A Model for Design and Implementation of a Laboratory Information-Management System Specific for Molecular Pathology Laboratory Operations.

The Molecular Pathology Section, Cleveland Clinic (Cleveland, OH), has undergone enhancement of its testing portfolio and processes. An Excel 2013- and paper-based data-management system was replaced with a commercially available laboratory information-management system (LIMS) software application, a separate bioinformatics platform, customized test-interpretation applications, a dedicated sample-accessioning service, and a results-releasing software application. The customized LIMS solution manages complex workflows, large-scale data packets, and process automation. A customized approach was required because, in a survey of commercially available off-the-shelf software products, none met the diverse and complex needs of this molecular diagnostics service. The project utilized the expertise of clinical laboratorians, pathologists, genetics counselors, bioinformaticians, and systems analysts in partnering with software-engineering consultants to design and implement a solution. Concurrently, Agile software-building best practices were formulated, which may be emulated for scalable and cost-effective laboratory-authored software.

Somatic mosaicism in adult-onset TNF receptor-associated periodic syndrome (TRAPS).

BACKGROUND: Somatic mosaicism is to date an uncommon finding in genetic autoinflammatory syndromes such as Cryopyrin-associated periodic syndrome, Blau syndrome, and TNF receptor-associated periodic syndrome (TRAPS). However, somatic mosaicism may be particularly important in adult-onset or atypical phenotypes of these conditions. Herein, we report a unique adult-onset TRAPS patient presenting with intermittent daily fever for 3 weeks, rash, peritonitis, and lymphadenopathy, who was found with hematopoietic mosaicism involving different white blood cell populations. METHODS: Patient's lymphocyte genomic DNA was initially analyzed by periodic fever seven-gene next-generation sequencing panel. Genomic DNAs extracted from patient's hair roots, buccal swab, and subpopulations of white blood cells were subsequently examined on the identified TNFRSF1A variant using Sanger sequencing. RESULTS: A de novo mosaic missense variant, c.265 T>C(p.Phe89Leu), in the TNFRSF1A gene was found in the patient's buccal swab, B cells, neutrophils, and NK cells but not detected in monocytes, T cells, and hair roots. CONCLUSION: These data provide additional information about somatic mosaicism in autoinflammatory conditions and provide new insights regarding cellular players that are indispensable for the phenotypic expression of TRAPS.

Characterization of tissue-specific and developmentally regulated alternative splicing of exon 64 in the COL5A1 gene.

The COL5A1 gene, a member of the clade B fibrillar collagen gene family, was recently shown to contain two alternatively spliced exons (64A and 64B) that encode 23 amino acids in the carboxyl-terminal propeptide. The two are identical in length, very similar in sequence, and used in a mutually exclusive fashion because of the small intron that separates them. Each COL5A1 allele uses both exons, but a given transcript will contain only one of the two exons. The sequences in other species are highly conserved at the amino acid level. The expression profile of the two isoforms was determined from analysis of RNA levels in a panel of murine tissues. While both isoforms were found in all tissues studied, actively proliferating tissues (liver, lung) used isoform B more often, while a less mitotically active tissue, brain, had a higher proportion of exon 64A. The high degree of conservation between the two exons is consistent with a regional genomic duplication. The presence of the two isoforms as far back as pufferfish (tetraodon) implies an important functional significance. The exact role determined by the two sequences is not known, but involvement in the determination of chain composition of mature type V collagen or regulation of cell activity is possible, given the differences in tissue distribution.

The origin of trisomy 13.

Trisomy 13 is one of the most common trisomies in clinically recognized pregnancies and one of the few trisomies identified in liveborns, yet relatively little is known about the errors that lead to trisomy 13. Accordingly, we initiated studies to investigate the origin of the extra chromosome in 78 cases of trisomy 13. Our results indicate that the majority of cases (>91%) are maternal in origin and, similar to other autosomal trisomies, the extra chromosome is typically due to errors in meiosis I. Surprisingly, however, a large number of errors also occur during maternal meiosis II ( approximately 37%), distinguishing trisomy 13 from other acrocentric and most nonacrocentric chromosomes. As with other trisomies, failure to recombine is an important contributor to nondisjunction of chromosome 13.

Synaptic defects at meiosis I and non-obstructive azoospermia.

BACKGROUND: Recent advances in immunofluorescence methodology have made it possible to directly monitor protein localization patterns in germ cells undergoing meiosis. We used this technology to examine the early stages of meiosis in testicular material obtained from men presenting for evaluation at infertility clinics. METHODS: Specifically, we compared meiotic progression, synapsis and recombination in 34 individuals with obstructive azoospermia ('controls') to 26 individuals with non-obstructive azoospermia (NOA) ('cases'). RESULTS: In 9 of the 26 cases, no germ cells were identified, but in the remaining 17, there was at least some progression through meiosis. Most of these individuals appeared to have normal levels of spermatogenic activity, with little evidence of meiotic impairment. However, in three individuals, we observed either complete or partial meiotic arrest associated with abnormalities in synapsis. CONCLUSIONS: This suggests that >10% of cases of unexplained NOA may be attributable to severe meiotic defects. The characterization of these meiotic arrest phenotypes may guide further research into the molecular basis of unexplained infertility.

Meiotic synapsis proceeds from a limited number of subtelomeric sites in the human male.

The formation of the synaptonemal complex (SC) is a crucial early step in the meiotic process, but relatively little is known about the establishment of the human SC. Accordingly, we recently initiated a study of synapsis in the human male, combining immunofluorescence and fluorescence in situ hybridization methodologies to analyze prophase spermatocytes from a series of control individuals. Our results indicate that synapsis is a tightly regulated process, with relatively little variation among individuals. On nonacrocentric chromosomes, there are two synaptic initiation sites, one on the distal short arm and one on the distal long arm, whereas acrocentric chromosomes exhibit a single site on the distal long arm. For both types of chromosomes, synapsis then proceeds toward the centromere, with little evidence that specific p- or q-arm sequences affect the process. However, the centromere appears to have an inhibitory effect on synapsis--that is, when one arm of a nonacrocentric chromosome is "zippered up" before the other, the centromere acts as a barrier to further movement from that arm.

Meiosis I arrest and azoospermia in an infertile male explained by failure of formation of a component of the synaptonemal complex.

OBJECTIVE: To characterize the early stages of meiosis in a male with unexplained azoospermia. DESIGN: Case report. SETTING: Case Western Reserve University and University Hospitals of Cleveland. PATIENT(S): A 30-year-old male with nonobstructive azoospermia. INTERVENTION(S): Immunostaining for components of the synaptonemal complex and recombination-associated proteins, fluorescence in situ hybridization (FISH) for specific chromosomes. MAIN OUTCOME MEASURE(S): Progression to and through pachytene of meiosis I in controls and in the patient. RESULT(S): We observed complete meiosis I arrest in the patient, associated with failure of formation of the mature, tripartite synaptonemal complex. CONCLUSION(S): Abnormalities in synaptonemal complex formation are responsible for a proportion of cases of unexplained male infertility.

Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system.

BACKGROUND: We have been studying an unusual mouse-the BALB/cWt (Wt) male-in which the Y chromosome is susceptible to high rates of mitotic non-disjunction, particularly at the first two cleavage divisions. As these are the same divisions that human embryos generated through assisted reproductive technology must complete in an artificial setting, analysis of the Wt Y chromosome allows us to examine the effect of fertilization and culture in vitro on mammalian chromosome segregation. METHODS: We performed standard mouse IVF, cultured embryos in 5% CO2 in air or in a lowered oxygen atmosphere, and used fluorescence in-situ hybridization to examine the sex chromosome constitutions of 2-, 4-, 8- and 16-cell stage Wt Y-bearing embryos. RESULTS: We observed a significant increase in mosaic sex chromosome aneuploidy at each embryonic stage in embryos cultured in 5% CO2 in air, but under lowered oxygen conditions mosaicism returned to control (in-vivo) levels. CONCLUSIONS: Our results demonstrate that slight alterations in in-vitro conditions may have a considerable impact on the genetic quality of assisted reproductive technology-derived embryos and suggest that the genetic quality of embryos should be a fundamental concern in the development of new culture systems for clinical use.

Covariation of synaptonemal complex length and mammalian meiotic exchange rates.

Analysis of recombination between loci (linkage analysis) has been a cornerstone of human genetic research, enabling investigators to localize and, ultimately, identify genetic loci. However, despite these efforts little is known about patterns of meiotic exchange in human germ cells or the mechanisms that control these patterns. Using recently developed immunofluorescence methodology to examine exchanges in human spermatocytes, we have identified remarkable variation in the rate of recombination within and among individuals. Subsequent analyses indicate that, in humans and mice, this variation is linked to differences in the length of the synaptonemal complex. Thus, at least in mammals, a physical structure, the synaptonemal complex, reflects genetic rather than physical distance.