The PMEL gene and merle (dapple) in the dachshund: cryptic, hidden, and mosaic variants demonstrate the need for genetic testing prior to breeding.

One of the most unique coat color patterns in the domestic dog is merle (also known as dapple in the dachshund breed), characterized by patches of normal pigmentation surrounded by diluted eumelanin pigment. In dogs, this striking variegated pattern is caused by an insertion of a SINE element into the PMEL gene. Differences in the length of the SINE insertion [due to a variable-length poly(A)-tail] has been associated with variation in the merle coat color and patterning. We previously performed a systematic evaluation of merle in 175 Australian shepherds and related breeds and correlated the length of the merle insertion variants with four broad phenotypic clusters designated as "cryptic", "atypical", "classic", and "harlequin" merle. In this study, we evaluated the SINE insertions in 140 dachshunds and identified the same major merle phenotypic clusters with only slight variation between breeds. Specifically, we identified numerous cases of true "hidden" merle in dachshunds with light/red (pheomelanin) coats with little to no black/brown pigment (eumelanin) and thus minimal or no observable merle phenotype. In addition, we identified somatic and gonadal mosaicism, with one dog having a large insertion in the harlequin size range of M281 that had no merle phenotype and unintentionally produced a double merle puppy with anophthalmia. The frequent identification of cryptic, hidden, and mosaic merle variants, which can be undetectable by phenotypic inspection, should be of particular concern to breeders and illustrates the critical need for genetic testing for merle prior to breeding to avoid producing dogs with serious health problems.

Canine factor VII deficiency: lessons learned in applying methods-based laboratory proficiency testing.

Canine inherited factor VII deficiency is a mild-to-moderate, inherited coagulopathy that affects several breeds of dog. We identified 2 polymorphisms near the disease-causing F7 gene mutation, one of which interfered with testing in several Beagles by causing allele dropout of the normal, wild-type allele. In the absence of an external proficiency program among veterinary genetic testing laboratories, implementation of an internal proficiency program, which requires 2 independent methods for genotyping dogs at any given locus, was further enhanced by ensuring minimally non-overlapping primer pairs between the 2 assays. After redesign of our clinical tests, all dogs were re-examined, and the correct genotypes were identified. These changes ensure higher accuracy in future testing of the F7 mutation.

Standards and guidelines for canine clinical genetic testing laboratories.

This publication represents a proposed approach to quality standards and guidelines for canine clinical genetic testing laboratories. Currently, there are no guidelines for laboratories performing clinical testing on dogs. Thus, there is no consensus set of protocols that set the minimal standards of quality among these laboratories, potentially causing variable results between laboratories, inconsistencies in reporting, and the inability to share information that could impact testing among organizations. A minimal standard for quality in testing is needed as breeders use the information from genetic testing to make breeding choices and irreversible decisions regarding spay, neuter or euthanasia. Incorrect results can have significant impact on the health of the dogs being tested and on their subsequent progeny. Because of the potentially serious consequences of an incorrect result or incorrect interpretation, results should be reviewed by and reported by individuals who meet a minimum standard of qualifications. Quality guidelines for canine genetic testing laboratories should include not only the analytical phase, but also the preanalytical and postanalytical phases, as this document attempts to address.

The PMEL Gene and Merle in the Domestic Dog: A Continuum of Insertion Lengths Leads to a Spectrum of Coat Color Variations in Australian Shepherds and Related Breeds.

Merle is a distinct coat color and pattern found in numerous species, including the domestic dog, characterized by patches of diluted eumelanin (black pigment) interspersed among areas of normal pigmentation. In dogs, this variegated pattern is caused by an insertion of a SINE element into the canine PMEL gene. Although variation in the length of the SINE insertion - due to a variable-length poly(A) tail - has been observed to be associated with variation in merle coat color and patterning, no systematic evaluation of this correlation has been conducted and published in the scientific literature. We performed high-resolution analysis of the SINE insertion lengths in 175 dogs (99 Australian shepherds, 45 miniature Australian shepherds, and 31 miniature American shepherds) and compared the genotypes with the coat phenotypes (when available). SINE insertion lengths varied from 201 to 277 bp, indicating that merle insertion variants can occur in virtually any size along the entire continuum. Genotype-phenotype correlation of 126 dogs with only a single SINE insertion (m/M) identified at least 4 major phenotypic clusters designated as "cryptic," "atypical," "classic," and "harlequin" merle. However, we found several phenotypic outliers that did not cluster within these major groupings, suggesting that insertion size is not the only factor responsible for merle phenotypic variability. In addition, we detected 25 dogs with 2 SINE insertions (M/M) and 24 dogs with more than 2 PMEL (merle) alleles, indicating mosaicism. Genotype-phenotype correlation of M/M dogs suggests that cryptic merle alleles often act like non-merle (m) alleles when combined with atypical, classic, and harlequin-sized alleles. The finding of mosaicism has important implications for the dog's phenotype and the ability to potentially transmit various alleles to its offspring. Furthermore, we identified examples of the SINE insertion poly(A)-tail expansion and contraction between generations, which also has important implications for breeding practices and determining mating pairs to avoid producing double merle dogs. These data demonstrate that there is a continuum of merle insertion lengths associated with a spectrum of coat color and patterns and that genotype-phenotype exceptions and overlap make it difficult to strictly assign certain insertion sizes with an expected coat color, although some generalizations are possible.

The development of a rapid assay for prenatal testing of common aneuploidies and microdeletion syndromes.

OBJECTIVE: To develop a novel, rapid prenatal assay for pregnancies with high likelihood of normal karyotypes, using BACs-on-Beads(™) technology, a suspension array-based multiplex assay that employs Luminex(®) xMAP(®) technology, for the detection of gains and losses in chromosomal DNA. METHODS: Fifteen relatively common microdeletions were selected that are not detectable, or may be missed, by karyotyping and usually do not present with abnormal ultrasound findings. Chromosomes 13, 18, 21, X, and Y were included. We validated the assay with 430 samples. RESULTS: All microdeletions and aneuploidies were correctly identified, except for a 69,XXX incorrectly identified as a normal female and a male with ∼20% maternal cell contamination (MCC) that could not be distinguished from 69,XXY. MCC became apparent at 20 to 30%. Mosaicism was identified at 30 to 35% abnormal cells. CONCLUSION: We have developed an alternative to fluorescence in situ hybridization (FISH) aneuploidy screening and microarray analysis in otherwise normal pregnancies undergoing invasive testing. We demonstrated that the assay will detect all microdeletions and aneuploidies of regions covered on the assay. We developed analytical software that displays results for well-characterized syndromes but not abnormalities of unclear clinical significance. This assay is likely to be preferred by women seeking testing beyond routine karyotyping but who desire more information than provided by aneuploidy FISH.

Effects of ozone exposure during microarray posthybridization washes and scanning.

The increasing prevalence of array-based comparative genomic hybridization in the clinical laboratory necessitates the implementation of quality control measures to attain accurate results with a high level of confidence. Environmental ozone is present in all industrialized cities and has been found to be detrimental to array data even at levels considered acceptable by US Environmental Protection Agency standards. In this study, we characterized the effect of ozone on microarray data on three different labeling platforms that use different fluorescent dyes (Cy3 and Cy5, Alexa Fluor 555 and Alexa Fluor 647, and Alexa Fluor 3 and Alexa Fluor 5) that are commonly used in array-based comparative genomic hybridization. We investigated the effects of ozone on microarray data by washing the array in variable ozone environments. In addition, we observed the effects of prolonged exposure to ozone on the microarray after washing in an ozone-free environment. Our results demonstrate the necessity of minimizing ozone exposure when washing and drying the microarray. We also found that washed microarrays produce the best results when immediately scanned; however, if a low-ozone environment is maintained, there will be little compromise in the data collected.

Y-specific sequences and polymorphisms in rainbow trout and Chinook salmon.

Improved methods for genetically sexing salmonids and for characterization of Y-chromosome homologies between species can contribute to understanding the evolution of sex chromosomes and sex-determining mechanisms. In this study we have explored 12.5 kb of Y-chromosome-specific sequence flanking the previously described OtY2 locus in Chinook salmon (Oncorhynchus tshawytscha) and 21 kb of homologous rainbow trout (Oncorhynchus mykiss) Y-chromosome-specific sequence. This is the first confirmed Y-specific sequence for rainbow trout. New Y-specific markers are described for Chinook salmon (OtY3) and rainbow trout (OmyY1), which are readily detected by PCR assays and are advantageous because they also produce autosomal control amplification products. Additionally, AFLP analysis of Chinook salmon yielded another potential Y-chromosome marker. These descriptions will facilitate genotypic sexing and should be useful for population studies of Y-chromosome polymorphisms and for future studies to characterize what appears to be a common sex-determining mechanism between these species.

Quantitative trait loci x maternal cytoplasmic environment interaction for development rate in Oncorhynchus mykiss.

Effects of maternal cytoplasmic environment (MCE) on development rate in rainbow trout were evaluated within a quantitative trait loci (QTL) analysis framework. Previous research had identified QTL for development rate in doubled haploid (DH) progeny produced from a cross between the Oregon State University (OSU) and the Swanson (SW) River rainbow trout clonal lines. In this study, progeny for QTL mapping were produced from a cross between the OSU and Clearwater (CW) River clonal lines. Doubled haploids were produced from the OSU x CW F1 by androgenesis using eggs from different females (or MCEs); with androgenesis, the maternal nuclear genome was destroyed by irradiation and diploidy was restored by blocking the first embryonic cleavage by heat shock. All embryos were incubated at the same temperature and development rate quantified as time to hatch. Using a linkage map constructed primarily with AFLP markers, QTL mapping was performed, including MCE covariates and QTL x MCE effects in models for testing. The major QTL for development rate in the OSU x SW cross overlaps with the major QTL found in this OSU x CW cross; effects at this locus were the same across MCEs. Both MCE and QTL x MCE effects contribute to variability in development rate, but QTL x MCE were minor and detected only at small-effect QTL.

Detection of low-level mosaicism by array CGH in routine diagnostic specimens.

The advent of microarray-based comparative genomic hybridization (array CGH) promises to revolutionize clinical cytogenetics because of its ability to rapidly screen the genome at an unprecedented resolution. Yet, the ability of array CGH to detect and evaluate low-level mosaicism is not known. Our laboratory has analyzed over 3,600 clinical cases with the SignatureChip which we developed for the detection of microdeletions, microduplications, aneuploidy, unbalanced translocations, and subtelomeric and pericentromeric copy number alterations. Here, we report 18 cases of mosaicism detected by array CGH in a routine diagnostic setting, 14 of which were not known to us at the time of the analysis. These 14 cases represent approximately 8% of all abnormal cases identified in our laboratory. For each case, fluorescence in situ hybridization (FISH) analysis was performed on PHA-stimulated cultures after mosaic chromosome abnormalities were suspected by array CGH. In all cases, FISH confirmed the mosaic chromosome abnormalities which included a variety of marker chromosomes, autosomal trisomies, terminal and interstitial deletions, and derivative chromosomes. Interestingly, confirmatory FISH analyses on direct blood smears indicated that the percentage of abnormal cells in unstimulated cultures was in some cases different than that found in PHA-stimulated cells. We also report the detection of a previously unsuspected case of an isochromosome 12p (associated with Pallister-Killian syndrome) by array CGH using genomic DNA extracted from peripheral blood. These results support a growing body of data that suggests that stimulated peripheral blood cultures likely distort the percentage of abnormal cells and may, for some chromosome abnormalities, make their detection unlikely by conventional analysis. Thus, array CGH, which is based on genomic DNA extracted directly from uncultured peripheral blood, may be more likely to detect low-level mosaicism for unbalanced chromosome abnormalities than traditional cytogenetic techniques.

Targeted genomic microarray analysis for identification of chromosome abnormalities in 1500 consecutive clinical cases.

OBJECTIVE: To assess the yield of array-based comparative genomic hybridization. STUDY DESIGN: The results of array comparative genomic hybridization were collected on 1500 consecutive clinical cases sent to our laboratory for a variety of developmental problems. Confirmation fluorescence in situ hybridization of metaphase or interphase cells, depending on the aberration, was performed. RESULTS: Of the 1500 cases, 134 (8.9%) showed an abnormality: 36 (2.4%) showed polymorphisms or familial variants, 14 (0.9%) showed alterations of unknown clinical significance, and 84 (5.6%) showed clinically relevant genomic alterations. These included subtelomeric deletions and unbalanced rearrangements, microdeletions and reciprocal duplications, rare abnormalities, and low-level trisomy mosaicism. CONCLUSIONS: A targeted array detects a substantial proportion of abnormalities even in those patients who have already had extensive cytogenetic and/or fluorescence in situ hybridization testing. This study, although not a controlled ascertainment of subjects with specific selection criteria, accurately reflects the reality of clinical cytogenetic practice and provides an estimate of the cytogenetic abnormalities that can be identified with a targeted microarray in a diagnostic laboratory. Microarray analysis likely doubles the current yield of abnormal results detected by conventional cytogenetic analysis.

Detecting sex chromosome anomalies and common triploidies in products of conception by array-based comparative genomic hybridization.

OBJECTIVES: In recent years, array-based comparative genomic hybridization (array CGH) has moved to the forefront of molecular cytogenetics with its ability to rapidly characterize chromosome abnormalities at resolutions much higher than routine chromosome banding. However, array CGH, like all CGH procedures, has heretofore been deemed unable to detect ploidy, a major cause of fetal demise and spontaneous miscarriage. METHOD: We recently developed a CGH microarray that is designed for detecting aneuploidy and unbalanced chromosome rearrangements. Here, we introduce the use of a Klinefelter male cell line (47,XXY) as a control for array CGH analyses on products of conception (POCs). RESULTS: This approach facilitates the detection of common trisomies and monosomies of the sex chromosomes by reducing the analysis to the identification of single copy gains or losses. Furthermore, in a blinded study, careful interpretation of the microarray results with particular attention to the sex chromosome ratios between the patient sample and the control allowed for the detection of some common triploidies. CONCLUSION: These results suggest that using a chromosomally abnormal cell line in array CGH analysis can be applied to other CGH platforms and that array CGH, when properly performed and analyzed, is a powerful tool that can detect most chromosomal abnormalities observed in a clinical setting including some polyploidies.

Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more?

Chromosome analysis is an important component to the diagnosis of congenital anomalies, developmental delay, and mental retardation. Routine chromosome analysis identifies aneuploidy and structural rearrangements greater than 5 Mb but cannot identify abnormalities of the telomeric regions or microdeletions reliably. Molecular cytogenetic techniques were developed to overcome these limitations. High-resolution comparative genomic hybridization (CGH)-based microarrays (array CGH) were developed to increase the resolution of chromosomal studies and to provide a comprehensive assay by using large-insert clones as the target for analysis. We constructed a microarray for the clinical diagnosis of medically significant and relatively common chromosomal alterations. Nine hundred six bacterial artificial chromosome (BAC) clones were chosen, the chromosomal locations of which were confirmed by fluorescence in situ hybridization (FISH). FISH-testing showed that 7% of the clones were mismapped based on map locations obtained from two publicly available databases (58 mapped to the wrong chromosome and three mapped to a different locus on the same chromosome), 16% cross-hybridized to other chromosomes, and 12% did not hybridize or showed poor hybridization signals under uniform FISH conditions. Thus, from a total of 906 BAC clones that were evaluated, only 589 (65%) were deemed adequate for arraying on this clinical device. The performance of this array was tested in a set of blinded experiments on a cohort of phenotypically normal individuals and on individuals with known chromosome abnormalities. The array identified deletion/duplication polymorphisms not seen by FISH in the phenotypically normal individuals and detected single copy dosage differences in all of the cases with known chromosomal abnormalities. All abnormalities detected by the array were confirmed by FISH with BACs from the appropriate loci. Our data demonstrate that the rigorous assessment of BACs and their use in array CGH is especially important when the microarray is used for clinical diagnosis. In addition, this study illustrates that when constructed carefully with proper attention to the quality of the BACs that are arrayed, array CGH is an effective and efficient tool for delineating chromosomal aberrations and an important adjunct to FISH and conventional cytogenetics.