Arthur B. Zinn

Delineation of the Marfan phenotype associated with mutations in exons 23–32 of the FBN1 gene

Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 gene in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling.

The Stable Isotope Dilution Method for Measurement of Methylmalonic Acid: a Highly Accurate Approach to the Prenatal Diagnosis of Methylmalonic Acidemia

Summary: We report a stable isotope dilution method for the accurate measurement of methylmalonic acid (MMA) in amniotic fluid and its successful application to the prenatal diagnosis of methylmalonic acidemia. The stable isotopically-labeled analogue of MMA, (2-[2H3]methyl)malonic acid, was synthesized and used as an internal standard. Samples were extracted, methylated, and analyzed by chemical ionization gas chromatography-mass spectrometry (CI-GC-MS) operated in the selected ion monitoring (SIM) mode. The analytical method is rapid (8 h), ultrasensitive (lower limit ~10 ng/ml), linear over three orders of magnitude, and reproducible. The concentration of MMA in amniotic fluids from normal pregnant women was 38 ± 15 ng/ml (mean ± 1 S.D.). This value is 20-fold less than our previous estimate by gas chromatographic analysis, indicating that interference by co-eluting materials was eliminated by use of SIM in the present method. Amniotic fluids from eight at-risk pregnancies were examined. MMA concentration in six of the pregnancies was in the normal range whereas the concentration in the remaining two pregnancies was elevated by greater than two orders of magnitude (125–250-fold). Analysis of urine from ten at-risk pregnancies showed a smaller (31/2–9-fold) but significant increase in urinary MMA excretion in two women carrying affected fetuses. A control group of normal pregnant women had a mean urinary MMA excretion of 1.7 ± 0.5 μg/mg creatinine, in excellent agreement with previous estimates. There was concordance in prenatal diagnosis of fetal status in all cases between amniotic fluid cell studies and MMA measurements in amniotic fluid and/or maternal urine. Amniotic fluid MMA determination by the stable isotope dilution technique provides a highly accurate technique for prenatal diagnosis of methylmalonic acidemia that is complementary to amniotic fluid cell studies, and may supplant such studies in selected instances.Speculation: The stable isotope dilution method may be extended to prenatal diagnosis of other inborn errors of metabolism in which low molecular weight metabolites accumulate in amniotic fluid. The concentrations of metabolites, which accumulate in some of these disorders, are below the sensitivity limits of currently available analytical techniques and it is thus impossible to distinguish differences that may be present in pregnancies carrying affected and unaffected fetuses. The enhanced sensitivity of the stable isotope dilution method may permit the desired discrimination.

Ethylmalonic/adipic aciduria: Effects of oral medium-chain triglycerides, carnitine, and glycine on urinary excretion of organic acids, acylcarnitines, and acylglycines

ABSTRACT: A 9-y-old girl with ethylmalonic/adipic aciduria was hospitalized to determine the possible therapeutic efficacy of oral carnitine and glycine supplementation. To provoke a mild metabolic stress, her diet was supplemented with 440 mg/kg/d of medium-chain triglycerides. She was treated successively with carnitine (100 mg/kg/d) for 5 d, neither carnitine nor glycine for 2 d, and then glycine (250 mg/kg/d) for 6 d. Consecutive 12-h urine collections were obtained throughout the entire period. The urinary excretion of eight organic acids, four acylglycines, and four acylcarnitines, which accumulate as a result of a metabolic block of five mitochondrial acyl-CoA dehydrogenases, were quantitatively determined by capillary gas chromatography, stable isotope dilution gas chromatography/mass spectrometry, and radioisotopic exchange HPLC, respectively. The excretion of each group of metabolites was calculated as the mean percentage of total output (μmol/24 h) during the four phases of the protocol (organic acids/acylglycines/acylcarnitines = 100.0%): 1) regular diet (3 d); 88.1/10.8/1.1; 2) medium-chain triglyceride supplementation (4); 82.5/15.6/1.9; 3) medium-chain triglycerides plus carnitine (5); 79.2/8.2/12.6; and 4) medium-chain triglycerides plus glycine (6); 81.0/18.7/0.3. Comparison between total and individual excretion of acylglycines and acylcarnitines indicates that oral glycine supplementation enhanced the conjugation and excretion of fatty acyl-CoA intermediates as efficiently as carnitine. We propose that oral glycine supplementation should be considered in the treatment of other inborn errors of metabolism associated with abnormal urinary excretion of acylglycines.

Molecular analysis of 46,XY females and regional assignment of a new Y-chromosome-specific probe.

SummaryThe relationship between Y-chromosome abnormalities and gonadal differentiation was investigated in six phenotypic females with a 46,XY karyotype and one patient with ambiguous genitalia secondary to apparently nonmosaic 46,XY mixed gonadal dysgenesis. No alterations were found in the Y chromosomes of six of these individuals by the use of either cytogenetic or molecular techniques. Cytogenetic analysis with high-resolution G-banding and Q-banding revealed a small deletion in the short arm of the Y chromosome in one female patient with some features of Turner syndrome. Southern hybridization with Y-specific probes showed a loss of DNA within deletion intervals 1, 2, and 3 of the Y chromosome. A new Y-chromosome-specific DNA probe that hybridizes to deletion interval 3 is described.

A 13-year-old boy with cognitive impairment, retinoblastoma, and Wilson disease

A developmentally delayed child manifested retinoblastoma at age 4 years and Wilson disease at age 11, a previously unreported association. Cytogenetic and molecular analysis showed an interstitial deletion in the long arm of the paternally derived homologue of chromosome 13 (13q14.2-13q22.2), which encompasses the retinoblastoma and Wilson disease loci. The authors postulate that the co-occurrence of retinoblastoma and Wilson disease was the consequence of an acquired somatic mutation at the retinoblastoma locus and an inherited mutation at the Wilson disease locus of the maternally derived chromosome 13, superimposed on the hemizygosity associated with the paternally derived deletion.