Double-blind, placebo-controlled crossover study of folinic acid (Leucovorin for the treatment of fragile X syndrome.

We conducted a randomized, double-blind, placebo-controlled crossover study of folinic acid therapy (dl-Leucovorin, 15 mg/day) or placebo for males with Fragile X (fra(x)) syndrome. Twenty-one patients were enrolled in the study. The treatment periods were 3 months in length. Patients were followed with chemistry panels and complete blood counts. No differences between placebo and treatment phases were noted in any laboratory parameter. Instruments to measure functioning were the Vineland Adaptive Behavioral Scales, Peabody Picture Vocabulary Test-Revised, Conners Parent and Teaching Rating Scales, the ADD-H: Comprehensive Teacher's Rating Scales (ACTeRS), and a questionnaire designed by the investigators. At the crossover point, 2 parents requested to withdraw from the study because they felt their children had made dramatic gains during the first half of the study and had lost those gains after the crossover point. Both parents had accurately predicted that their sons were receiving folinic acid during the first half of the study. However, no statistically significant differences could be demonstrated between the treatment and placebo phases of the study with any instrument when the results were averaged over the entire cohort. After the conclusion of the study, approximately one-half of the parents believed that their children had benefitted from the folinic acid therapy and elected to continue treatment. Thus far, no significant side effects have been noted from long-term folinic acid therapy so we are offering all Fragile X patients a 3-month trial of medication.

Structure of the type II collagen gene.

In summary, the exon/intron structure of the chicken type II collagen gene is identical with that of the chicken alpha 2(I) collagen gene and differs at only one known position from the human and mouse alpha 1(I) genes. However, the chicken type II gene is different from the chicken alpha 2(I) gene in that it is considerably shorter because of a much smaller average intron size and in that the G+C composition of the introns is much higher. The codon usage of the type II genes also shows characteristic differences. There is a single copy of the chick type II gene per haploid genome.

Chronic vaginal candidiasis responsive to biotin therapy in a carrier of biotinidase deficiency.

BACKGROUND: Many patients experience recurrent or persistent episodes of vaginal candidiasis. Some of these women might be carriers of an inborn error of biotin metabolism (either biotinidase deficiency or holocarboxylase synthetase activity). These women might benefit from administration of pharmacologic amounts of biotin. CASE: A 38-year-old gravida 2, para 2 carrier of biotinidase deficiency presented with a 14-month history of persistent vaginal candidiasis, despite appropriate therapy. After 3 months of pharmacologic doses of biotin, her symptoms resolved completely. CONCLUSION: Given that 1 in every 123 individuals is predicted to be a carrier of biotinidase deficiency, there might be other women with chronic vaginal candidiasis who will respond to biotin administration.

Mode of delivery and risk of respiratory diseases in newborns.

OBJECTIVE: To determine whether there is an increased incidence of persistent pulmonary hypertension in neonates delivered by cesarean, with or without labor, compared with those delivered vaginally. METHODS: We did a computerized retrospective review of 29,669 consecutive deliveries over 7 years (1992-1999). The incidences of persistent pulmonary hypertension of the newborn, transient tachypnea of the newborn, and respiratory distress syndrome (RDS) were tabulated for each delivery mode. Cases of persistent pulmonary hypertension were reviewed individually to determine delivery method and whether labor had occurred. The three groups defined were all cesarean deliveries, all elective cesareans, and all vaginal deliveries. RESULTS: Among 4301 cesareans done, 17 neonates had persistent pulmonary hypertension (four per 1000 live births). Among 1889 elective cesarean deliveries, seven neonates had persistent pulmonary hypertension (3.7 per 1000 live births). Among 21,017 vaginal deliveries, 17 neonates had persistent pulmonary hypertension (0.8 per 1000 live births). chi2 analysis showed an odds ratio 4.6 and P <.001 for comparison of elective cesarean and vaginal delivery for that outcome. CONCLUSION: The incidence of persistent pulmonary hypertension of the newborn was approximately 0.37% among neonates delivered by elective cesarean, almost fivefold higher than those delivered vaginally. The findings have implications for informed consent before cesarean and increased surveillance of neonates after cesarean.

Pediatrician attendance at cesarean delivery: necessary or not?

OBJECTIVE: To determine whether it is necessary for a pediatrician to attend all cesarean deliveries. METHODS: We analyzed a database of 17,867 consecutive deliveries to determine the rates of low Apgar scores in the following three groups of patients: those with vaginal delivery, cesarean delivery using regional anesthesia without fetal indication, and cesarean delivery for fetal indications or using general anesthesia. RESULTS: There was a significantly higher rate of low Apgar scores in the fetal indications or general anesthesia group when compared with vaginal deliveries. Specifically, 35 (5.8%) of 596 cesareans for fetal heart rate abnormality or using general anesthesia had 1-minute Apgars under 4 in contrast to 115 of 10,270 (1.1%) of vaginal deliveries. There was no significantly increased risk for low Apgar scores in the group of cesareans using regional anesthesia for nonfetal indications (33 of 2057, 1.6%). Results were similar for Apgar scores under 7 at 5 minutes. CONCLUSION: Because there is no higher incidence of low Apgar scores in cesarean deliveries using regional anesthesia for nonfetal indications compared with vaginal deliveries, there is no convincing need for pediatrician attendance at such deliveries.

Hypoplastic pulmonary arteries and aorta with obstructive uropathy in 2 siblings.

Two sisters who presented with diffuse hypoplasia of pulmonary arteries, relative hypoplasia of ascending aorta, obstructive uropathy, bilateral ureteral reflux, and hydronephrosis, are described. The subsequent course was characterized by progressive and gradual onset of right heart failure, failure to thrive, chronic malabsorption and systemic hypertension. The syndrome which appears to be transmitted by autosomal recessive inheritance can possibly represent a generalized hypoplasia and growth failure of a part or the entire arterial system. Peripheral pulmonary stenosis can occur as an isolated lesion or in association with other congenital cardiac anomalies, as well as in rubella syndrome and the syndrome of supravalvular aortic stenosis. This communication reports two siblings with a hitherto unreported combination of hypoplastic pulmonary arteries, and aorta with identical genito-urinary tract abnormalities.

Decreasing risk of pregnancy loss following chorionic villus sampling. Elimination of transabdominal chorionic villus sampling during the ninth week of pregnancy.

Chorionic villus sampling (CVS) is a method of obtaining fetal cells in the first trimester of pregnancy for genetic analysis. The transcervical (TC) approach was the first technique to be widely used. In the National Institute of Child Health and Human Development collaborative study the absolute loss rate following CVS (the total number of spontaneous abortions and neonatal deaths following CVS) was 4%. More recently the transabdominal (TA) approach has been introduced. This study compares the loss rates for the two approaches at various gestational ages for three 6-month periods following the addition of the TA approach with each other and with the loss rates prior to the introduction of TA CVS. We found that the percentage of pregnancy losses following TA CVS during the ninth week of gestation (63-69 days) was consistently higher than for TC CVS performed at the same gestational age. The loss rate for TC CVS has steadily decreased since the introduction of TA CVS after remaining the same for the two years prior to the introduction of the TA approach. After minimizing the number of TA CVS performed during the ninth week of gestation, the overall loss rate during the most recent 6-month period has been reduced to 0.94%. We conclude that the lowest loss rate following CVS can be obtained if both the TA and TC methods are available, and that the number of TA procedures performed during the ninth week of gestation is minimized.

Use of nested PCR to identify charred human remains and minute amounts of blood.

Reliable single cell PCR requires nested or heminested PCR and careful optimization of conditions. This report describes the successful use of nested PCR for gender identification and reverse paternity testing in a forensic case where the only available materials consisted of charred human remains and a minute quantity of blood that were unsuitable for standard PCR. Use of nested PCR allowed the blood and burned tissue to be identified as human female. Analysis of two PCR length polymorphisms (AMPFLP) was successful on the blood sample and reverse paternity testing yielded a 98% probability that the blood spot was from the victim. The defendant was convicted of murder following a bench trial and the verdict was upheld by the Appellate court.

The importance of ß globin deletion analysis in the evaluation of patients with ß thalassemia.

INTRODUCTION: Beta globin deletion/duplication analysis may serve as a useful adjunct to sequence analysis. Our purpose was to develop a robust assay for beta globin deletion/duplication analysis and determine its role in evaluating patients with beta thalassemia. METHODS: A single tube semi-quantitative fluorescent PCR assay capable of detecting deletions and duplications in the beta globin cluster and the associated locus control region (LCR) was developed and validated. RESULTS: Six hundred seventy one de-identified samples submitted for beta globin sequence analysis were tested for deletions and duplications of the beta globin cluster. Twenty-two deletions were detected (3%, 22/671). Seventeen of the 22 (82%) deletion samples were negative for mutations in the whole gene sequencing assay. For 5 of the samples, homozygous point mutations were inferred by beta globin sequencing. Among the deletions detected, 11 (50%) involved only the beta globin gene (5 covering the entire gene, 2 spanning the 5' end of the gene and 4 encompassing the 3' end of the gene). Ten samples (45%) were heterozygous delta-beta deletions spanning both the delta globin and beta globin genes. One patient with a single deletion had Hb Lepore. CONCLUSION: Beta globin deletion/duplication analysis is necessary to correctly identify the genotype in some patients being evaluated for beta thalassemia.

Analysis of three restriction fragment length polymorphisms in the human type II procollagen gene.

Cloned genomic DNA sequences corresponding to various regions of the human type II procollagen gene were used to analyze the DNA from 78 normal volunteers. Southern hybridization experiments detected polymorphic HindIII, BamHI, and EcoRI sites. The presence of the polymorphic HindIII site results in a 7.0-kilobase (kb) band, and the absence of this site results in a 14.0-kb band. When present, the BamH1 polymorphic site yields a 4.8-kb band, and when absent, yields a 7.2-kb band. The presence of the EcoRI polymorphic site results in a 3.7-kb band, and its absence results in a 7.0-kb band. Each polymorphic site was mapped. Analyses of the data demonstrated that the sites are present in overall gene frequencies of .39 for HindIII, .04 for BamHI, and .02 for EcoRI. Gene frequencies of the polymorphic sites were also studied with respect to race. The polymorphic sites are present in a Hardy-Weinberg distribution in the study population. Study of an extended family demonstrated that the segregation of the HindIII polymorphic site is consistent with Mendelian inheritance.

Isolation and characterization of genomic clones corresponding to the human type II procollagen gene.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.

Distribution of 5-bromodeoxyuridine and thymidine in the DNA of developing chick cartilage.

In order to study the mechanism of the irreversible effects of BrdUrd on the differentiation of limb bud mesenchyme to cartilage, the reannealing behavior of DNA obtained from such cells was examined. Cells incubated with [3H]thymidine ([3H]dThd) during days 1 and 2 of culture incorporated label into repetitive, moderately repetitive, and unique classes of DNA. In contrast, when 5-bromo-2'-[3H]deoxyuridine ([3H]Brd Urd) was added during the first 48 hr (in the presence of 32 muM BrdUrd), the label was preferentially incorporated into a late moderately repetitive region. Simultaneous incubation of unlabeled BrdUrd and [3H]dThd revealed a selective inhibition of [3H]dThd incorporation into moderately repetitive regions. Cultures incubated during days 3 and 4 with [3H]dThd incorporated label into all three classes of DNA; however, when [3H]dThd was present during days 3 and 4 in cultures previously incubated with BrdUrd during days 1 and 2, the [3H]dThd was incorporated preferentially in the late moderately repetitive region. The melting behavior of this reannealed DNA was identical with that of the reannealed 1-2 day [3H]BrdUrd-labeled, late moderately repetitive DNA. Turnover experiments revealed that whereas there was no loss of [3H]deoxycytidine or [3H]dThd, 37% of [3H]BrdUrd activity was lost from the DNA in 2 days after removal of the isotopes.

Amplification of moderately repetitive DNA sequences during chick cartilage differentiation.

A 5-bromo-2'-[3H]deoxyuridine (BrdUrd) probe was isolated to analyze DNAs obtained from various chick tissues and cell types. [3H]BrdUrd-substituted DNA, prepared from limb bud cultures, was sheared and freed from palindromic DNA. Nonradioactive DNA was prepared from embryonic liver, undifferentiated limb bud mesenchyme, sternal cartilage, differentiated limb bud cultures, and BrdUrd-blocked cultures, and was sheared. These DNAs were used in 100-fold excess to drive the reassociation of the [3H]-BrdUrd-DNA probe. Purified mature cartilage DNAs of embryonic sternae or differentiated limb bud cultures drove the reassociation of the probe approximately two times faster than did DNA from liver, undifferentiated limb bud, or BrdUrd-blocked cells. These data indicate that cartilage DNA contains a greater number of sequences complementary to the BrdUrd probe than do DNAs of noncartilage or undifferentiated precartilage cells. Calculations determined an average substitution of 10% of thymidine residues by BrdUrd in purified probe, whereas CsCl density gradients of unsheared probe revealed radioactive peaks of greater than 20% substitution. The BrdUrd appears to be clustered in the genome.

Localization of human type II procollagen gene (COL2A1) to chromosome 12.

DNA was prepared from 39 human-mouse somatic cell hybrid lines, and mouse and human parental cell lines. The DNA was digested to completion with EcoRI and Southern filters were prepared. These filters were hybridized at high stringency conditions to the human genomic subclone phHCol(II) A which corresponds to the human alpha 1(type II) procollagen gene (COL2A1). The mouse DNA yielded a single band at greater than 10 kb, whereas the human DNA had the expected single band at 4.8kb. Analyses of these human-mouse cell hybrids demonstrated that the human alpha 1(type II) procollagen gene segregates with chromosome 12.

Reliability of gender determination using the polymerase chain reaction (PCR) for single cells.

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic diseases. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment.

Reliability of polymerase chain reaction (PCR) analysis of single cells for preimplantation genetic diagnosis.

PURPOSE: We investigated the reliability of polymerase chain reaction (PCR) genotype analyses performed on single cells for the purposes of preimplantation genetic analysis. METHODS: We performed blind analysis of 130 single skin fibroblasts heterozygous for the delta-F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene and 73 single skin fibroblasts from an individual heterozygous for the XbaI polymorphic site of the Factor VIII gene. RESULTS: Amplification was successful for 116 cells and 52 cells respectively and in all but one case (a CFTR analysis) both alleles were amplified. The incidence of diagnostic error was 1 out of 203 analyses or 0.0043. We conclude that PCR is a reliable method for determining the genotype of single cells for the purposes of preimplantation genetic analysis.

Nonrandom association of a type II procollagen genotype with achondroplasia.

Achondroplasia is an autosomal dominant disorder that involves defective endochondral bone formation. Type II collagen is the predominant collagen of cartilage. We found a HindIII polymorphic site in the normal Caucasian population by using the type II procollagen gene probe pgHCol(II)A. The presence of this site yields a 7.0-kilobase (kb) band; its absence yields a 14.0-kb band. We found a significant deviation in genotype distribution and allele frequencies in a population of unrelated individuals with sporadic achondroplasia, compared with the normal control population. The HindIII genotype frequencies in 32 individuals with achondroplasia are 0.41 for the 7/7 genotype (controls, 0.08), 0.34 for the 7/14 genotype (controls, 0.54), and 0.25 for the 14/14 genotype (controls, 0.37). The apparent equilibrium excess of the "7" allele in individuals with achondroplasia may reflect either a predisposition for the mutation that causes achondroplasia or it could be the result of the achondroplasia-causing mutation. In either case, these findings suggest an association of the type II procollagen gene with achondroplasia.

Amplification of DNA sequences during chicken cartilage and neural retina differentiation.

[3H]BrdUrd-substituted DNA probes were prepared from organ cultures of differentiating chicken neural retina and cell cultures of stage 24 chicken limb buds. Reassociation reactions using the neural retina probe demonstrated amplification of DNA sequences during differentiation of neural retina. This probe also contained sequences present in greater numbers in heart DNA than in DNA from undifferentiated neural retina. Reassociation reactions of both differentiated cartilage and differentiated neural retina DNA with both the neural retina probe and the cartilage probe demonstrated that at least part of the amplified sequences are tissue specific.

Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling).

PURPOSE: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. METHODS: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. RESULTS: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. CONCLUSIONS: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.