of hippocampus in male diabetic rats. Insulin therapy prevented all diabetes-induced changes. In conclusion, these results indicated for the first time that, DM altered the expression of aromatase both in central and peripheral nervous systems. Peripheral nervous system is more vulnerable to damage than central nervous system in diabetes. These effects of diabetes differ with gender and compensatory neuroprotective mechanisms are more efficient in female rats.     

Archaeological mitogenomes illuminate the historical ecology of sea otters (Enhydra lutris) and the viability of reintroduction

Genetic analyses are an important contribution to wildlife reintroductions, particularly in the modern context of extirpations and ecological destruction. To address the complex historical ecology of the sea otter (Enhydra lutris) and its failed 1970s reintroduction to coastal Oregon, we compared mitochondrial genomes of pre-extirpation Oregon sea otters to extant and historical populations across the range. We sequenced, to our knowledge, the first complete ancient mitogenomes from archaeological Oregon sea otter dentine and historical sea otter dental calculus. Archaeological Oregon sea otters (n = 20) represent 10 haplotypes, which cluster with haplotypes from Alaska, Washington and British Columbia, and exhibit a clear division from California haplotypes. Our results suggest that extant northern populations are appropriate for future reintroduction efforts. This project demonstrates the feasibility of mitogenome capture and sequencing from non-human dental calculus and the diverse applications of ancient DNA analyses to pressing ecological and conservation topics and the management of at-risk/extirpated species.     

Methylenetetrahydrofolate reductase C677T mutation in patients with alopecia areata in Turkish population

Objective

Methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme regulating folate metabolism and it is thought to influence DNA methylation and nucleic acid synthesis. Mutations in the MTHFR gene have been associated with several autoimmune disorders in previous studies. Alopecia areata (AA) is considered to be a tissue-specific autoimmune disease as the hair follicle has been targeted and antibodies to their own hair follicle structures have been developed. Since there is a common shared pathway between AA and other autoimmune disorders, we aimed to investigate a possible association between the MTHFR gene C677T mutation and AA susceptibility in the Turkish population.

Methods

The study included 136 patients affected by AA and 130 healthy controls. Genomic DNA was isolated and genotyped using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay for the MTHFR gene C677T mutation.

Results

The distributions of genotype and allele frequencies of MTHFR gene C677T mutation were statistically different between AA patients and the control group (p = 0.036 and p = 0.011, respectively). High differences were also observed when the patients and controls were compared according to CC versus CT + TT (p = 0.012). CT + TT genotypes and T allele of MTHFR gene C677T mutation were found to be a susceptibility factor for AA in the Turkish population.

Conclusion

The results suggest that MTHFR gene C677T mutation may have an effect on the risk of alopecia areata in the Turkish population. This is the first study reporting the association between the MTHFR (C677T) genotype and AA.     

Loss of the BMP antagonist, SMOC-1, causes Ophthalmo-acromelic (Waardenburg Anophthalmia) syndrome in humans and mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ～10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.