[Follow-up of the patient with pacemakers: Interrogation and basic programming]. / Seguimiento del paciente con marcapasos: interrogatorio y programación básica.

Permanent pacemakers are a frequently used therapeutic modality. Its use has had a great impact on the morbidity and mortality and quality of life of patients with heart rhythm disturbances, with an exponential increase observed in recent decades. The use of this strategy presents different phases, in which follow-up throughout the useful life of the device is a fundamental and determinant pillar of the efficacy and safety of this therapeutic modality. This review seeks to provide a clear and structured update of the fundamental aspects to consider in the follow-up of all patients with pacemakers. The follow-up of the patient with a pacemaker must follow a complete, systematic and periodic protocol, evaluating aspects and parameters related to the patient and the pacemaker, in order to ensure the proper and safe operation of the device adapted to the person.

Los marcapasos permanentes son una modalidad terapéutica de uso frecuente. Su empleo ha tenido un gran impacto en la morbimortalidad y calidad de vida de los pacientes con alteraciones del ritmo cardiaco, observándose en las últimas décadas un incremento exponencial. El empleo de esta estrategia presenta diferentes fases, que inician con la indicación de la estimulación, la selección del sistema de marcapaso apropiado, el procedimiento de implantación, la programación inicial y el seguimiento posterior, en la cual el seguimiento a lo largo de la vida útil del dispositivo es un pilar fundamental y determinante de la eficacia y seguridad de esta modalidad terapéutica. La presente revisión busca proporcionar una actualización clara y estructurada de los aspectos fundamentales a considerar en el seguimiento de todo paciente portador de marcapasos. El seguimiento del paciente con marcapasos debe seguir un protocolo completo, sistemático y periódico, evaluando aspectos y parámetros relacionado con el paciente y el marcapaso, con la finalidad de garantizar un funcionamiento adecuado y seguro del dispositivo adaptado a la persona.

CMA analysis identifies homozygous deletion of MCPH1 in 2 brothers with primary Microcephaly-1.

BACKGROUND: Homozygous mutations and deletions of the microcephalin gene (MCPH1; OMIM *607117) have been identified as a cause of autosomal recessive primary microcephaly and intellectual disability (MIM #251200). Previous studies in families of Asian descent suggest that the severity of the phenotype may vary based on the extent of the genomic alteration. We report chromosome microarray (CMA) findings and the first described family study of a patient with primary microcephaly in a consanguineous Hispanic family. CASE PRESENTATION: The proband, a boy born at full-term to consanguineous parents from Mexico, presented at 35 months of age with microcephaly, abnormal brain MRI findings, underdeveloped right lung, almond-shaped eyes, epicanthal folds, bilateral esotropia, low hairline, large ears, smooth philtrum, thin upper lip, and developmental delay. MRI of the brain showed a small dermoid or lipoma (without mass effect) within the interpeduncular cistern and prominent arachnoid granulation. The underdeveloped right lung was managed with long-acting inhaled corticosteroids. Otherwise the proband did not have any other significant medical history. The proband had 2 older brothers, ages 14 and 16, from the same consanguineous parents. The 14-year-old brother had a phenotype similar to that of the proband, while both parents and the oldest brother did not have the same phenotypic findings as the proband. The SNP-based CMA analysis of the proband detected a homozygous 250-kb microdeletion at 8p23.2p23.1, extending from 6,061,169 to 6,310,738 bp [hg19]. This genomic alteration encompasses the first 8 exons of MCPH1. Follow-up studies detected the same homozygous deletion in the affected brother, segregating with microcephaly and intellectual disability. Regions of homozygosity (ROHs) were also observed in the affected brother. Since ROHs are associated with an increased risk for recessive disorders, presence of ROH may also contribute to the phenotype of the affected brothers. The parents were both hemizygous for the deletion. CONCLUSION: Here we report a homozygous deletion of multiple exons of the MCPH1 gene that was associated with primary microcephaly and intellectual disability in a Hispanic family. In the context of previous studies, our results support the idea that deletions involving multiple exons cause a more severe phenotype than point mutations.

Enrichment of small pathogenic deletions at chromosome 9p24.3 and 9q34.3 involving DOCK8, KANK1, EHMT1 genes identified by using high-resolution oligonucleotide-single nucleotide polymorphism array analysis.

BACKGROUND: High-resolution oligo-SNP array allowed the identification of extremely small pathogenic deletions at numerous clinically relevant regions. In our clinical practice, we found that small pathogenic deletions were frequently encountered at chromosome 9p and 9q terminal regions. RESULTS: A review of 531 cases with reportable copy number changes on chromosome 9 revealed142 pathogenic copy number variants (CNVs): 104 losses, 31 gains, 7 complex chromosomal rearrangements. Of 104 pathogenic losses, 57 were less than 1 Mb in size, enriched at 9p24.3 and 9q34.3 regions, involving the DOCK8, KANK1, EHMT1 genes. The remaining 47 cases were due to interstitial or terminal deletions larger than 1 Mb or unbalanced translocations. The small pathogenic deletions of DOCK8, KANK1 and EHMT1 genes were more prevalent than small pathogenic deletions of NRXN1, DMD, SHANK3 genes and were only second to the 16p11.2 deletion syndrome, 593-kb (OMIM #611913). CONCLUSIONS: This study corroborated comprehensive genotype-phenotype large scale studies at 9p24.3 and 9q24.3 regions for a better understanding of the pathogenicity caused by haploinsufficiency of the DOCK8, KANK1 and EHMT1 genes. TRIAL REGISTRATION NUMBER: None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.

Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD.

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Regions of homozygosity identified by oligonucleotide SNP arrays: evaluating the incidence and clinical utility.

Copy neutral segments with allelic homozygosity, also known as regions of homozygosity (ROHs), are frequently identified in cases interrogated by oligonucleotide single-nucleotide polymorphism (oligo-SNP) microarrays. Presence of ROHs may be because of parental relatedness, chromosomal recombination or rearrangements and provides important clues regarding ancestral homozygosity, consanguinity or uniparental disomy. In this study of 14 574 consecutive cases, 832 (6%) were found to harbor one or more ROHs over 10 Mb, of which 651 cases (78%) had multiple ROHs, likely because of identity by descent (IBD), and 181 cases (22%) with ROHs involving a single chromosome. Parental relatedness was predicted to be first degree or closer in 5%, second in 9% and third in 19%. Of the 181 cases, 19 had ROHs for a whole chromosome revealing uniparental isodisomy (isoUPD). In all, 25 cases had significant ROHs involving a single chromosome; 5 cases were molecularly confirmed to have a mixed iso- and heteroUPD15 and 1 case each with segmental UPD9pat and segmental UPD22mat; 17 cases were suspected to have a mixed iso- and heteroUPD including 2 cases with small supernumerary marker and 2 cases with mosaic trisomy. For chromosome 15, 12 (92%) of 13 molecularly studied cases had either Prader-Willi or Angelman syndrome. Autosomal recessive disorders were confirmed in seven of nine cases from eight families because of the finding of suspected gene within a ROH. This study demonstrates that ROHs are much more frequent than previously recognized and often reflect parental relatedness, ascertain autosomal recessive diseases or unravel UPD in many cases.

Concurrent triplication and uniparental isodisomy: evidence for microhomology-mediated break-induced replication model for genomic rearrangements.

Whole-genome oligonucleotide single-nucleotide polymorphism (oligo-SNP) arrays enable simultaneous interrogation of copy number variations (CNVs), copy neutral regions of homozygosity (ROH) and uniparental disomy (UPD). Structural variation in the human genome contributes significantly to genetic variation, and often has deleterious effects leading to disease causation. Co-occurrence of CNV and regions of allelic homozygosity in tandem involving the same chromosomal arm are extremely rare. Replication-based mechanisms such as microhomology-mediated break-induced replication (MMBIR) are recent models predicted to induce structural rearrangements and gene dosage aberrations; however, supportive evidence in humans for one-ended DNA break repair coupled with MMBIR giving rise to interstitial copy number gains and distal loss of heterozygosity has not been documented. We report on the identification and characterization of two cases with interstitial triplication followed by uniparental isodisomy (isoUPD) for remainder of the chromosomal arm. Case 1 has a triplication at 9q21.11-q21.33 and segmental paternal isoUPD for 9q21.33-qter, and presented with citrullinemia with a homozygous mutation in the argininosuccinate synthetase gene (ASS1 at 9q34.1). Case 2 has a triplication at 22q12.1-q12.2 and segmental maternal isoUPD 22q12.2-qter, and presented with hearing loss, mild dysmorphic features and bilateral iris coloboma. Interstitial triplication coupled with distal segmental isoUPD is a novel finding that provides human evidence for one-ended DNA break and replication-mediated repair. Both copy number gains and isoUPD may contribute to the phenotype. Significantly, these cases represent the first detailed genomic analysis that provides support for a MMBIR mechanism inducing copy number gains and segmental isoUPD in tandem.

Characterization of a complex chromosomal rearrangement using chromosome, FISH, and microarray assays in a girl with multiple congenital abnormalities and developmental delay.

Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes (3, 7 and 12), seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient's developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient.

Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results.

Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome (MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1 (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses.

Abnormalities in spontaneous abortions detected by G-banding and chromosomal microarray analysis (CMA) at a national reference laboratory.

BACKGROUND: Cytogenetic evaluation of products of conception (POC) for chromosomal abnormalities is central to determining the cause of pregnancy loss. We compared the test success rates in various specimen types and the frequencies of chromosomal abnormalities detected by G-banding analysis with those found by Oligo-SNP chromosomal microarray analysis (CMA). We evaluated the benefit of CMA testing in cases of failed culture growth. METHODS: Conventional cytogenetic results of 5457 consecutive POC specimens were reviewed and categorized as placental villi, fetal parts, and unspecified POC tissue. The CMA was performed on 268 cases. Of those, 32 cases had concurrent G-banding results. The remaining 236 cases included 107 cases with culture failure and 129 cases evaluated by CMA alone. RESULTS: The overall POC culture success rate was 75%, with the lowest for fetal parts (37.4%) and the highest for placental villi (81%). The abnormality rate was 58% for placental villi, but only 25% for fetal parts. Of the abnormalities detected, the most common were aneuploidies, including trisomy 16, triploidy, monosomy X, trisomy 22, trisomy 21 and trisomy 15, while the least encountered aneuploidies were trisomy 1, trisomy 19 and monosomies (except monosomy 21). Overall, POC specimens studied by CMA were successful in 89.6% of cases and yielded a 44.6% abnormality rate. CONCLUSIONS: Placental villi yielded higher rates of culture success and a higher percentage of abnormal karyotypes than did other specimen types. The Oligo-SNP CMA method has demonstrated a viable alternative to the G-banding method in view of its advantages in detection of submicroscopic genomic aberrations, shorter turnaround time due to elimination of time required for culture and a higher test success rate.

Short stature, digit anomalies and dysmorphic facial features are associated with the duplication of miR-17 ~ 92 cluster.

MicroRNAs (miRNAs) are key regulators of gene expression, playing important roles in development, homeostasis, and disease. Recent experimental evidence indicates that mutation or deregulation of the MIR17HG gene (miR-17 ~ 92 cluster) contributes to the pathogenesis of a variety of human diseases, including cancer and congenital developmental defects. We report on a 9-year-old boy who presented with developmental delay, autism spectrum disorder, short stature, mild macrocephaly, lower facial weakness, hypertelorism, downward slanting palpebral fissures, brachydactyly, and clinodactyly. SNP-microarray analysis revealed 516 kb microduplication at 13q31.3 involving the entire MIR17HG gene encoding the miR-17 ~ 92 polycistronic miRNA cluster, and the first five exons of the GPC5 gene. Family study confirmed that the microduplication was maternally inherited by the proband and one of his five half-brothers; digit and other skeletal anomalies were exclusive to the family members harboring the microduplication. This case represents the smallest reported microduplication to date at 13q31.3 and provides evidence supporting the important role of miR-17 ~ 92 gene dosage in normal growth and skeletal development. We postulate that any dosage abnormality of MIR17HG, either deletion or duplication, is sufficient to interrupt skeletal developmental pathway, with variable outcome from growth retardation to overgrowth.

Genotype-phenotype analysis of recombinant chromosome 4 syndrome: an array-CGH study and literature review.

BACKGROUND: Recombinant chromosome 4, a rare constitutional rearrangement arising from pericentric inversion, comprises a duplicated segment of 4p13~p15→4pter and a deleted segment of 4q35→4qter. To date, 10 cases of recombinant chromosome 4 have been reported. RESULT: We describe the second case in which array-CGH was used to characterize recombinant chromosome 4 syndrome. The patient was a one-year old boy with consistent clinical features. Conventional cytogenetics and FISH documented a recombinant chromosome 4, derived from a paternal pericentric inversion, leading to partial trisomy 4p and partial monosomy of 4q. Array-CGH, performed to further characterize the rearranged chromosome 4 and delineate the breakpoints, documented a small (4.36 Mb) 4q35.1 terminal deletion and a large (23.81 Mb) 4p15.1 terminal duplication. Genotype-phenotype analysis of 10 previously reported cases and the present case indicated relatively consistent clinical features and breakpoints. This consistency was more evident in our case and another characterized by array-CGH, where both showed the common breakpoints of p15.1 and q35.1. A genotype-phenotype correlation study between rec(4), dup(4p), and del(4q) syndromes revealed that urogenital and cardiac defects are probably due to the deletion of 4q whereas the other clinical features are likely due to 4p duplication. CONCLUSION: Our findings support that the clinical features of patients with rec(4) are relatively consistent and specific to the regions of duplication or deletion. Recombinant chromosome 4 syndrome thus appears to be a discrete entity that can be suspected on the basis of clinical features or specific deleted and duplicated chromosomal regions.

B-lymphoblastic leukemia/lymphoma associated with t(8;13)(p11;q12)/ ZMYM2 (ZNF198)-FGFR1 : rare case and review of the literature.

Myeloid and lymphoid neoplasms with fibroblastic growth factor receptor-1 (FGFR1) abnormalities originate from mutated pluripotent stem cells and have a heterogeneous clinical presentation. There are 12 identified partner genes commonly involved in FGFR1 translocation at an 8p11 breakpoint. In FGFR1-related neoplasms, T-lymphoblastic lymphoma with eosinophilia is the most common clinical scenario, whereas acute B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is rare. To date, only 7 cases of B-ALL/LBL with FGFR1 abnormalities have been reported. Here, we report an additional case of a 64-year-old gentleman with leukocytosis, eosinophilia and diffuse mediastinal and general lymphadenopathy. Bone marrow examination showed patchy infiltrates of immature precursors/blasts, along with myeloid/eosinophilic hyperplasia. Immunophenotyping confirmed increased B lymphoblasts (30-40%). Karyotyping revealed cytogenetic abnormalities, including t(8;13)(p11;q12)/ZMYM2 (ZNF198)-FGFR1 and trisomy 21. The patient did not respond to hyper-CVAD chemotherapy and within 4 months developed acute myelomonocytic leukemia and expired 11 months after the initial diagnosis. Similar cases from the literature are reviewed.

Neocentric X-chromosome in a girl with Turner-like syndrome.

BACKGROUND: Neocentromeres are rare human chromosomal aberrations in which a new centromere has formed in a previously non-centromeric location. We report the finding of a structurally abnormal X chromosome with a neocentromere in a 15-year-old girl with clinical features suggestive of Turner syndrome, including short stature and primary amenorrhea. RESULT: G-banded chromosome analysis revealed a mosaic female karyotype involving two abnormal cell lines. One cell line (84% of analyzed metaphases) had a structurally abnormal X chromosome (duplication of the long arm and deletion of the short arm) and a normal X chromosome. The other cell line (16% of cells) exhibited monosomy X. C-banding studies were negative for the abnormal X chromosome. FISH analysis revealed lack of hybridization of the abnormal X chromosome with both the X centromere-specific probe and the "all human centromeres" probe, a pattern consistent with lack of the X chromosome endogenous centromere. A FISH study using an XIST gene probe revealed the presence of two XIST genes, one on each long arm of the iso(Xq), required for inactivation of the abnormal X chromosome. R-banding also demonstrated inactivation of the abnormal X chromosome. An assay for centromeric protein C (CENP-C) was positive on both the normal and the abnormal X chromosomes. The position of CENP-C in the abnormal X chromosome defined a neocentromere, which explains its mitotic stability. The karyotype is thus designated as 46,X,neo(X)(qter- > q12::q12- > q21.2- > neo- > q21.2- > qter)[42]/45,X[8], which is consistent with stigmata of Turner syndrome. The mother of this patient has a normal karyotype; however, the father was not available for study. CONCLUSION: To our knowledge, this is the first case of mosaic Turner syndrome involving an analphoid iso(Xq) chromosome with a proven neocentromere among 90 previously described cases with a proven neocentromere.

Spectral Karyotyping for identification of constitutional chromosomal abnormalities at a national reference laboratory.

Spectral karyotyping is a diagnostic tool that allows visualization of chromosomes in different colors using the FISH technology and a spectral imaging system. To assess the value of spectral karyotyping analysis for identifying constitutional supernumerary marker chromosomes or derivative chromosomes at a national reference laboratory, we reviewed the results of 179 consecutive clinical samples (31 prenatal and 148 postnatal) submitted for spectral karyotyping. Over 90% of the cases were requested to identify either small supernumerary marker chromosomes (sSMCs) or chromosomal exchange material detected by G-banded chromosome analysis. We also reviewed clinical indications of those cases with marker chromosomes in which chromosomal origin was identified by spectral karyotyping. Our results showed that spectral karyotyping identified the chromosomal origin of marker chromosomes or the source of derivative chromosomal material in 158 (88%) of the 179 clinical cases; the identification rate was slightly higher for postnatal (89%) compared to prenatal (84%) cases. Cases in which the origin could not be identified had either a small marker chromosome present at a very low level of mosaicism (< 10%), or contained very little euchromatic material. Supplemental FISH analysis confirmed the spectral karyotyping results in all 158 cases. Clinical indications for prenatal cases were mainly for marker identification after amniocentesis. For postnatal cases, the primary indications were developmental delay and multiple congenital anomalies (MCA). The most frequently encountered markers were of chromosome 15 origin for satellited chromosomes, and chromosomes 2 and 16 for non-satellited chromosomes. We were able to obtain pertinent clinical information for 47% (41/88) of cases with an identified abnormal chromosome. We conclude that spectral karyotyping is sufficiently reliable for use and provides a valuable diagnostic tool for establishing the origin of supernumerary marker chromosomes or derivative chromosomal material that cannot be identified with standard cytogenetic techniques.

Inherited and de novo 22q11.2 distal duplications in two patients with autistic features, speech delay and no dysmorphology.

In a screen of patients by fluorescence in-situ hybridization and array comparative genomic hybridization in the past two years (July 2007--July 2009), we identified two patients with duplications in the 22q11.22-23, occurring outside the common DiGeorge syndrome/valocardiofacial syndrome region. Fluorescent in-situ hybridization, multiplex ligation-dependent probe amplification and high density bacterial artificial chromosomes and oligo arrays were used to identify the extent of the duplications. In one patient the duplication extended from LCR22-E/5 to LCR22-H/8, which is similar to recently described 22q11.2 distal duplications, while in the second patient, a de novo duplication was identified extending between LCR22-E/5 to LCR22-F/6. The second proband also harbored a de novo 15q14 duplication, complicating phenotype interpretation. The patients were affected with speech delay and autistic features, but neither reported cardiac concern or dysmorphic features.

Fluorescence in situ hybridization and K-ras analyses improve diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration of solid pancreatic masses.

OBJECTIVES: Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) is the main diagnostic modality for pancreatic mass lesions. However, cytology is often indeterminate, leading to repeat FNAs and delay in care. Here, we evaluate whether combining routine cytology with fluorescence in situ hybridization (FISH) and K-ras/p53 analyses improves diagnostic yield of pancreatic EUS-FNA. METHODS: Fifty EUS-FNAs of pancreatic masses in 46 patients were retrospectively analyzed. Mean follow-up was 68 months. Thirteen initial cytologic samples (26%) were benign, 23 malignant (46%), and 14 atypical (28%). We performed FISH for p16, p53, LPL, c-Myc, MALT1, topoisomerase 2/human epidermal growth factor receptor 2, and EGFR, as well as K-ras/p53 mutational analyses. RESULTS: On final diagnosis, 11 (79%) of atypical FNAs were malignant, and 3 benign (21%). Fluorescence in situ hybridization was negative in all benign and all atypical samples with final benign diagnosis. Fluorescence in situ hybridization plus K-ras analysis correctly identified 60% of atypical FNAs with final malignant diagnosis. Combination of routine cytology with positive FISH and K-ras analyses yielded 87.9% sensitivity, 93.8% specificity, 96.7% positive predictive value, 78.9% negative predictive value, and 89.8% accuracy. CONCLUSIONS: Combining routine cytology with FISH and K-ras analyses improves diagnostic yield of EUS-FNA of solid pancreatic masses. We propose to include these ancillary tests in the workup of atypical cytology from pancreatic EUS-FNA.

Clinical array comparative genomic hybridization: a new paradigm.

BACKGROUND: Although the clinical utility of array comparative genomic hybridization (aCGH) is undisputed, the implementation of this technology is a unique experience for each laboratory. OBJECTIVE: Endeavors to construct a bacterial artificial chromosome (BAC)-based CGH microarray targeting microdeletion and duplication syndromes related to mental retardation and developmental delay are described. METHOD: Covering each chromosome at the 650-band level, the array comprises 1360 BAC clones with emphasis on the subtelomeric and pericentromeric regions and enrichment of genomic hot spots containing genes associated with specific constitutional disorders. During development of the array, fluorescence in situ hybridization (FISH) and end-sequencing analysis eliminated 24% of BACs that were mismapped or cross-hybridized, underscoring the need rigorously to assess arrayed elements. Performance of the BACs was tested further with chromosome-specific add-in experiments. CONCLUSION: Of the first 500 clinical cases, 54 (11%) showed chromosome abnormalities, which were confirmed by FISH with BACs from the aberrant loci or by conventional cytogenetics. Array CGH is a powerful tool that is now being implemented in the realm of diagnostic testing.

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype.

PURPOSE: The study's purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles. METHOD: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans. RESULTS: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype. CONCLUSION: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.

A large deletion in the CFTR gene in CBAVD.

PURPOSE: Most cystic fibrosis mutation screening methods do not detect large exon deletions or duplications in the cystic fibrosis transmembrane regulator gene. We looked for such mutations in congenital bilateral absence of the vas deferens patients in whom routine screening assays had identified only one or no cystic fibrosis transmembrane regulator gene mutations. METHODS: DNA samples from 48 men with congenital bilateral absence of the vas deferens were tested for exonic deletions and duplications in the cystic fibrosis transmembrane regulator gene using a laboratory-developed semiquantitative fluorescent PCR assay. RESULTS: Semi-quantitative fluorescent PCR identified a large deletion in one (2%) of the 48 patients. This patient, previously characterized as carrying only the IVS8-5T mutation, was found to have a deletion of exons 22-24 of the cystic fibrosis transmembrane regulator gene. In a second patient with the IVS8-5T mutation, we identified a one-base pair insertion in exon 17b that disrupted the reading frame. CONCLUSIONS: Analysis of the cystic fibrosis transmembrane regulator gene for exon deletions and duplications should be included for complete study of CBAVD patients, especially those considering assisted reproduction.