Designing pharmacogenetic projects in industry: practical design perspectives from the Industry Pharmacogenomics Working Group.

Pharmacogenetic association studies have the potential to identify variations in DNA sequence which impact drug response. Identifying these DNA variants can help to explain interindividual variability in drug response; this is the first step in personalizing dosing and treatment regimes to a patient's needs. There are many intricacies in the design and analysis of pharmacogenetic association studies, including having adequate power, selecting proper endpoints, detecting and correcting the effects of population stratification, modeling genetic and nongenetic covariates accurately, and validating the results. At this point there are no formal guidelines on the design and analysis of pharmacogenetic studies. The Industry Pharmacogenomics Working Group has initiated discussions regarding potential guidelines for pharmacogenetic study design and analyses (http://i-pwg.org) and the results from these discussions are presented in this paper.

Detection of chromosomal abnormalities in the dysmorphic fetus using fluorescence in situ hybridization: evaluation for monosomy X genotype.

The cytogenetic abnormalities of dysmorphic fetuses who died in utero cannot be analyzed reliably by karyotyping. To overcome this obstacle, the authors applied fluorescence in situ hybridization (FISH) to formalin-fixed, paraffin-embedded tissue of two female infants and 13 female fetuses whose phenotypic features suggested possible Turner's syndrome. Previous cytogenetic evaluation of the amnionic fluid showed five were 45,XO karyotype; two were 46,XX karotype; and eight were of unknown karyotype. Karyotyping had been attempted on four of the unknown cases without success. The copy number of X chromosomes were correctly identified by FISH in all previously karyotyped cases. Of the remaining eight cases with unknown karyotype, three appeared to be XX, and five cases were identified as monosomy XO, confirming the suspicion of Turner's syndrome genotype. FISH is useful for confirming suspected Turner's syndrome genotypes in infants and macerated fetuses in which a karyotype cannot be obtained otherwise.

Sponge artifact in biopsy specimens.

We describe a sponge-induced artifact in histologic sections of small biopsy specimens. The artifacts are angulated, often triangular holes within the tissue. They appear to be introduced as individual sponge barbs become embedded in the perimeter of biopsy specimens during tissue processing. The artifact is generally of little importance, but in certain specimens, such as needle biopsies of the kidney or liver, it may occasionally obscure important information. Other methods, such as lens paper wrapping, may be superior in these situations. The utility of the tissue cassette sponge, in most situations, outweighs the artifact.

Genetic parameters among weight, prolificacy, and wool traits of Columbia, Polypay, Rambouillet, and Targhee sheep.

Genetic parameters for Columbia, Polypay, Rambouillet, and Targhee sheep were estimated using REML with animal models for prolificacy, weight, and wool traits. All bivariate analyses included a covariance between additive genetic effects for the two traits plus appropriate additional covariances. Number of observations by breed ranged from 5,140 to 7,095 for prolificacy traits, from 7,750 to 9,530 for weight traits, and from 4,603 to 34,746 for wool traits. Heritability estimates ranged from .03 to .11 for prolificacy traits (litter size at birth and litter size at weaning), from .09 to .26 for weight traits (birth weight and average daily gain), and from .25 to .53 for wool traits (fleece weight, fleece grade and staple length). Estimates of direct genetic correlations among prolificacy and among weight traits were positive and ranged from .58 to 1.00 and .18 to 1.00, respectively. Estimates of direct genetic correlation between fleece weight and staple length were positive (.50 to .70) but were negative between fleece weight and fleece grade (-.60 to -.34) and between staple length and fleece grade (-.72 and -.40). Prolificacy and wool traits were essentially uncorrelated. Weight and prolificacy traits were slightly positively correlated. Weight traits had a moderate positive direct genetic correlation with fleece weight and staple length, but were uncorrelated with fleece grade. These estimates of genetic parameters between prolificacy, weight, and wool traits can be used to construct multiple-trait selection indexes for dual-purpose sheep.

Genetic correlations for litter weight weaned with growth, prolificacy, and wool traits in Columbia, Polypay, Rambouillet, and Targhee sheep.

Total litter weight weaned at 120 d postpartum per ewe lambing is often believed to be a measure of range ewe productivity. Genetic correlations for litter weight weaned at 120 d with prolificacy, growth, and wool traits for Columbia, Polypay, Rambouillet, and Targhee sheep were estimated using REML with animal models. Observations per breed ranged from 5,140 to 7,083 for litter weight weaned, from 5,140 to 7,095 for prolificacy traits, from 7,750 to 9,530 for growth traits, and from 4,603 to 18,443 for wool traits. Heritability estimates for litter weight weaned were low and ranged from 0.02 to 0.11. Fraction of variance due to permanent environmental effects averaged 0.05 and, due to effects of mates, averaged 0.01. Estimates of genetic correlations with litter weight weaned varied from breed to breed. The ranges were as follows: 0.42 to 0.65 with litter size born, 0.80 to 0.99 with litter size weaned, -0.22 to 0.28 with birth weight, -0.07 to 0.23 with average daily gain to weaning, -0.56 to 0.19 with fleece weight, -0.15 to 0.02 with fleece grade, and -0.11 to 0.08 with staple length. Results suggest that, if selection were practiced on litter weight weaned, the average correlated responses would be expected to be favorable or neutral for prolificacy, growth, and wool traits although responses might vary from breed to breed.

Usefulness of subjective ovine milk scores: I. Associations with range ewe characteristics and lamb production.

Range ewes are commonly evaluated for milking ability by producers to determine the ewe's ability to rear lamb(s). The U.S. Sheep Experiment Station has subjectively scored (low, average, high) a ewe's milking ability within 24 h of lambing for many years. The relationship of subjective milk scores with lamb production was investigated using lambing records of Columbia (n = 1,731), Polypay (n = 1,129), Rambouillet (n = 1,704), and Targhee (n = 1,638) ewes. The incidence of high milk scores increased from less than 10% at first parity to 29 to 40% at second and greater parities. At maturity, Columbia ewes (38%) had the highest percentage of high milk scores. A positive association existed between ewe BW and her milk score at third and later parities. Ewes with high milk scores gave birth to heavier lambs (P < 0.05), whereas ewes with low milk scores were associated with lighter (P < 0.05) lambs at birth. Ewes with low milk scores weaned less (P < 0.05) total weight than ewes with better milk scores across all age groups for all breeds. Lighter weaned litter weights from ewes with low milk scores were linked to lighter birth weights and fewer weaned lambs. Differences for litter weight weaned between ewes with average and high milk scores were generally observed at 2 and 3 yr of age, when litter weights were heavier among ewes with high milk scores (P < 0.05) for all breeds. Between the ages of 1 and 3 yr, Columbia, Polypay, Rambouillet, and Targhee ewes with an average milk score weaned heavier (P < 0.05) litters (average differences of 10, 9, 13, and 12%, respectively) than ewes with low milk scores. For all breeds at all ages, individual lamb weaning weights were heavier (P < 0.05) when they were reared by ewes with high milk scores compared to lambs reared by ewes with low milk scores. Results suggest that milk score is an economically important trait in these four breeds and should be considered in management and breeding objectives; at a minimum, the incidence of low milk scores should be kept as small as possible.

Usefulness of subjective ovine milk scores: II. Genetic parameter estimates.

Genetic parameters for a subjective milk score given to ewes within 24 h of parturition were estimated to determine the usefulness of milk score as a selection trait to improve milk production, which influences total litter weight weaned. Heritability of milk score and the genetic correlation of milk score with litter weight weaned were estimated by REML separately for four sheep breeds, Rambouillet (n = 1,731), Targhee (n = 1,638), Columbia (n = 1,731), and Polypay (n = 1,129). Litter weight weaned was the total weight of lambs weaned at approximately 120 d of age under a western range production system. Observed heritability estimates for milk score at first parity were moderate and similar among breeds, ranging from 0.18 to 0.32. Heritability estimates adjusted for a binomial distribution of milk scores at first parity were high (Columbia, 0.43; Polypay, 0.35; Rambouillet, 0.50; Targhee, 0.84). Estimates of observed heritability for second-parity milk score were moderate to high, ranging from 0.23 to 0.46. Milk score at first or second parity was genetically correlated with milk score records at maturity (third parity and greater), with estimates ranging from 0.69 to 1.00. Milk score and litter weight weaned were genetically correlated at first or second parity in Rambouillet (r(g) = 1.00) and Targhee breeds (r(g) = 1.00 and 0.61, respectively), but not in the Columbia and Polypay breeds. Estimates of heritability for lifetime records for milk score ranged from 0.16 to 0.26 across breeds. Estimates of genetic correlations of annual lifetime milk score records with litter weight weaned were high (Columbia, 1.00; Polypay, 0.81; Rambouillet, 1.00; and Targhee, 0.77). Repeatability estimates for milk score were similar across breeds, 0.23 for Columbia, Rambouillet, and Targhee ewes and 0.28 for Polypay ewes. Milk score measured at first or second parity may be a good predictor of future potential milking ability. Further, milk score can be used as a selection trait to improve maternal ability for increasing litter weight weaned. The need for increasing ewe milking performance and lamb growth rate at first parity in commercial range sheep production systems may be addressed by selection for milk score at first parity.

Genotypic expression at different ages: I. Prolificacy traits of sheep.

Genetic parameters for prolificacy traits for Columbia (COLU), Polypay (POLY), Rambouillet (RAMB), and Targhee (TARG) breeds of sheep were estimated with REML using animal models. Traits were number of live births (LAB), litter size at birth (LSB) and weaning (LSW), and litter weight weaned (LWW). Numbers of observations ranged from 5,140 to 7,095 for prolificacy traits and from 5,101 to 8,973 for litter weight weaned for the four breeds. For single-trait analyses, ewes were classified as young (1 yr old), middle-aged (2 and 3 yr old), or older (> 3 yr old). After single-trait analyses, three-trait analyses were done for each characteristic with traits defined by age class. Generally, heritability estimates from single-trait analyses were low and ranged from .01 to .17 for LAB and LSB and from .00 to .10 for LSW. Heritability estimates obtained for LWW ranged from low to moderate (.00 to .25) and were less for older ewes. Heritability estimates from the three-trait analyses were generally similar to estimates from single-trait analyses. Heritabilities for LAB and LSB were similar, and, for three-trait analyses, they ranged across age groups from .07 to .13 for COLU, .13 to .16 for POLY, .10 to .16 for RAMB, and .01 to .16 for TARG. Estimates for LSW from three-trait analyses ranged from .07 to .12 for COLU, .04 to .09 for POLY, .01 to .11 for RAMB, and .03 to .11 for TARG. For LWW, heritabilities ranged from .00 to .21 for COLU, .05 to .08 for POLY, .12 to .15 for RAMB, and .18 to .29 for TARG. Genetic correlations for LAB, LSB and LSW among age-defined traits ranged from .25 to 1.00. Genetic correlations for LAB and LSB between young and middle and between young and older age classes were less than .80 in COLU, POLY, and RAMB breeds. Only genetic correlations between middle and older age classes for these breeds were greater than .80. For TARG, genetic correlations among all age classes were greater than .80 (.88 to 1.00) for those traits. All genetic correlations among ages for LSW were greater than .80 for POLY and TARG. For RAMB, only the correlation between young and older age classes for LSW was less than .80 (.45). None was greater than .80 for COLU. For LWW, genetic correlations among all age classes in POLY and RAMB were greater than .80 (.82 to 1.00). For COLU, genetic correlation between young and middle was low (.07), between young and older was high (.88), and between middle and older classes was moderately high (.54). For TARG, genetic correlations were .49, .65, and .98 for young-middle, young-older, and middle-older age classes, respectively. Results indicate that more progress could be made in selection programs for prolificacy traits in some sheep breeds by considering age of ewe as a part of the trait rather than by simply adjusting for ages of ewes.

Genotypic expression at different ages: II. Wool traits of sheep.

Genetic parameters for wool traits for Columbia, Polypay, Rambouillet, and Targhee breeds of sheep were estimated with single- and multiple-trait analyses using REML with animal models. Traits considered were fleece grade, fleece weight, and staple length. Total number of observations ranged from 11,673 to 34,746 for fleece grade and fleece weight and from 3,500 to 11,641 for staple length for the four breeds. For single-trait analyses, data were divided by age of ewe: young ages (age of 1 yr), middle ages (ages of 2 and 3 yr), and older ages (age greater than 3 yr). Heritability estimates averaged over breeds for fleece grade decreased from .42 at a young age to .37 for older ages. For fleece weight, heritability estimates averaged .52, .57, and .55 within the successively older groups. Heritability estimates for staple length averaged .54 for young and middle age classes. Few older ewes had staple length measurements. After single-trait analyses, new data sets were created for three-trait analyses with traits defined by three age classes when animals were measured. Heritability estimates with three-trait analyses, except for a few cases, were somewhat greater than those from single-trait analyses. For fleece grade, the genetic correlations averaged over breeds were .72 for young with middle, .42 for young with older, and .86 for middle with older age classes. For fleece weight, the average genetic correlations were .81, .83, and .98. For staple length, the average genetic correlation for young with middle age classes was .82. Estimates of genetic correlations across ages varied considerably among breeds. The average estimates of correlations suggest that fleece grade may need to be defined by age, especially for the Columbia and Rambouillet breeds. For fleece weight and staple length, however, the average correlations suggest no need to define those traits by age.

Genotypic expression with different ages of dams: III. Weight traits of sheep.

Correlations between genetic expression in lambs when dams were young (1 yr), middle-aged (2 and 3 yr), or older (older than 3 yr) were estimated with three-trait analyses for weight traits. Weights at birth (BWT) and weaning (WWT) and ADG from birth to weaning were used. Numbers of observations were 7,731, 9,518, 9,512, and 9,201 for Columbia (COLU), Polypay (POLY), Rambouillet (RAMB), and Targhee (TARG) breeds of sheep, respectively. When averaged, relative estimates for WWT and ADG were similar across breeds. Estimates were variable across breeds. On average, direct heritability was greater when environment was young dams (.44 for BWT and .34 for WWT) than when environment was dams of middle age or older (.24 and .28 for BWT and .20 and .16 for WWT, respectively). Maternal heritability was greater when dams were middle-aged or older (.28 and .22 vs .18) for BWT but was greater when dams were younger (.10 vs .05 and .04) for WWT. The estimates of genetic correlations for direct effects across age of dam environments averaged .32 for birth weight and averaged .70 for weaning weight. Average estimates of maternal genetic correlations across age of dam classes were .36 or less for both BWT and WWT. Average estimates of correlations among maternal permanent environmental effects were .49 or less across age of dam classes. Total maternal effects accounted for .33 to .42 of phenotypic variance for BWT and for .09 to .26 of phenotypic variance for WWT. The average estimates of genetic correlations between expressions of the same genotypes with different ages of dams suggest that measurements of BWT of lambs with dams in young, middle, and older age classes should be considered to be separate traits for genetic evaluation and that for WWT measurements with young age of dam class and combined middle and older age of dam classes should be considered to be separate traits for genetic evaluation.

Immunohistochemical distinction of classic and chondroid chordomas.

Chondroid chordomas are cartilage-rich neoplasms, most often located in the spheno-occipital region, that have a better prognosis than classic chordomas. The immunohistochemical features of 19 classic and chondroid chordomas were studied retrospectively using avidin-biotin-complex (ABC) immunoperoxidase histochemistry on formalin-fixed, paraffin-embedded tissue. Of the 19 tumors, all located in the spheno-occipital region, 5 exhibited predominantly chondroid morphologic features. The 14 classic chordomas showed the following pattern of antigen expression (percent of tumors positive): epithelial membrane antigen (EMA) 100%, AE 1/3 (a "cocktail" of monoclonal antibodies directed against low and high molecular weight epidermal cytokeratins) 100%, DP keratin (DPK) 100%, vimentin 100%, S100 86%, neuron specific enolase (NSE) 100%, carcinoembryonic antigen (CEA) 57%, and HMB-45 (an anti-melanoma-associated antibody) 57%. The five chondroid chordomas exhibited the following pattern: EMA 0%, AE 1/3 0%, DPK 0%, vimentin 100%, S100 100%, NSE 100%, CEA 0%, and HMB-45 0%. The focal, weak HMB-45 positivity (performed on the index case because of a clinical concern of metastatic melanoma) seen in 57% of the classic chordomas is a previously unreported finding. This finding suggests either that classic chordomas are capable of HMB-45 expression or that this antibody has broader reactivity than previously recognized. The lack of cytokeratin, EMA, and CEA expression by the chondroid chordomas is similar to chondrosarcomas as reported in the literature and dissimilar to the classic chordoma group. These immunohistochemical findings suggest that chondroid chordomas may more validly be classified as low grade chondrosarcomas.

Chordoma: incidence and survival patterns in the United States, 1973-1995.

BACKGROUND: Chordoma, a rare tumor arising from notochordal remnants, has been described to date only by single-institution case series or small population-based surveys. METHODS: We used data from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, 1973-1995, to calculate age-adjusted incidence and survival rates for 400 cases of microscopically confirmed chordoma and to derive information regarding case distribution and risk of second cancer. RESULTS: The age-adjusted chordoma incidence rate (IR) of 0.08 per 100,000 was age-dependent, more common in males (IR 0.10) than females (IR 0.06) and rare among patients aged <40 years and blacks. Within the axial skeleton 32% of cases were cranial, 32.8% spinal and 29.2% sacral. Young age (<26 years; p = 0.0001) and female sex (p = 0.037) were associated with greater likelihood of cranial presentation. There was no overall increased risk for second primary cancers after chordoma. Median survival was 6.29 years; 5- and 10-year relative survival rates were 67.6% and 39.9%, respectively. Comparison with other bone sarcomas revealed racial disparities in incidence for the two developmental tumors, chordoma and Ewing's sarcoma. CONCLUSIONS: This study provides new data regarding incidence and survival patterns of chordoma in the US. Additional epidemiologic studies are required to elucidate the genetic and environmental determinants underlying this rare, distinctive neoplasm.

Intramedullary and spinal canal tumors in patients with neurofibromatosis 2: MR imaging findings and correlation with genotype.

PURPOSE: To determine the appearance of spinal tumors on magnetic resonance (MR) images of patients with neurofibromatosis 2 (NF2), to assess the biologic behavior of these tumors, and to determine the correlation between NF2 germline mutations and these tumors. MATERIALS AND METHODS: Spinal MR images in 49 patients with NF2 were reviewed retrospectively. Intramedullary and intradural extramedullary tumors were counted, and imaging features and growth patterns of intramedullary tumors were determined. Medical records were reviewed for spinal tumor surgery. Data on spinal tumors and NF2 germline mutations in 37 patients from 19 families were analyzed for genotype-phenotype correlation. RESULTS: Thirty-one patients (63%) had spinal tumors: Twenty-six (53%) had intramedullary tumors, 27 (55%) had intradural extramedullary tumors, and 22 (45%) had at least one tumor of each type. Three (12%) patients with intramedullary tumors versus 16 (59%) with extramedullary tumors had undergone surgery for the respective types of tumors. Compared with patients with all other types of mutations, a higher percentage of patients with nonsense and frameshift mutations had intramedullary tumors (P <.025); these patients also had higher mean numbers of all tumors (P <.001), intramedullary tumors (P <.001), and nerve sheath tumors (NSTs) (P <.001). CONCLUSION: In patients with NF2 and spinal tumors, extramedullary tumors (predominantly NSTs) were present in higher numbers and were associated with more surgery than were intramedullary tumors. Our data suggest that the association between nonsense and frameshift mutations and severe NF2 may extend to specific categories of spinal tumors.

Prognostic factors and secondary malignancies in childhood medulloblastoma.

PURPOSE: Little is known of the outcome of long-term survivors of childhood medulloblastoma, one of the most common pediatric malignancies. To determine the potential for secondary malignancies, a retrospective outcome evaluation in 88 consecutive cases of childhood medulloblastoma was performed. PATIENTS AND METHODS: The records of all patients with childhood medulloblastoma diagnosed at Children's National Medical Center in Washington, DC from 1969 through 1997 were reviewed. RESULTS: The median follow-up time was 92 months (range 6-257 months). Overall survival was 59% at 5 years and 52% at 10 years. Univariate analysis showed that age at diagnosis, extent of surgical resection, presence of metastatic disease (M stage), ventriculoperitoneal shunt placement within 30 days from diagnosis, posterior fossa radiation therapy dose, and adjuvant chemotherapy significantly affected survival. Although based on small numbers, the risk of second neoplasms was significantly increased in this cohort. Multiple basal cell carcinomas developed in the areas of radiation therapy in two patients; these patients also had nevoid basal cell carcinoma syndrome (NBCCS) diagnosed. One other patient died of glioblastoma multiforme 8 years after treatment of medulloblastoma. A meningioma developed in another patient 10 years after radiation therapy. CONCLUSION: As survival of medulloblastoma patients improves, increased surveillance regarding secondary malignancies is required, especially because radiation-induced tumors may occur many years after treatment. These two cases of NBCCS also illustrate the importance of considering the concomitant diagnosis of NBCCS in young patients with medulloblastoma. In those patients, alternative therapy should be considered to minimize radiation therapy-related sequelae.