A randomized trial of single versus double high-level disinfection of duodenoscopes and linear echoendoscopes using standard automated reprocessing.

BACKGROUND AND AIMS: In a pilot study, we demonstrated that current guidelines for duodenoscope and linear echoendoscope (DLE) reprocessing using a single cycle of high-level disinfection (HLD) in an automated reprocessor may be inadequate. In August 2015, the U.S. Food and Drug Administration offered double HLD as a possible response to address this concern. As a result, Providence Health and Services adopted double HLD as standard procedure for DLEs, but no rigorous clinical studies supported this practice. We undertook a quality improvement study to compare single HLD versus double HLD at 4 of our 34 hospitals. METHODS: HLD of DLE was randomized, separately in each facility, to either single HLD or double HLD on weekdays, with standard double HLD on weekends or holidays. There was 99.7% compliance with the randomization scheme. Daily qualitative surveillance cultures of dried, post-HLD DLEs were collected for 6 months (1 swab sample from the elevator mechanism and 1 combined brush sample from the suction and working channels for each encounter), and each sample was incubated for 48 hours. Positivity rates of any microbial growth and growth of high-concern pathogens (potentially pathogenic enteric flora) were compared between the 2 study arms. RESULTS: Altogether, 5850 surveillance culture specimens were obtained during 2925 encounters from the 45 DLEs in clinical use in the participating hospitals. Of these, 3052 (52.2%) were from endoscopes cleaned by double HLD. Double HLD demonstrated no benefit over single HLD because similar positivity rates were observed (all P > .05). The elevator mechanism was more frequently colonized than the biopsy channel (5.2% vs 2.9%, P < .001). Among the 224 encounters with positive growth, 140 (62.5%) recovered microbes from only the elevator mechanism specimens, 73 (32.6%) recovered microbes from only the channel specimens, and 11 (4.9%) recovered microbes from both types of specimens. Double HLD failed to improve contamination rates for either sample site at any of the 4 endoscopy facilities, although there were significant overall differences in contamination rates among the facilities (P < .001), as observed in our previous study. Only 8 high-concern pathogens were recovered from 5 DLEs, all from the elevator mechanism. Persistent growth was observed on 2 duodenoscopes. One grew Enterococcus spp (not vancomycin-resistant enterococci) on 3 occasions, and Escherichia coli was present on 2 of these occasions, 1 of which was a multidrug-resistant organism. The other grew different enteric flora on 2 specimens. CONCLUSIONS: Our prospectively randomized study, involving 4 separate endoscopy facilities and standard automated endoscope reprocessing, showed that double HLD did not reduce culture positivity rates compared with single HLD in facilities with an already low positive culture rate. Alternative risk mitigation strategies will be assessed in an ongoing effort to reduce endoscope contamination.

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.

Diagnosis of cryptic chromosomal syndromes by fluorescence in situ hybridization (FISH).

This unit describes the various methods by which cytogeneticists detect chromosome abnormalities. The unit offers guidance for detecting such abnormalities with fluorescence in situ hybridization (FISH), as well as the benefits, limitations, and other applications of FISH.

Prenatal diagnosis using array CGH.

Microarray-based comparative genomic hybridization (array CGH) is a fast and high-resolution approach to the diagnosis of a large number of genetic syndromes associated with loss or gain of the human genome. This technology has proven to be useful in several clinical settings, including postnatal diagnosis of mental retardation, developmental delay, and congenital malformation syndromes. We describe the use of array CGH for prenatal diagnosis of a range of chromosomal syndromes associated with congenital malformations visible by ultrasound. The procedure is reproducible in a clinical setting and provides reliable results in a short period (approximately 5 days). Thus, depending on the array used, array CGH may develop into an excellent tool for prenatal diagnosis.

Genomic analysis of the chromosome 15q11-q13 Prader-Willi syndrome region and characterization of transcripts for GOLGA8E and WHCD1L1 from the proximal breakpoint region.

BACKGROUND: Prader-Willi syndrome (PWS) is a neurobehavioral disorder characterized by neonatal hypotonia, childhood obesity, dysmorphic features, hypogonadism, mental retardation, and behavioral problems. Although PWS is most often caused by a paternal interstitial deletion of a 6-Mb region of chromosome 15q11-q13, the identity of the exact protein coding or noncoding RNAs whose deficiency produces the PWS phenotype is uncertain. There are also reports describing a PWS-like phenotype in a subset of patients with full mutations in the FMR1 (fragile X mental retardation 1) gene. Taking advantage of the human genome sequence, we have performed extensive sequence analysis and molecular studies for the PWS candidate region. RESULTS: We have characterized transcripts for the first time for two UCSC Genome Browser predicted protein-coding genes, GOLGA8E (golgin subfamily a, 8E) and WHDC1L1 (WAS protein homology region containing 1-like 1) and have further characterized two previously reported genes, CYF1P1 and NIPA2; all four genes are in the region close to the proximal/centromeric deletion breakpoint (BP1). GOLGA8E belongs to the golgin subfamily of coiled-coil proteins associated with the Golgi apparatus. Six out of 16 golgin subfamily proteins in the human genome have been mapped in the chromosome 15q11-q13 and 15q24-q26 regions. We have also identified more than 38 copies of GOLGA8E-like sequence in the 15q11-q14 and 15q23-q26 regions which supports the presence of a GOLGA8E-associated low copy repeat (LCR). Analysis of the 15q11-q13 region by PFGE also revealed a polymorphic region between BP1 and BP2. WHDC1L1 is a novel gene with similarity to mouse Whdc1 (WAS protein homology region 2 domain containing 1) and human JMY protein (junction-mediating and regulatory protein). Expression analysis of cultured human cells and brain tissues from PWS patients indicates that CYFIP1 and NIPA2 are biallelically expressed. However, we were not able to determine the allele-specific expression pattern for GOLGA8E and WHDC1L1 because these two genes have highly related sequences that might also be expressed. CONCLUSION: We have presented an updated version of a sequence-based physical map for a complex chromosomal region, and we raise the possibility of polymorphism in the genomic orientation of the BP1 to BP2 region. The identification of two new proteins GOLGA8E and WHDC1L1 encoded by genes in the 15q11-q13 region may extend our understanding of the molecular basis of PWS. In terms of copy number variation and gene organization, this is one of the most polymorphic regions of the human genome, and perhaps the single most polymorphic region of this type.

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.

Comparative genomic hybridization by microarray for the detection of cytogenetic imbalance.

Chromosomal abnormalities often result in the improper dosage of genes in a particular chromosome or chromosome segment, which may cause specific and complex clinical phenotypes. Comparative genomic hybridization by microarray (array CGH) is a high-throughput and high-resolution method for the detection of microscopic and submicroscopic chromosome abnormalities, some of which may not be detectable by conventional cytogenetic techniques. In addition, with the human genome sequenced and publicly available, array CGH allows for the direct correlation between chromosomal anomalies and genomic sequence. Properly constructed, microarrays have the potential to be a valuable tool for the detection of chromosomal abnormalities in cancer and genetic disease.

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.

Recurrent biparental hydatidiform mole: additional evidence for a 1.1-Mb locus in 19q13.4 and candidate gene analysis.

OBJECTIVES: A maternal autosomal recessive mutation causing recurrent biparentally inherited complete hydatidiform moles (BiCHM) in affected women was previously mapped to a 12.4-cM interval in 19q13.4, which was recently further narrowed to a smaller 1.1-Mb region at the centromeric end. It is believed that the mutant gene in this condition is a major contributor to the regulation of imprinting in the maternal germline. To confirm and possibly narrow the critical interval we studied additional rare familial and recurrent cases. METHODS: Using polymorphic marker analysis, we first confirmed biparental inheritance on the studied molar tissues. We then performed targeted homozygosity mapping with markers in 19q13.4 on DNA from affected women of a new large consanguineous pedigree, an additional potentially familial case, and three cases with sporadic recurrent CHM. Direct sequencing of coding exons and Southern analysis with a coding-region probe for one candidate gene (NALP5) was also performed. RESULTS: Biparental inheritance was confirmed for those molar tissues available for analysis. All women, except for one of the isolated cases, were homozygous for markers in the identified 1.1-Mb region in 19q13.4. No mutations or large genomic rearrangements were found in NALP5 (MATER), a gene with oocyte-specific expression. Heterozygosity for a single-nucleotide polymorphism in exon 13 of NALP5 in one patient may refine the candidate region to 1.0 Mb. CONCLUSIONS: The reported candidate region for BiCHM in 19q13.4 was confirmed in additional families, further establishing it as the major locus that harbors a gene mutated in this condition.

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.

Delineation of mechanisms and regions of dosage imbalance in complex rearrangements of 1p36 leads to a putative gene for regulation of cranial suture closure.

Structural chromosome abnormalities have aided in gene identification for over three decades. Delineation of the deletion sizes and rearrangements allows for phenotype/genotype correlations and ultimately assists in gene identification. In this report, we have delineated the precise rearrangements in four subjects with deletions, duplications, and/or triplications of 1p36 and compared the regions of imbalance to two cases recently published. Fluorescence in situ hybridization (FISH) analysis revealed the size, order, and orientation of the duplicated/triplicated segments in each subject. We propose a premeiotic model for the formation of these complex rearrangements in the four newly ascertained subjects, whereby a deleted chromosome 1 undergoes a combination of multiple breakage-fusion-bridge (BFB) cycles and inversions to produce a chromosome arm with a complex rearrangement of deleted, duplicated and triplicated segments. In addition, comparing the six subjects' rearrangements revealed a region of overlap that when triplicated is associated with craniosynostosis and when deleted is associated with large, late-closing anterior fontanels. Within this region are the MMP23A and -B genes. We show MMP23 gene expression at the cranial sutures and we propose that haploinsufficiency results in large, late-closing anterior fontanels and overexpression results in craniosynostosis. These data emphasize the important role of cytogenetics in investigating and uncovering the etiologies of human genetic disease, particularly cytogenetic imbalances that reveal potentially dosage-sensitive genes.

Acute lymphoblastic leukemia in a patient with Greig cephalopolysyndactyly and interstitial deletion of chromosome 7 del(7)(p11.2 p14) involving the GLI3 and ZNFN1A1 genes.

Greig cephalopolysyndactyly (GCPS; OMIM 175700) is an autosomal dominant condition caused by mutations of the gene GLI3, located on 7p13. To date, several cases of deletions and/or translocations involving this locus have been reported in patients with GCPS. GLI3 is a transcription factor from the GLI-Kruppel gene family that has been implicated in three distinct entities: GCPS, Pallister-Hall syndrome, and postaxial polydactyly type A. The zinc finger protein, subfamily 1, member 1 gene (ZNFN1A1; OMIM 603023), on 7p12, codes for a lymphoid-restricted zinc finger transcription factor, ZNFN1A1, also called IKAROS, that regulates lymphocyte differentiation and has been associated with the development of childhood leukemia. We present the case of a 9-year-old Latin-American boy who was referred for stem cell transplantation because of recurrent acute lymphoblastic leukemia (ALL). On evaluation, he was found to have dysmorphic features consistent with GCPS, including a prominent forehead, down-slanting palpebral fissures, 1-2-3 toe syndactyly, broad thumbs and first toes, and mild developmental delay. He had developed ALL at 5 years of age. Chromosome analysis of bone marrow and fibroblastic cells showed an interstitial deletion of chromosome arm 7p, del(7)(p11.2p14), in 74% and 44% of the cells, respectively. We performed FISH analysis with a BAC clone containing the ZNFN1A1 gene and demonstrated that it is contained in the deleted segment. To our knowledge, this is the first report of a patient with GCPS and leukemia. We hypothesize that constitutional deletion of the ZNFN1A1 gene in this patient may have resulted in an increased risk of lymphoid malignancy.

A mixed epigenetic/genetic model for oligogenic inheritance of autism with a limited role for UBE3A.

The genetic contribution to autism is often attributed to the combined effects of many loci (ten or more). This conclusion is based in part on the much lower concordance for dizygotic (DZ) than for monozygotic (MZ) twins, and is consistent with the failure to find strong evidence for linkage in genome-wide studies. We propose that the twin data are compatible with oligogenic inheritance combined with a major, genetic or epigenetic, de novo component to the etiology. Based on evidence that maternal but not paternal duplications of chromosome 15q cause autism, we attempted to test the hypothesis that autism involves oligogenic inheritance (two or more loci) and that the Angelman gene (UBE3A), which encodes the E6-AP ubiquitin ligase, is one of the contributing genes. A search for epigenetic abnormalities led to the discovery of a tissue-specific differentially methylated region (DMR) downstream of the UBE3A coding exons, but the region was not abnormal in autism lymphoblasts or brain samples. Based on evidence for allele sharing in 15q among sib-pairs, abnormal DNA methylation at the 5'-CpG island of UBE3A in one of 17 autism brains, and decreased E6-AP protein in some autism brains, we propose a mixed epigenetic and genetic model for autism with both de novo and inherited contributions. The role of UBE3A may be quantitatively modest, but interacting proteins such as those ubiquitinated by UBE3A may be candidates for a larger role in an oligogenic model. A mixed epigenetic and genetic and mixed de novo and inherited (MEGDI) model could be relevant to other "complex disease traits".

Shuffling of genes within low-copy repeats on 22q11 (LCR22) by Alu-mediated recombination events during evolution.

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der(22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5' or 3' ends of them. A possible stimulus for the 5' or 3' terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.

Development of a comparative genomic hybridization microarray and demonstration of its utility with 25 well-characterized 1p36 deletions.

Chromosomal abnormalities, such as deletions and duplications, are characterized by specific and often complex phenotypes resulting from an imbalance in normal gene dosage. However, routine chromosome banding is not sensitive enough to detect subtle chromosome aberrations (<5-10 Mb). Array-based comparative genomic hybridization (array CGH) is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by co-hybridizing differentially labeled test and control DNAs to a microarray of genomic clones. We used a previously assembled contig of large-insert clones that span 10.5 Mb of the most distal region of 1p36 to design a microarray. The array includes 97 clones from 1p36, 41 clones from the subtelomeric regions of all human chromosomes, and three clones from each of the X and Y chromosomes. We used this microarray to study 25 subjects with well-characterized deletions of 1p36. All array CGH results agree with the deletion sizes and locations of the breakpoints in these subjects as determined previously by FISH and microsatellite analyses. Terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements were also identified. We anticipate that array CGH will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.

Monosomy 1p36 breakpoint junctions suggest pre-meiotic breakage-fusion-bridge cycles are involved in generating terminal deletions.

Terminal deletions of 1p36 result in a mental retardation syndrome that is presumably caused by haploinsufficiency of a number of genes. Although monosomy 1p36 is the most commonly observed terminal deletion syndrome in humans, the molecular mechanism(s) that generates and stabilizes terminal deletions of 1p36 is not completely understood. Our previous molecular analysis of a large cohort of monosomy 1p36 subjects demonstrated that deletion sizes vary widely from approximately 1 Mb to >10.5 Mb in the most distal portion of 1p36 with no single common breakpoint. In this report, we have identified the precise breakpoint junctions in three subjects with apparently pure terminal deletions of 1p36 ranging from 2.5 to 4.25 Mb. These junctions revealed one deletion to be stabilized by telomeric repeat sequences and two to have terminal deletions associated with cryptic interrupted inverted duplications at the ends of the chromosomes. These interrupted inverted duplication/deletion breakpoints are reminiscent of those seen in tumor cell lines that have undergone breakage-fusion-bridge (BFB) cycles leading to gene amplification. We propose a pre-meiotic model for the formation of these deletions in which a terminally deleted chromosome is generated in the germ line and passes through at least one BFB cycle to produce gametes with terminal deletions associated with interrupted inverted duplications. These data suggest that, on a molecular level, seemingly pure terminal deletions visualized cytogenetically may be more complex, and BFB cycles may play an important role in generating terminal deletions associated with genetic disease in humans.

Frequent translocations occur between low copy repeats on chromosome 22q11.2 (LCR22s) and telomeric bands of partner chromosomes.

The chromosome 22q11.2 region is susceptible to rearrangements, mediated by low copy repeats (LCR22s). Deletions and duplications are mediated by homologous recombination events between LCR22s. The recurrent balanced constitutional translocation t(11;22)(q23;q11) breakpoint occurs in an LCR22 and is mediated by double strand breaks in AT-rich palindromes on both chromosomes 11 and 22. Recently, two cases of a t(17;22)(q11;q11) were reported, mediated by a similar mechanism (21). Except for these constitutional translocations, the molecular basis for non-recurrent, reciprocal 22q11.2 translocations is not known. To determine whether there are specific mechanisms that could mediate translocations, we analyzed cell lines derived from 14 different individuals by genotyping and FISH mapping. Somatic cell hybrid analysis was carried out for four cell lines. In five cell lines, the translocation breakpoints occurred in the same LCR22 as for the t(11;22) translocation, suggesting that similar molecular mechanisms are responsible. An additional three occurred in other LCR22s, and six were in non-LCR22 regions, mostly in the proximal half of the 22q11.2 region. The translocation breakpoints on the partner chromosomes were all located in the telomeric bands, proximal to the most telomeric unique sequence probe, in eight cell lines and distal to those loci in six. Therefore, several of the breakpoints were found to occur in the vicinity of highly dynamic regions of the genome, 22q11.2 and telomeric bands. We hypothesize that these regions are more susceptible to breakage and repair, resulting in translocations.

Physical map of 1p36, placement of breakpoints in monosomy 1p36, and clinical characterization of the syndrome.

Monosomy 1p36 is the most common terminal deletion syndrome. This contiguous gene deletion syndrome is presumably caused by haploinsufficiency of a number of genes. We have constructed a contig of overlapping large-insert clones for the most distal 10.5 Mb of 1p36, evaluated the deletion sizes in 61 subjects with monosomy 1p36 from 60 families, and created a natural deletion panel. We found pure terminal deletions, interstitial deletions, derivative chromosomes, and more complex rearrangements. Breakpoints were "binned" into 0.5-Mb regions. Analyses revealed some clustering of breakpoints but no single common breakpoint. Determination of the parental origin showed that 60% of de novo 1p36 terminal deletions arose from the maternally inherited chromosome. Of the 61 subjects, 30 were examined systematically through a protocol at the Texas Children's Hospital General Clinical Research Center. Specifically, we report hearing evaluations, palatal and ophthalmological examinations, echocardiograms, neurological assessments, and thyroid function tests. To our knowledge, this systematic molecular and clinical characterization of monosomy 1p36 is the largest and most comprehensive study of this deletion syndrome to date. Many cytogenetically visible, apparent terminal deletions are more complex than anticipated by cytogenetics, as revealed at the molecular level by our study. Our clinical findings allow for the more accurate recognition of the syndrome and for proper medical evaluation.

Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluorescence in situ hybridization and biochemical analysis.

It has been previously reported that a low or absent maternal serum unconjugated estriol (uE3) level is associated with placental steroid sulfatase (STS) deficiency. Here we report a correlation between patients who present with a very low or absent maternal serum uE3 and a deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH). We studied nine prenatal cases that presented to the clinical laboratory with an abnormal triple screen, specifically low or absent maternal serum uE3 and a 46,XY karyotype. FISH analysis showed complete deletion of a probe containing the STS gene in six cases and one case had a partial deletion (reduced but not absent signal). The remaining two cases were not deleted for the STS probe. All mothers tested whose fetus showed a deletion were shown to be STS deletion carriers using FISH. Biochemical analysis was performed on 7/9 prenatal specimens. All fetuses deleted for the STS probe were also found to be deficient for STS by biochemical analysis of cultured amniotic fluid (5/5). Of the two fetuses not deleted for the STS probe, one was deficient for STS activity, while the other had a normal result. The abnormal result of enzyme deficiency by biochemical analysis in a non-deletion case likely represents a mutation in the STS gene, not detectable by this FISH assay. Postnatal FISH confirmation of the STS deletion was performed in 1/7 cases. Clinical follow-up was available for 4/9 cases following birth.