Bruce A. Hamilton

The diastrophic dysplasia gene encodes a novel sulfate transporter: Positional cloning by fine-structure linkage disequilibrium mapping

Diastrophic dysplasia (DTD) is a well-characterized autosomal recessive osteochondrodysplasia with clinical features including dwarfism, spinal deformation, and specific joint abnormalities. The disease occurs in most populations, but is particularly prevalent in Finland owing to an apparent founder effect. DTD maps to distal chromosome 5q and, based on linkage disequilibrium studies in the Finnish population, we had previously predicted that the DTD gene should lie about 64 kb away from the CSF1R locus. Here, we report the positional cloning of the DTD gene by fine-structure linkage disequilibrium mapping. The gene lies in the predicted location, approximately 70 kb proximal to CSF1R, and encodes a novel sulfate transporter. Impaired function of its product is likely to lead to undersulfation of proteoglycans in cartilage matrix and thereby to cause the clinical phenotype of the disease. These results demonstrate the power of linkage disequilibrium mapping in isolated populations for positional cloning.

The vibrator Mutation Causes Neurodegeneration via Reduced Expression of PITPα: Positional Complementation Cloning and Extragenic Suppression

The mouse vibrator mutation causes an early-onset progressive action tremor, degeneration of brain stem and spinal cord neurons, and juvenile death. We cloned the vibrator mutation using an in vivo positional complementation approach and complete resequencing of the resulting 76 kb critical region from vibrator and its parental chromosome. The mutation is an intracisternal A particle retroposon insertion in intron 4 of the phosphatidylinositol transfer protein alpha gene, causing a 5-fold reduction in RNA and protein levels. Expression of neurofilament light chain is also reduced in vibrator, suggesting one signaling pathway that may underlie vibrator pathology. The vibrator phenotype is suppressed in one intercross. We performed a complete genome scan and mapped a major suppressor locus (Mvb-1) to proximal chromosome 19.

IOC consensus paper on the use of platelet-rich plasma in sports medicine

PRP and its variant forms were originally used in clinical practice as an adjunct to surgery to assist in the healing of various tissues. PRP has also been used in prosthetic surgery to promote tissue healing and implant integration, and to control blood loss. 4 5 Furthermore, the application of activated PRP has an effect on pain and pain medication use following open subacromial decompression surgery. 5

RORα coordinates reciprocal signaling in cerebellar development through Sonic hedgehog and calcium-dependent pathways

The cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORalpha block Purkinje cell differentiation with a secondary loss of afferent granule cells. We show that early transcriptional targets of RORalpha include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORalpha acts through recruitment of gene-specific sets of transcriptional cofactors, including beta-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORalpha-deficient cerebellum. Our results suggest a link between RORalpha and beta-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORalpha expression in coordinating expression of genes required for reciprocal signals in cerebellar development.

Catecholamine release-inhibitory peptide catestatin (chromogranin A352-372): Naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension

Background— Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release–inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure. Methods and Results— Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by ≈47%) and downward deflections (by ≈44%), increased cardiac parasympathetic index (by ≈2.4-fold), and decreased cardiac sympathetic index (by ≈26%). Renal norepinephrine excretion was diminished by ≈26% and epinephrine excretion by ≈34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to ≈70 000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by ≈5 to 6 mm Hg, and the polymorphism accounted for ≈1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men. Conclusions— The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions.

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

Organic anion and cation transporters occur in pairs of similar and similarly expressed genes.

Organic anion and cation transporters (OATs, OCTs, OCTNs, and ORCTLs), transmembrane proteins essential to renal xenobiotic excretion, are encoded by a group of related genes. As yet there have been no studies of the transcriptional regulation of this important gene family. While such studies have traditionally been labor-intensive, comparative genomics approaches are now available that have proven reliable guides to critical regulatory elements. We report here the genomic sequencing of murine OAT1 (the cDNA of which was originally cloned by us as NKT) and OAT3 (Roct), and derivation of phylogenetic footprints (evolutionarily conserved non-coding sequences) by comparison to the human genome. We find binding sites within these footprints for several transcription factors implicated in kidney development, including PAX1, PBX, WT1, and HNF1. Additionally, we note that OATs and OCTs occur in the human and mouse genomes as tightly linked pairs (OAT1 and OAT3, UST3 and OAT5, OAT4 and URAT1/RST, OCT1 and 2, OCTN1 and 2, ORCTL3 and 4) that are also close phylogenetic relations, with Flipt1 and 2, and OAT2 the only unpaired family members. Finally, we find that pair-members have similar tissue distributions, suggesting that the pairing might exist to facilitate the co-regulation of the genes within each pair.

C-reactive protein, an 'intermediate phenotype' for inflammation: Human twin studies reveal heritability, association with blood pressure and the metabolic syndrome, and the influence of common polymorphism at catecholaminergic/β-adrenergic pathway loci

Background C-reactive protein (CRP) both reflects and participates in inflammation, and its circulating concentration marks cardiovascular risk. Here we sought to understand the role of heredity in determining CRP secretion. Methods CRP, as well as multiple facets of the metabolic syndrome, were measured in a series of 229 twins, both monozygotic (MZ) and dizygotic (DZ), to estimate trait heritability (h2). Single nucleotide polymorphism (SNP) genotyping was done at adrenergic pathway loci. Haplotypes were inferred from genotypes by likelihood methods. Association of CRP with hypertension and the metabolic syndrome was studied in a larger series of 732 individuals, including 79 with hypertension. Results MZ and DZ twin variance components indicated substantial h2 for CRP, at ∼56 ± 7% (P < 0.001). CRP was significantly associated (P < 0.05) with multiple features of the metabolic syndrome in twins, including body mass index (BMI), blood pressure (BP), leptin and lipids. In established hypertension, elevated CRP was associated with increased BP, BMI, insulin, HOMA (index of insulin resistance), leptin, triglycerides and norepinephrine. Twin correlations indicated pleiotropy (shared genetic determination) for CRP with BMI (P = 0.0002), leptin (P < 0.001), triglycerides (P = 0.002) and systolic blood pressure (SBP) (P = 0.042). Approximately 9800 genotypes (43 genetic variants at 17 loci) were scored within catecholaminergic pathways: biosynthetic, receptor and signal transduction. Plasma CRP concentration in twins was predicted by polymorphisms at three loci in physiological series within the catecholamine biosynthetic/β-adrenergic pathway: TH (tyrosine hydroxylase), ADRB1 (β1-adrenergic receptor) and ADRB2 (β2-adrenergic receptor). In the TH promoter, common allelic variation accounted for up to ∼6.6% of CRP inter-individual variance. At ADRB1, variation at Gly389Arg predicted ∼2.8% of CRP, while ADRB2 promoter variants T-47C and T-20C also contributed. Particular haplotypes and diplotypes at TH and ADRB1 also predicted CRP, though typically no better than single SNPs alone. Epistasis (gene-by-gene interaction) was demonstrated for particular combinations of TH and ADRB2 alleles, consistent with their actions in a pathway in series. In an illustration of pleiotropy, not only CRP but also plasma triglycerides were predicted by polymorphisms at TH (P = 0.0053) and ADRB2 (P = 0.027). Conclusions CRP secretion is substantially heritable in humans, demonstrating pleiotropy (shared genetic determination) with other features of the metabolic syndrome, such as BMI, triglycerides or BP. Multiple, common genetic variants in the catecholaminergic/β-adrenergic pathway contribute to CRP, and these variants (especially at TH and ADRB2) seem to interact (epistasis) to influence the trait. The results uncover novel pathophysiological links between the adrenergic system and inflammation, and suggest new strategies to probe the role and actions of inflammation within this setting.

Both Rare and Common Polymorphisms Contribute Functional Variation at CHGA, a Regulator of Catecholamine Physiology

The chromogranin/secretogranin proteins are costored and coreleased with catecholamines from secretory vesicles in chromaffin cells and noradrenergic neurons. Chromogranin A (CHGA) regulates catecholamine storage and release through intracellular (vesiculogenic) and extracellular (catecholamine release-inhibitory) mechanisms. CHGA is a candidate gene for autonomic dysfunction syndromes, including intermediate phenotypes that contribute to human hypertension. Here, we show a surprising pattern of CHGA variants that alter the expression and function of this gene, both in vivo and in vitro. Functional variants include both common alleles that quantitatively alter gene expression and rare alleles that qualitatively change the encoded product to alter the signaling potency of CHGA-derived catecholamine release-inhibitory catestatin peptides.

Mice Lacking Phosphatidylinositol Transfer Protein-α Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITPα function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITPα–/– mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITPα in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes.