Searching for genes underlying normal variation in human adiposity

A primary challenge in biomedical research today is the elucidation of the underlying genetic architecture of complex conditions such as obesity. In contrast to simple Mendelian disorders that result from a mutation in a single gene, complex phenotypes are the product of the action (as well as interaction) of multiple genes and environmental factors. The genetic configuration of these genes can range from effectively polygenic (i.e., many genes each with a relatively small contribution) to oligogenic (i.e., a few genes with relatively large measurable effects often expressed on a residual additive genetic background). While the task at hand is complicated, it is not intractable; however, it does require consideration of the nature of the disease and definition of its associated phenotypes in selecting the most appropriate study design. Here we will discuss the characteristics of obesity and its related phenotypes, which must be considered in designing analyses to identify the genes involved as well as reviewing what these approaches have provided in the search for genes influencing adiposity in humans     

Variations in regional sweat composition in normal human males

This project aimed to quantify the regional distribution of sweat composition over the skin surface and to determine whether sweat constituent concentrations collected from regional sites can estimate whole-body concentrations. Ten males cycled for 90 min in a 20 degrees C (50% relative humidity) environment at 45% peak aerobic power. Sweat was collected from eleven skin regions and the whole body, using a wash-down technique. Strong relationships were evident between the regional and whole-body sweat [Na+] and [Cl-], such that the thigh and calf exhibited greater correlation coefficients than area-weighted means derived from four and eight skin regions. Therefore, in this particular protocol the whole-body sweat [Na+] and [Cl-] could be predicted from regional sweat collections. Relationships between sweat constituents were evident for sweat [Na+] and pH, and sweat [K+] and [lactate] when data were pooled between skin regions and subjects. To our knowledge this is the first investigation to report a positive relationship between sweat [K+] and [lactate]. The exact mechanism responsible for the positive relationship between sweat [K+] and [lactate] is uncertain although it is speculated to occur at the secretory coil.     

Studies of biological properties of the recombinants between human influenza and fowl plague viruses as related to genome composition

Some biological properties of recombinants obtained by crossing of fowl plague and human influenza viruses were studied. The capacity of the recombinants to reproduce in chick embryo fibroblast cultures was in reverse correlation to the number of genes coding for P proteins derived from the human influenza virus. The genome composition was of importance for the expression of ts-phenotype of the recombinants in different systems. Substitution of at least one gene in the fowl plague virus genome by a corresponding human influenza virus gene resulted in the decrease of virulence for 1-day-old chickens. The presence of three P genes from human influenza virus genome in the genome of the recombinant proved to be insufficient for the capability of the recombinant to reproduce in organ cultures of human origin.     

Molecular variation of the human elastin (ELN) gene in a normal human population

DNA sequence diversity in the human elastin genomic region has been estimated by RFLP analysis in a normal human population. The proportion of polymorphic nucleotides and the degree of nucleotide diversity were 0·0034 and 0·0018 respectively. It is argued that the estimate of nucleotide diversity does not indicate strong purifying selection in the region. A total of 144 restriction sites were sampled in each of 80 independent chromosomes representing the screening of 58080 bp overall. Six main haplotypes were constructed; they represent at least 84% of the 80 chromosomes sampled. Analysis for linkage disequilibrium revealed two statistically significant comparisons out of 54 tests, approximately the proportion that would be statistically significant at the 5% level by chance. A higher order quadrigenic disequilibrium was detected. The relationship between the physical distance separating polymorphic restriction sites and linkage disequilibrium is discussed. The development of elastin haplotypes and knowledge of the pattern of linkage disequilibrium should aid the study of elastin related disease and human evolution.     

Genetic determination of human facial morphology: links between cleft-lips and normal variation

Recent genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with non-syndromic cleft lip with or without cleft palate (NSCL/P), and other previous studies showed distinctly differing facial distance measurements when comparing unaffected relatives of NSCL/P patients with normal controls. Here, we test the hypothesis that genetic loci involved in NSCL/P also influence normal variation in facial morphology. We tested 11 SNPs from 10 genomic regions previously showing replicated evidence of association with NSCL/P for association with normal variation of nose width and bizygomatic distance in two cohorts from Germany (N=529) and the Netherlands (N=2497). The two most significant associations found were between nose width and SNP rs1258763 near the GREM1 gene in the German cohort (P=6 × 10(-4)), and between bizygomatic distance and SNP rs987525 at 8q24.21 near the CCDC26 gene (P=0.017) in the Dutch sample. A genetic prediction model explained 2% of phenotype variation in nose width in the German and 0.5% of bizygomatic distance variation in the Dutch cohort. Although preliminary, our data provide a first link between genetic loci involved in a pathological facial trait such as NSCL/P and variation of normal facial morphology. Moreover, we present a first approach for understanding the genetic basis of human facial appearance, a highly intriguing trait with implications on clinical practice, clinical genetics, forensic intelligence, social interactions and personal identity.     

Butyrate as a bioactive human milk protective component against food allergy

Background

Food allergy (FA) is a growing health problem worldwide. Effective strategies are advocated to limit the disease burden. Human milk (HM) could be considered as a protective factor against FA, but its mechanisms remain unclear. Butyrate is a gut microbiota-derived metabolite able to exert several immunomodulatory functions. We aimed to define the butyrate concentration in HM, and to see whether the butyrate concentration detected in HM is able to modulate the mechanisms of immune tolerance.

Methods

HM butyrate concentration from 109 healthy women was assessed by GS-MS. The effect of HM butyrate on tolerogenic mechanisms was assessed in in vivo and in vitro models.

Results

The median butyrate concentration in mature HM was 0.75 mM. This butyrate concentration was responsible for the maximum modulatory effects observed in all experimental models evaluated in this study. Data from mouse model show that in basal condition, butyrate up-regulated the expression of several biomarkers of gut barrier integrity, and of tolerogenic cytokines. Pretreatment with butyrate significantly reduced allergic response in three animal models of FA, with a stimulation of tolerogenic cytokines, inhibition of Th2 cytokines production and a modulation of oxidative stress. Data from human cell models show that butyrate stimulated human beta defensin-3, mucus components and tight junctions expression in human enterocytes, and IL-10, IFN-γ and FoxP3 expression through epigenetic mechanisms in PBMCs from FA children. Furthermore, it promoted the precursors of M2 macrophages, DCs and regulatory T cells.

Conclusion

The study's findings suggest the importance of butyrate as a pivotal HM compound able to protect against FA.

     

Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variation

Human physical pigmentation is determined by the type and amount of melanin and the process of pigmentation production probably involves more than 100 genes. A failure to synthesize melanin results in oculocutaneous albinism (OCA). A recently identified form of OCA results from mutations in the Membrane Associated Transporter Protein (MATP) gene. The role of MATP in human pigmentation is not clear. We investigated the role of two nonpathogenic nonsynonymous single nucleotide polymorphisms (SNPs) in the MATP gene to determine if they are associated with normal human skin, hair, and eye color variation. A total of 608 individuals from four different population groups (456 Caucasians, 31 Asians, 70 African-Americans, and 51 Australian Aborigines) were genotyped for c.814G>A (p.Glu272Lys) and c.1122C>G (p.Phe374Leu). Results indicate that the allele frequencies of both polymorphisms are significantly different between population groups. The two alleles, 374Leu and 272Lys, are significantly associated with dark hair, skin, and eye color in Caucasians. The odds ratios (ORs) of the LeuLeu genotype for black hair and olive skin are 25.63 and 28.65, respectively, and for the LysLys genotype are 43.23 and 8.27, respectively. The OR for eye color is lower at 3.48 for the LeuLeu and 6.57 for LysLys genotypes. This is the first report of this highly significant association of MATP polymorphisms with normal human pigmentation variation.     

A comprehensive analysis of normal variation and disease-causing mutations in the human DSPP gene

Within nine dentin dysplasia (DD) (type II) and dentinogenesis imperfecta (type II and III) patient/families, seven have 1 of 4 net -1 deletions within the approximately 2-kb coding repeat domain of the DSPP gene while the remaining two patients have splice-site mutations. All frameshift mutations are predicted to change the highly soluble DSPP protein into proteins with long hydrophobic amino acid repeats that could interfere with processing of normal DSPP and/or other secreted matrix proteins. We propose that all previously reported missense, nonsense, and splice-site DSPP mutations (all associated with exons 2 and 3) result in dominant phenotypes due to disruption of signal peptide-processing and/or related biochemical events that also result in interference with protein processing. This would bring the currently known dominant forms of the human disease phenotype in agreement with the normal phenotype of the heterozygous null Dspp (-/+) mice. A study of 188 normal human chromosomes revealed a hypervariable DSPP repeat domain with extraordinary rates of change including 20 slip-replication indel events and 37 predominantly C-to-T transition SNPs. The most frequent transition in the primordial 9-basepair (bp) DNA repeat was a sense-strand CpG site while a CpNpG (CAG) transition was the second most frequent SNP. Bisulfite-sequencing of genomic DNA showed that the DSPP repeat can be methylated at both motifs. This suggests that, like plants and some animals, humans methylate some CpNpG sequences. Analysis of 37 haplotypes of the highly variable DSPP gene from geographically diverse people suggests it may be a useful autosomal marker in human migration studies.     

Sensitization to human milk

Background Allergy to milk is one of the earliest manifestations of IgE‐mediated allergies and affects about 2.5% of newborn children. Several reports indicate that milk‐allergic patients may be sensitized also to human milk proteins. Objective To analyse the specificity and possible biological relevance of IgE reactivity to human milk antigens in milk‐allergic patients. Methods The specificity of IgE reactivity to cow's milk and human milk antigens was analysed with sera from milk‐allergic children and adults by IgE immunoblotting. IgE cross‐reactivity between milk antigens was studied by immunoblot inhibition experiments. That IgE reactivity to human milk antigens is not due to alloreactivity or due to the transmission of foreign antigens into mother's milk was demonstrated through the analysis of milk samples from genetically unrelated mothers before and after intake of dietary milk products. The biological relevance of IgE reactivity to human milk was confirmed by skin testing. Results IgE antibodies to human milk were found in more than 80% of the tested milk‐allergic patients. Cross‐reactive IgE‐reactive human antigens such as α‐lactalbumin and non‐cross‐reactive human milk antigens were identified. Immediate‐type skin reactions could be elicited with human milk samples in patients with IgE reactivity to human milk. Conclusion IgE reactivity to human milk in milk‐allergic patients can be due to cross‐ sensitization and genuine sensitization to human milk and may cause allergic symptoms. IgE‐mediated sensitization to human milk is common in milk‐allergic patients and may require diagnostic testing and monitoring.     

Effects of milking procedure on rat milk composition

The effects of serial milking on the zinc concentration of rat milk and in liver of the suckling young are reported. With consecutive milkings at 7, 14, and 21 days of lactation, the zinc content of rat milk on Day 21 was almost three times as high as that found in rats milked only once (on Day 21). This effect was also reflected in the sucklings; liver zinc concentrations found in pups from serially milked dams were twice those found in pups from dams not milked serially. The results show that serial milking cannot be used if the objective is the procurement of physiologically “normal” rat milk.     

Immunoglobulin localization in the normal human mammary gland: variation with the menstrual cycle.

Differences in immunoglobulin localization (IgG, IgM, IgA) in benign versus malignant lesions of the human mammary gland have been previously demonstrated. The present investigation was undertaken to evaluate the influence of menstrual cycling on these immunoglobulins in the breast. Tissues were studied from 53 normal breasts. The patients were 16 to 51 years old, with a mean age of 35 +/- 9 years, and were documented as having regular menstrual cycling, lack of debilitating disease, and abstinence from use of hormones or from medications that might influence the pituitary-ovarian axis. Immunoglobulin localization in the tissue section was determined by direct immunofluorescence with antibodies specific for IgG, IgA, IgM, and IgA secretory component. The breast tissues were categorized into 5 histologically distinct menstrual cycle phases. No significant IgG localization was noted. IgA concentration was noted principally in the preovulatory phase of the cycle (P less than 0.03). IgM showed a similar variance, and IgA secretory component paralleled IgA localization. No correlation between IgA and IgM localization and plasma cells infiltration nor between plasma cell infiltration and menstrual cycle phase could be documented. These data suggest that menstrual cycling influences the human breast as part of the secretory immune system.