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Background: Melanoma risk factors include fair pigmentation, multiple nevi, low DNA repair capacity, and CDKN2A or CDK4 mutations. Variants of the melanocortin-1 receptor (MC1R) gene have been associated with fair pigmentation and melanoma risk, and a polymorphism of the Agouti Signaling Protein (ASIP) gene has been associated with dark pigmentation. We examined MC1R and ASIP genotypes in relation to phenotypic characteristics, sporadic and familial melanoma risk, and melanoma thickness as an indicator of disease progression in a Mediterranean population. Methods: We studied 267 melanoma patients and 382 control subjects from a case-control study and a family study in northeastern Italy. Host factors were assessed by physical examination, questionnaire, spectrophotometer, and minimal erythema dose measurement. MC1R was sequenced, ASIP was genotyped, and DNA repair capacity was measured by the host-cell reactivation assay. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression models. Effect modification of the association between MC1R and melanoma risk by phenotypic characteristics and DNA repair capacity was also assessed. All statistical tests were two-sided. Results: Carrying MC1R variant alleles was associated with a two- to fourfold increase in risk of both sporadic and familial melanoma compared with carrying wild-type MC1R, particularly in individuals carrying multiple variant alleles (OR = 3.9; 95% CI = 3.3 to 4.6). This association was stronger in individuals with fewer additional risk factors (those with dark skin or few nevi). MC1R variant allele carriers were also three to four times more likely than were non-carriers to have thick melanomas. The ASIP polymorphism was not associated with pigmentation, nevi, or melanoma risk. Conclusions: MC1R was associated with melanoma risk and progression in a Mediterranean population, particularly in the absence of other strong risk factors, such as freckling or many nevi.

Diagnosing and screening for tumors through non-invasive means represents an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease including follow up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of circulating tumor cells and circulating tumor DNA are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of circulating tumor cells and circulating tumor DNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect circulating tumor cells and some of the intricacies of the characterization of the circulating tumor DNA will also be provided.

Purpose: Although neonatal screening (or newborn screening) for cystic fibrosis (CF) is commonly practiced, systematic methods for accurate risk calculations are currently lacking.Methods and Results: We evaluated characteristics of the immunoreactive trypsinogen (IRT) test using the published data. The probability that a neonate has a positive IRT test, if the neonate is affected, a carrier, or a noncarrier, is ˜ 1, 0.041, or 0.011, respectively. We provide methods to calculate genetic risks for a variety of commonly encountered scenarios in which neonates are positive by the IRT test.Conclusion: Our Bayesian methods permit CF disease probabilities to be calculated accurately, taking into account all relevant information.

Although neonatal screening (or newborn screening) for cystic fibrosis (CF) is commonly practiced, systematic methods for accurate risk calculations are currently lacking.

Medulloblastoma is the most common malignant central nervous system tumor of childhood and can occur sporadically or in association with inherited cancer susceptibility syndromes such as the nevoid basal cell carcinoma syndrome (NBCCS). To determine whether an association existed between the risk of developing medulloblastoma and undiagnosed syndromes, we retrospectively reviewed clinical data on 33 patients with medulloblastoma from a single institution and compared them with their unaffected relatives (n = 46). Six patients had tumors showing desmoplastic histology. Two of the six met diagnostic criteria for NBCCS. One NBCCS patient had a missense mutation of patched‐1 (PTCH1); the other had no identifiable PTCH1 mutation. Two patients with isolated desmoplastic medulloblastoma had an insertion and splice site mutation, respectively, in suppressor of fused (SUFU). All patients with nondesmoplastic medulloblastoma histology received molecular testing for SUFU. None of these patients had an identifiable mutation in PTCH1 or SUFU. We performed a clinical evaluation for Greig cephalopolysyndactyly syndrome (GCPS) in four medulloblastoma families, who exhibited macrocephaly as the only finding consistent with the diagnosis of GCPS. Molecular analysis of GLI3 in these four families was negative. There was a paucity of clinical findings among the majority of medulloblastoma patients in this study group to suggest a definable cancer genetic syndrome. We conclude that clinically recognizable syndromes are uncommon among patients with medulloblastoma, however, PTCH1 and SUFU mutations are present at a low but significant frequency. Published 2005 Wiley‐Liss, Inc.

Purpose: Risk assessment is an essential component of genetic counseling and testing, and Bayesian analysis plays a central role in complex risk calculations. We previously developed generalizable Bayesian methods to calculate the autosomal recessive disease risk of a fetus when one or no mutation is detected, and another, independent risk factor is present. Our methods are particularly useful for calculating the CF disease risk for a fetus with echogenic bowel. In genetics practice, however, there are other scenarios for which our previous methods are inadequate.

Reverse allele specific oligonucleotide assays provide a robust method for the molecular characterization of high-mutation spectrum disorders. Commercial tests have been developed for human leukocyte antigens class I and class II regions of human chromosome 6, the cystic fibrosis transmembrane conductance regulator at 7q31 and strains of human Hepatitis B and C virus. In their most developed form, these assays rely upon highly multiplexed PCR reactions containing biotinylated primers providing a substrate for nonradioactive detection systems. Sophisticated reverse dot–blot technology involves mechanized covalent attachment of activated primary amine-conjugated oligonucleotides to carboxylated nylon membranes or bovine serum albumin. Subsequent to line or dot printing, membranes are stored or sold dry in preparation for hybridization. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminescence after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film. The entire procedure from blood specimen receipt to result usually requires less than 1 day. Because of the simplicity, speed, and generally high sensitivity and specificity, large numbers of individuals can be rapidly screened using this technology. Rapid turnaround is often required in prenatal diagnosis of cystic fibrosis, β-thalassemia and hemoglobinopathies, giving this technology has special applicability in those genetic diseases. Commercial instruments are available which automate the hybridization and color development. In addition, scanning software can capture the probe reactivity pattern and interpret it in terms of a genotype.

We report on a generally useful, partially automated, human mutation detection method based upon printing moderate density oligonucleotide arrays using a biorobot on activated nylon membranes. The Beckman Biomek 2000 was adapted to this task through fabrication of aluminum membrane filter holders and the development of an addressable Tool Command Language (Tcl) program, which can be invoked through BioScript. During program execution, a robot arm is moved along the x, y,and zaxes to expel liquid, without dripping, from disposable barrier pipette tips and then to touch the drops on preactivated membranes. Printed arrays consist of alternating rows of oligonucleotides containing normal and mutant sequences. Hybridization of biotin labeled polymerase chain reaction products derived from human patient genomic DNA samples are visualized using chemiluminescent or chromogenic indicators. This technique allows unequivocal genotyping of 32 mutations at the β-thalassemia locus (11p15.5) and of 34 mutations and one polymorphism at the cystic fibrosis transconductance membrane regulator locus (7p35).

Background:Comprehensive assessment of thrombophilia risk includes characterization of the R506Q (Leiden) mutation in factor V in many patients. Although activated protein C resistance is often assessed by means of a coagulation test, molecular interrogation of the G1691A mutation provides confirmation and interpretive utility in patients undergoing anticoagulation. Many molecular methods are available to provide genotyping at this locus. Among these, PCR[mdash ]restriction fragment length polymorphism (RFLP) is widely used. Unfortunately, because this common mutation is 11 bp from the 3' end of exon 10, one PCR primer often anneals within intron 10. As a consequence, polymorphism can confound test results. Method and Results:In the course of a clinical diagnostic test of 15,301 patients, two samples repeatedly showed two different unusual electrophoretic mobilities after PCR and restriction enzyme digestion. After stripping patient identifiers and entering a research protocol, the amplicons from these DNAs were sequenced in parallel with normal and heterozygous G1691A control genomic DNA samples. This sequencing showed two novel polymorphisms, each mapping to intron 10. Conclusion:PCR-RFLP[mdash ]based methods rely on sequence conservation in the interrogated region. Amplification of mutated loci adjacent to introns present a special risk for confounding restriction patterns. Sequencing amplicons with reproducibly unusual restriction patterns resolved the paradoxical restriction pattern in this case.

Background:Unequivocal molecular characterization of the FMR- 1triplet expansion region requires the combined use of PCR to amplify normal- and premutation-length alleles and Southern analysis to detect fully expanded alleles and assess methylation. We provide a detailed laboratory protocol, which can be generalized, for the preparation and use of a digoxigenin (DIG)-labeled probe for Southern analysis of genomic DNA digested with EcoR I and EagI.

Following intratesticular injection of [35S]methionine or [3H]leucine, four testicular cell types (pachytene spermatocytes, round spermatids, elongating spermatids and residual bodies) were purified from mouse testicular cell suspensions by unit gravity sedimentation and equilibrium density gradient centrifugation through Percoll. Measurement of the amount of radiolabeled amino acid incorporated into protein in the testicular cells revealed that for a constant number of cells, pachytene spermatocytes incorporated 5.4 times more isotope than round spermatids, which incorporated 2.4 times more isotope than elongating spermatids. Analysis by two-dimensional gel electrophoresis of the polypeptides synthesized in vivo in the four testicular cell types demonstrated qualitative and quantitative changes in protein synthesis during spermatogenesis. At the level of detection provided by the electrophoretic methods used, pachytene spermatocytes and round spermatids synthesized approximately equivalent numbers of polypeptides while the number of polypeptides synthesized in elongating spermatids and residual bodies was decreased. Quantitative changes for polypeptides ranging in molecular weight from 16,500 to 82,000 were detected during spermatogenesis. For each cell type examined, a minimumof 5% of the polypeptides appear to be either unique or greatly enriched. These studies indicate that the expression of a sizable number of polypeptides is specific to certain stages of spermatogenesis.

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