Comparative assessment of gene-specific variant distribution in prenatal and postnatal cohorts tested for Noonan syndrome and related conditions.

PURPOSE: To compare the pattern of gene-specific involvement and the spectrum of variants observed in prenatal and postnatal (mean ± SD, 8.9 ± 9.4 years) cohorts tested for Noonan syndrome and related conditions. METHODS: Outcomes of sequencing panel testing were compared between prenatal (n = 845) and postnatal (n = 409) cohorts. RESULTS: PTPN11 and SOS1 harbored the majority of observed variants in both prenatal and postnatal cohorts, and BRAF, HRAS, KRAS, MAP2K1, MAP2K2, RAF1, and SHOC2 had similarities in their pattern of involvement in both cohorts. PTPN11 was the largest contributor of pathogenic variants and had the lowest frequency of variants of uncertain significance (VUS). SOS1 had the highest VUS frequency in both cohorts. The overall VUS frequency was twice as high in prenatal specimens (58.1 vs. 29.3%). PTPN11 and SOS1 had a 1.5-fold higher VUS frequency in the prenatal cohort (10.7 vs. 7.4% and 95 vs. 61.1%, respectively). The diagnostic yield was 3.7% for prenatal samples, with a higher yield of 12.3% in fetuses with cystic hygroma as a sole finding, and 21.3% for postnatal. CONCLUSION: Comparison of prenatal versus postnatal specimens demonstrates that the pattern of specific gene involvement is similar, whereas the classification spectrum of observed variants differs considerably.

Improved detection of CFTR mutations in Southern California Hispanic CF patients.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a common autosomal recessive disease in Caucasians. The broad mutation spectrum varies among different patient groups. Current molecular diagnoses are designed to detect 80-97% of CF chromosomes in Caucasians and Ashkenazi Jews but have a much lower detection rate in Hispanic CF patients. Grebe et al. [1994] reported a 58% detection rate in Hispanic patients. Since then, there has been no large-scale, complete mutational analysis of Hispanic CF patients. In this study, the mutations in 62 Hispanic patients from southern California were investigated. The entire coding and flanking intronic regions of the CFTR gene were analyzed by temporal temperature gradient gel electrophoresis (TTGE) followed by sequencing to identify the mutations. Eleven novel mutations were discovered in this patient group: 3876delA, 406-1G>A, 935delA, 663delT, 3271delGG, 2105-2117del13insAGAAA, 3199del6, Q179K, 2108delA, 3171delC, and 3500-2A>T. Among the mutations, seven were out-of-frame insertions and deletions that result in truncated proteins, two were splice-site mutations, one was an in-frame 6 bp deletion, and one was a missense mutation that involved the non-conservative change of glutamine-179 to lysine. All patients presented severe classical clinical course with pancreatic insufficiency and poor growth, consistent with the nature of truncation mutation. The results indicate that TTGE screening following the analysis of recurrent mutations will substantially improve the mutation detection rate for Hispanic CF patients from southern California.

Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel.

PURPOSE: To determine the comparative frequency of 93 CFTR mutations in U.S. individuals with a clinical diagnosis of cystic fibrosis (CF). METHODS: A total of 5,840 CF chromosomes from Caucasians, Ashkenazi Jews, Hispanics, African Americans, Native Americans, Asians, and individuals of mixed race were analyzed using a pooled ASO hybridization strategy. RESULTS: Sixty-four mutations provided a sensitivity of 70% to 95% in all ethnic groups except Asians, and at least 81% when the U.S. population was considered as a whole. CONCLUSIONS: For population-based carrier screening for CF in the heterogeneous U.S. population, which is characterized by increasing admixture, a pan-ethnic mutation panel of 50 to 70 CFTR mutations may provide a practical test that maximizes sensitivity.

Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease.

The CFTR intron 8 variable length polythymidine tract modulates the cystic fibrosis (CF) phenotype associated with the mutation R117H. To explore whether other mutations reside on multiple intron 8 backgrounds with discernible impacts on phenotype, we developed an allele-specific PCR assay to characterize this locus. Our approach types samples rapidly without the use or radioisotopes. Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789 + 5 G > A, 3849 + 10kb C > T), and/or located at hypermutable CpG loci (R117H, 3845 + 10kb C > T, R553X, R334W, S945L and R75Q). R117H was detected in cis with each of three alleles (5T, 7T, 9T) at the intron 8 locus. The novel R117H-9T association was detected in a 10-month African-American male with borderline-to-mildly elevated sweat chloride values (approximately 50-66 mEq/L). All other mutations studied were associated with 7T except 3849 + 10kb C > T, which was detected on both 7T and 9T backgrounds, but not 5T. Three individuals with a delta F508/3849 + 10kb C > T genotype were 9T,9T and had pancreatic sufficiency and normal sweat chloride values, whereas 15 others who carried 3849 + 10kb C > T on a 7T background had variable pancreatic function (sufficient, n = 12, insufficient, n = 3), and variable sweat chloride values (normal, n = 12, elevated, n = 3). Surprisingly, when not associated with known CFTR mutations, 5T was detected with elevated frequency among individuals with sinopulmonary disease of ill-defined etiology, but with some characteristics of variant CF. In summary, the 5T allele was not found in cis with CF-causing mutations besides R117H, but an elevated 5T allele frequency in variant CF patients suggests 5T may be associated with disease in some situations.

Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.

Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

Effects of diarylsulfonylurea antitumor agents on the function of mitochondria isolated from rat liver and GC3/c1 cells.

Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pKa values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.

Cephaloridine-induced renal pathological and biochemical changes in female rabbits and isolated proximal tubules in suspension.

Cephaloridine (Cld) is a nephrotoxic cephalosporin antibiotic. The intracellular biochemical changes that occur leading to Cld-induced nephrotoxicity may involve lipid peroxidation and/or mitochondrial injury. The purpose of this report was to examine and correlate the biochemical changes induced by Cld in vivo and in vitro with the observed pathological changes in an attempt to understand better the mechanisms of beta-lactam-induced nephrotoxicity. Cld treatment (500 mg/kg sc) caused elevations in blood urea nitrogen and decreases in the accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) by renal cortical slices. Histopathological alterations, characterized by individual cell necrosis of tubular epithelial cells, were first seen 6 hr after treatment in the pars recta of the outer stripe of the medulla. Ultrastructural alterations involved the straight (S2 and S3) segments of the proximal tubules. Mitochondrial morphology was, for the most part, unaffected by Cld exposure. Cld did not cause any significant changes in tissue malondialdehyde (MDA) content in vivo at any of the time points examined, but it did cause a depletion of GSH to approximately 40% of control by 1 hr after dosing that recovered toward control by 6 hr. Significant changes were observed in renal ATP content beginning at 6 hr after treatment; however, this change mirrored the onset of histological evidence of necrosis. In isolated tubules in vitro, the onset of glutathione (GSH) depletion and MDA formation clearly preceded lactate dehydrogenase (LDH) leakage, whereas ATP depletion was a mirror image of cell death. These data demonstrate that isolated proximal tubules in vitro are a reasonable model for Cld nephrotoxicity in vivo. Cld-induced mitochondrial alterations leading to ATP depletion and cell injury were not observed in this study.

Study of 30 DNA markers in three southern African populations.

Thirty anonymous DNA markers were investigated in Southern African Caucasoid, Negroid and San populations. Sixteen of these are new markers that were developed in our laboratory; the remainder are closely linked to the cystic fibrosis locus on chromosome 7. Average heterozygosity in the Caucasoid and Negroid populations was calculated at the loci identified by each of the anonymous probes, using two approaches, and was found to be .0020 and .0030 for the Caucasoid population and .0023 and .0025 for the Negroid population. Variation between populations (measured by FST) and between markers was calculated from allele frequency data gathered for all markers in the three populations. Significant differences in allele frequency between the populations were observed for the cystic fibrosis markers MET D, MET H and 7C22, with little or no variation observed in the Negroid and San populations. Mean heterozygosity (D) was found to be considerably lower in San (.250) than in Caucasoid (.373) and Negroid populations (.0320) and possible explanations for this are provided. The smallest genetic distance (60 x 10(-3)) was found between the Negroid and San populations, and the greatest distance between the Caucasoid and San populations (167 x 10(-3)).

The tyrosinase-positive oculocutaneous albinism locus is not linked to the beta-globin locus in man.

Twenty informative families have been studied. and linkage between the tyrosinase-positive oculocutaneous albinism locus and the beta-globin locus has been excluded with a maximum lod score of -9.85 at 0 = 0.05. In lower mammals there is linkage between the p locus (considered to be equivalent to the human tyrosinase-positive oculocutaneous albinism) and the beta-globin locus.