The frequency and clinical impact of synonymous HTT loss-of-interruption and duplication-of-interruption variants in a diverse HD cohort.

PURPOSE: To determine the frequency and clinical impact of loss-of-interruption (LOI) and duplication-of-interruption modifier variants of the HTT CAG and CCG repeat in a cohort of individuals with Huntington disease (HD). METHODS: We screened symptomatic HD participants from the UBC HD Biobank and 5 research sites for sequence variants. After variant identification, we examined the clinical impact and frequency in the reduced penetrance range. RESULTS: Participants with CAG-CCG LOI and CCG LOI variants have a similar magnitude of earlier onset of HD, by 12.5 years. The sequence variants exhibit ancestry-specific differences. Participants with the CAG-CCG LOI variant also have a faster progression of Total Motor Score by 1.9 units per year. Symptomatic participants with the CAG-CCG LOI variant show enrichment in the reduced penetrance range. The CAG-CCG LOI variant explains the onset of 2 symptomatic HD participants with diagnostic repeats below the pathogenetic range. CONCLUSION: Our findings have significant clinical implications for participants with the CAG-CCG LOI variant who receive inaccurate diagnoses near diagnostic cutoff ranges. Improved diagnostic testing approaches and clinical management are needed for these individuals. We present the largest and most diverse HTT CAG and CCG sequence variant cohort and emphasize their importance in clinical presentation in HD.

Length of Uninterrupted CAG, Independent of Polyglutamine Size, Results in Increased Somatic Instability, Hastening Onset of Huntington Disease.

Huntington disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Although the length of this repeat is inversely correlated with age of onset (AOO), it does not fully explain the variability in AOO. We assessed the sequence downstream of the CAG repeat in HTT [reference: (CAG)n-CAA-CAG], since variants within this region have been previously described, but no study of AOO has been performed. These analyses identified a variant that results in complete loss of interrupting (LOI) adenine nucleotides in this region [(CAG)n-CAG-CAG]. Analysis of multiple HD pedigrees showed that this LOI variant is associated with dramatically earlier AOO (average of 25 years) despite the same polyglutamine length as in individuals with the interrupting penultimate CAA codon. This LOI allele is particularly frequent in persons with reduced penetrance alleles who manifest with HD and increases the likelihood of presenting clinically with HD with a CAG of 36-39 repeats. Further, we show that the LOI variant is associated with increased somatic repeat instability, highlighting this as a significant driver of this effect. These findings indicate that the number of uninterrupted CAG repeats, which is lengthened by the LOI, is the most significant contributor to AOO of HD and is more significant than polyglutamine length, which is not altered in these individuals. In addition, we identified another variant in this region, where the CAA-CAG sequence is duplicated, which was associated with later AOO. Identification of these cis-acting modifiers have potentially important implications for genetic counselling in HD-affected families.

Frequency of the loss of CAA interruption in the HTT CAG tract and implications for Huntington disease in the reduced penetrance range.

PURPOSE: In some Huntington disease (HD) patients, the "loss of interruption" (LOI) variant eliminates an interrupting codon in the HTT CAG-repeat tract, which causes earlier age of onset (AOO). The magnitude of this effect is uncertain, since previous studies included few LOI carriers, and the variant also causes CAG size misestimation. We developed a rapid LOI detection screen, enabling unbiased frequency estimation among manifest HD patients. Additionally, we combined published data with clinical data from newly identified patients to accurately characterize the LOI's effect on AOO. METHODS: We developed a LOI detection polymerase chain reaction (PCR) assay, and screened patients to estimate the frequency of the LOI variant and its effect on AOO. RESULTS: Mean onset for LOI carriers (n = 49) is 20.4 years earlier than expected based on diagnosed CAG size. After correcting for CAG size underestimation, the variant is still associated with onset 9.5 years earlier. The LOI is present in 1.02% of symptomatic HD patients, and in 32.2% of symptomatic reduced penetrance (RP) range patients (36-39 CAGs). CONCLUSION: The LOI causes significantly earlier onset, greater than expected by CAG length, particularly in persons with 36-39 CAG repeats. Detection of this variant has implications for HD families, especially for those in the RP range.

Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein.

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1-84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescence-associated tumor suppressor proteins p16(INK4a) and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.

Co-treatment of PTH with osteoprotegerin or alendronate increases its anabolic effect on the skeleton of oophorectomized mice.

UNLABELLED: We examined the effects of 60 days of co-treatment of PTH with either OPG or alendronate in oophorectomized mice. Compared with PTH alone, co-treatment of PTH with either of these two mechanistically distinct anti-catabolics improved bone volume, mechanical strength, and appendicular and axial mineralization and prolonged the beneficial effect of PTH on BMD. INTRODUCTION: Conflicting evidence exists as to whether the anabolic effect of PTH is inhibited by the action of anti-catabolics. To examine this issue, we assessed the effects of alendronate and osteoprotegerin (OPG), two anti-catabolics with different modes of action, on the anabolic activity of PTH(1-34) in the skeleton of 4-month-old oophorectomized mice. MATERIALS AND METHODS: Mice treated with vehicle alone (PBS), alendronate alone (100 microg/kg/week), OPG alone (10 mg/kg twice a week), or PTH alone (80 microg/kg/day) were compared with each other and with animals administered PTH plus alendronate or PTH plus OPG. We assessed lumbar spine and femoral BMD at 0, 30, and 60 days. Contact radiography, histology, and histomorphometry, three-point bending assay of the femur, and serum osteocalcin and TRACP5b assays were performed at 2 months. RESULTS: Although alendronate and OPG each suppressed bone turnover, at the doses used, this was more profound with OPG. Increases in lumbar spine and femoral BMD and in trabecular bone volume were at least as great with OPG as with alendronate, and mechanical indices of femoral bone strength improved only with OPG. Both produced a plateau in spine and femoral BMD increases by 30 days. Co-treatment of PTH with each anti-catabolic produced additive increases in BMD in the femur and supra-additive increases in the lumbar spine with no plateau effects. Neither anti-catabolic impeded the PTH-induced increase in bone volume or the increase in mechanical strength of the femur. CONCLUSIONS: These studies show that the highly potent anti-catabolic OPG can produce dramatic increases in BMD and bone strength; that the temporal pattern of activity of bone formation and resorption modulators may have major influence on net skeletal accrual; and that, depending on timing, inhibition of osteoclastic activity may markedly augment the anabolic action of PTH.

Pretreatment with anticatabolic agents blunts but does not eliminate the skeletal anabolic response to parathyroid hormone in oophorectomized mice.

Previous studies have indicated that bisphosphonate pretreatment can inhibit the anabolic actions of PTH. We examined the capacity of two anticatabolic agents with different mechanisms of action, alendronate and osteoprotegerin (OPG), to influence the anabolic activity of PTH. Oophorectomized mice were pretreated for 30 d with alendronate or OPG and then treated for 30 d with the respective anticatabolic alone or the respective anticatabolic plus PTH(1-34). Bones were analyzed by bone mineral density (BMD), microcomputed tomography, histology and histomorphometry, and biochemical bone markers. OPG pretreatment produced a greater inhibition of bone turnover and a greater increase in bone than alendronate. Increases in bone were sustained during subsequent treatment with vehicle or continued administration of the anticatabolic. Pretreatment with each anticatabolic blunted the capacity of PTH to increase BMD and bone volume and continued treatment with each anticatabolic agent also reduced the effectiveness of PTH. Although both anticatabolics decreased the maximal PTH effect, BMD and bone volume increased more when PTH was added than when only anticatabolics were used. These results demonstrate that mechanistically distinct anticatabolics may reduce PTH efficacy, that the characteristics of this inhibition may reflect the different modes of action of the anticatabolics, but that the addition of PTH still provides a skeletal benefit even if the anabolic effect is submaximal.

Inheritance of the photoperiodic response controlling imaginal summer diapause in the cabbage beetle, Colaphellus bowringi.

Adults of the cabbage beetle Colaphellus bowringi display a summer diapause in response to the exposure of their larvae to long photoperiods. In the present study, the inheritance of the photoperiodic response controlling summer diapause in C. bowringi by crossing a high diapause strain (D strain) with a laboratory selected nondiapause strain (N strain) was investigated under different photoperiods at 22, 25 and 28°C. The beetles in both reciprocal crosses and backcrosses showed a clear short-day response for the induction of diapause at all temperatures, similar to that of the D strain, suggesting that photoperiodic response of this beetle is heritable. The diapause incidences in the progeny from all the crosses under LD 15:9 or LD 12:12 at 25°C suggest that genetic and genetic-environmental interactions are involved in diapause induction. The incidence of diapauses in F(1) progeny was significantly lower than that in the D×D strain but significantly higher than that in the N×N strain, indicating that the diapause capability is inherited in an incomplete dominant manner. The incidence of diapause was greater among the offspring of hybrid females when those females had a D strain mother or grandmother rather than a N strain mother or grandmother, indicating that maternal effects on diapause induction are stronger than paternal effects. The laboratory selected nondiapause strain also showed a short-day photoperiodic response at a low temperature of 22°C, indicating that the photoperiodic photoreceptor and photoperiodic clock still function in the nondiapause strain.

Exogenous PTH and endogenous 1,25-dihydroxyvitamin D are complementary in inducing an anabolic effect on bone.

PTH and 1,25(OH)(2)D each exert dual anabolic and catabolic skeletal effects. We assessed the potential interaction of PTH and 1,25(OH)(2)D in promoting skeletal anabolism by comparing the capacity of exogenous, intermittently injected PTH(1-34) to produce bone accrual in mice homozygous for the 1 alpha(OH)ase-null allele [1 alpha(OH)ase(-/-) mice] and in wildtype mice. In initial studies, 3-mo-old wildtype mice were either injected once daily (40 microg/kg) or infused continuously (120 microg/kg/d) with PTH(1-34) for up to 1 mo. Infused PTH reduced BMD, increased the bone resorption marker TRACP-5b, and raised serum calcium but did not increase serum 1,25(OH)(2)D. Injected PTH increased serum 1,25(OH)(2)D and BMD, raised the bone formation marker osteocalcin more than did infused PTH, and did not produce sustained hypercalcemia as did PTH infusion. In subsequent studies, 3-mo-old 1 alpha(OH)ase(-/-) mice, raised on a rescue diet, and wildtype littermates were injected with PTH(1-34) (40 microg/kg) either once daily or three times daily for 1 mo. In 1 alpha(OH)ase(-/-) mice, baseline bone volume (BV/TV) and bone formation (BFR/BS) were lower than in wildtype mice. PTH administered intermittently increased BV/TV and BFR/BS in a dose-dependent manner, but the increases were always less than in wildtype mice. These studies show that exogenous PTH administered continuously resorbs bone without raising endogenous 1,25(OH)(2)D. Intermittently administered PTH can increase bone accrual in the absence of 1,25(OH)(2)D, but 1,25(OH)(2)D complements this PTH action. An increase in endogenous 1,25(OH)(2)D may therefore facilitate an optimal skeletal anabolic response to PTH and may be relevant to the development of improved therapeutics for enhancing skeletal anabolism.

Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis.

A ubiquitin protein ligase (E3), E3(Histone)/LASU1 (Mule/ARF-BP1/HUWE1), was recently identified that mediates ubiquitination of core histones, the Mcl-1 anti-apoptotic protein, and the p53 tumor suppressor protein. However, the expression of E3(Histone)/LASU1 remains poorly studied. Because we identified E3(Histone)/LASU1 from the testis, we explored its regulation during spermatogenesis. In the first wave of rat spermatogenesis, E3(Histone)/LASU1 mRNA and protein had peak expression at days 10 and 20, respectively, and decreased with age. Consistent with these findings, immunohistochemistry revealed that E3(Histone)/LASU1 was highly expressed in nuclei from spermatogonia to mid-pachytene spermatocytes. There was no obvious staining in spermatids, when histones are ubiquitinated and degraded. E3(Histone)/LASU1 was also expressed in other tissues. However, except in neuronal cells of the brain, expression was cytoplasmic. Thus, E3(Histone)/LASU1 may play a role in chromatin modification in early germ cells of the testis, but also has functions in other tissues.