Vicki Park

Naturally Occurring Dominant Resistance Mutations to Hepatitis C Virus Protease and Polymerase Inhibitors in Treatment-Naïve Patients

Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000‐fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment‐naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT‐C) in the population, we analyzed HCV genome sequences from 507 treatment‐naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication‐competent, drug‐resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN‐191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG‐021541; and to the NS4A antagonist ACH‐806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug‐resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. Conclusion: Naturally occurring dominant STAT‐C resistance mutations are common in treatment‐naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin. (HEPATOLOGY 2008;48:1769–1778.)

Hypercoagulable state mutation analysis in white patients with early first-trimester recurrent pregnancy loss.

OBJECTIVE Antiphospholipid antibodies (APA) and other coagulation abnormalities have been associated with an increased risk of venous, arterial, and placental thrombosis and recurrent pregnancy loss (RPL). Factor V Leiden (a point mutation [1691G-->A] in the factor V gene), the prothrombin 20210G-->A mutation, and homozygosity for a common polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene (677C-->T) have been associated with arterial and venous thrombosis and arterial occlusive disease. We explored an association between these markers of thrombophilic states and RPL. DESIGN Prospective case-control evaluation. SETTING University-associated private practice. PATIENT(S) Fifty nonpregnant women with three or more pregnancy losses and 50 healthy, nonpregnant controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Anticardiolipin and antiphosphatidylserine antibodies were detected in serum by ELISA. Polymerase chain reaction was performed to identify the factor V Leiden (1691G-->A) mutation, the thermobile MTHFR (677C-->T) mutation, and the prothrombin 20210G-->A mutation. RESULT(S) The following were identified by restriction fragment-linked polymorphism analyses: 1 (2%) factor V Leiden heterozygosity; 1 (2%) prothrombin 20210G-->A heterozygosity; and 4 (8%) thermolabile MTHFR homozygosity. None of these mutation frequencies in women with RPL were statistically significantly different from controls. CONCLUSION(S) These data suggest that factor V Leiden, thermolabile MTHFR (677C-->T), and prothrombin 20210G-->A are not found at an increased frequency in women with a history of early RPL.

Neurofibromatosis type 1 (NF1): a protein truncation assay yielding identification of mutations in 73% of patients.

Neurofibromatosis type 1 (NF1) is caused by mutations in a tumour suppressor gene located on chromosome 17 (17q11.2). Disease causing mutations are dispersed throughout the gene, which spans 350 kilobases and includes 59 exons. A common consequence of NF1 mutations is introduction of a premature stop codon, and the majority of mutant genes encode truncated forms of neurofibromin. We used a protein truncation assay to screen for mutations in 15 NF1 patients and obtained positive results in 11 of them (73%). Sequencing of cDNA and genomic DNA yielded identification of 10 different mutations, including four splicing errors, three small deletions, two nonsense mutations, and one small insertion. Nine mutations were predicted to cause premature termination of translation, while one mutation caused in frame deletion as a result ofexon skipping. In one other case involving abnormal splicing, five different aberrantly spliced transcripts were detected. One germline nonsense mutation (R1306X, 3916C>T) corresponded to the same base change that occurs by mRNA editing in normal subjects. The second nonsense mutation (R2496X) was the sole germline mutation that has been previously described. The subjects studied represented typically affected NF1 patients and no correlations between genotype and phenotype were apparent. A high incidence of ocular hypertelorism was observed.

Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling

OBJECTIVE Our purpose was to determine the risk of fetal mosaicism when placental mosaicism is found on chorionic villus sampling. STUDY DESIGN We present a case of mosaic trisomy 22 detected on chorionic villus sampling and subsequently found in the fetus. A review of comprehensive chorionic villus sampling studies with emphasis on follow-up for fetal mosaicism was conducted. RESULTS Among 13 studies reviewed, 469 cases of placental mosaicism are presented; fetal mosaicism was found in 50 (10.7%). Factors associated with fetal mosaicism are (1) mosaicism on mesenchymal core culture and (2) type of chromosome abnormality involved--specifically, marker chromosomes (26.7%) and common autosomal trisomies (19.0%). Amniocentesis predicted fetal genotype in 93% to 100% of cases of placental mosaicism, depending on the cell type in which mosaicism was diagnosed. CONCLUSIONS Although mosaicism is usually confined to the placenta, the fetus is involved in about 10% cases. Patients should be counseled about this risk and the accuracy of follow-up amniocentesis.

Comparison Between Southern Blots and qPCR Analysis of Leukocyte Telomere Length in the Health ABC Study

Only a few studies, primarily limited to small samples, have examined the relationship between leukocyte telomere length (LTL) data generated by Southern blots, expressed in kilobases, versus quantitative PCR data, expressed in the telomere product/a single gene product (T/S). In the present study, we compared LTL data generated by the two methods in 681 elderly participants (50% African Americans, 50% of European origin, 49.2% women, mean age 73.7±2.9 years) in the Health Aging and Body Composition Study. The correlation between the data generated by the two methods was modest (R (2) = .27). Both methods captured the age effect on LTL and the longer LTL in women than in men. However, only the Southern blot method showed a significantly longer LTL in African Americans than in European decent individuals, which might be attributed to the larger measurement error of the quantitative PCR-based method than the Southern blots.

Preliminary Findings: 25(OH)D Levels and PTH Are Indicators of Rapid Bone Accrual in Pubertal Children

Objective: The objective of this study was to evaluate the role of serum levels of 25(OH)D and PTH on the accumulation of whole body bone mass in a cohort of children. Methods: This was a longitudinal study (1.98 ± 0.07 y) of sixty-nine children (89% Caucasian, 44% male) enrolled in a calcium supplementation trial. Bone area, bone mineral content (BMC) and density (BMD) of the whole body and radius were assessed using a QDR 2000 (Hologic, Inc) dual energy x-ray absorptiometer. Serum PTH and 25(OH)D were measured using radioimmunoassays. Results: Vitamin D stores were inversely related gain in bone area (p < 0.002), BMC (p < 0.002) BMD (p < 0.027), as well as to PTH levels (p < 0.0001). Compared to those with adequate vitamin D stores (>34 ng/ml), those who had consistently low vitamin D stores (18 ng/ml) had a 8% larger gain in bone area (p < 0.05); 11% in BMC (p < 0.05) and no differences in gain in BMD; after adjusting for baseline bone measurements, race, gender, season measured, Tanner stage, and calcium intake. Conclusions: High normal PTH with low-normal 25(OH)D stores and moderate to high calcium intake may be beneficial to accruing larger bone size and BMC during puberty.

A jumping Robertsonian translocation : a molecular and cytogenetic study

Abstract We report a patient with mosaicism for two different Robertsonian translocations, both involving chromosome 21. She carries an unbalanced cell line with an i(21q) and a balanced cell line with a rob(21q22q). She is phenotypically normal but has two children who inherited the i(21q) and have Down syndrome. We demonstrate that both abnormal chromosomes are dicentric and that the proband’s 21/21 rearrangement is an isochromosome formed from a maternally derived chromosome 21. We propose a model in which the i(21q) is the progenitor rearrangement in the proband, which subsequently participated in a nonreciprocal rearrangement characteristic of a jumping translocation. In addition, we review other cases of constitutional mosaicism involving jumping translocations.

Sex chromosome markers: Characterization using fluorescence in situ hybridization and review of the literature

Fluorescence in situ hybridization (FISH) using biotin labeled X- and Y-chromosome DNA probes was utilized in the analysis of 23 sex chromosome-derived markers. Specimens were obtained through prenatal diagnosis, because of a presumptive diagnosis of Ullrich-Turner syndrome, mental retardation, and minor anomalies or ambiguous genitalia; three were spontaneous abortuses. Twelve markers were derived from the X chromosome and eleven from the Y chromosome; this demonstrates successfully the value and necessity of FISH utilizing DNA probes in the identification of sex chromosome markers. Both fresh and older slides, some of which had been previously G-banded, were used in these determinations. We have also reviewed the literature on sex chromosome markers identified using FISH.

Alternative splicing of exons 29 and 30 in the neurofibromatosis type 1 gene.

Alternative splicing of exons 29 and 30 of the human neurofibromatosis type 1 (NF1) gene was detected by reverse transcription/polymerase chain reaction (RT-PCR). Three different isoforms that omitted either one or both exons were identified (ex29–, ex30–, and ex29/30–). The alternatively spliced transcripts exhibited tissue-specific differences, with the ex30– variant apparent only in brain. All three isoforms altered the reading frame and introduced a stop codon in the adjacent downstream exon. Alternative splicing of this region of the NF1 gene also was detected in RNA from rats, although only the ex30– variant was observed. RNA from mice revealed only constitutive expression in this region of the NF1 gene. This study adds a new site of alternative processing to the complex expression of NF1.

Double non-disjunction in maternal meiosis II giving rise to a fetus with 48,XXX,+21.

We describe a prenatally detected case of double trisomy involving chromosome 21 and the X chromosome (48,XXX,+21) along with determination of the segregation errors responsible for the double aneuploidy. The patient was ascertained as a result of an abnormal maternal serum analyte screen showing an increased risk for fetal Downs syndrome. Following determination of the abnormal karyotype, pregnancy termination was elected. Microsatellite polymorphisms and cytogenetic heteromorphisms were used to determine that both aneuploidies arose as a result of non-disjunction in maternal meiosis II. These results support hypotheses that a segregation defect at a cellular level may cause non-disjunction involving more than one chromosome.