3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of α (biotin-containing) and β subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCCα and MCCβ subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted “CGA” and “CGB.” We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCCα. Two MCCB missense mutations and one splicing defect mutation leading to early MCCβ truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCCβ truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the α and β subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

The nemaline myopathies are characterized by weakness and eosinophilic, rodlike (nemaline) inclusions in muscle fibers. Amish nemaline myopathy is a form of nemaline myopathy common among the Old Order Amish. In the first months of life, affected infants have tremors with hypotonia and mild contractures of the shoulders and hips. Progressive worsening of the proximal contractures, weakness, and a pectus carinatum deformity develop before the children die of respiratory insufficiency, usually in the second year. The disorder has an incidence of ∼1 in 500 among the Amish, and it is inherited in an autosomal recessive pattern. Using a genealogy database, automated pedigree software, and linkage analysis of DNA samples from four sibships, we identified an ∼2-cM interval on chromosome 19q13.4 that was homozygous in all affected individuals. The gene for the sarcomeric thin-filament protein, slow skeletal muscle troponin T (TNNT1), maps to this interval and was sequenced. We identified a stop codon in exon 11, predicted to truncate the protein at amino acid 179, which segregates with the disease. We conclude that Amish nemaline myopathy is a distinct, heritable, myopathic disorder caused by a mutation in TNNT1.

The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, gamma-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated.

To establish a genetic linkage between highly polymorphic microsatellite loci and the disease locus responsible for an autosomal recessive neurodegenerative syndrome that causes posterior column ataxia and retinitis pigmentosa.

Glutaric aciduria type I (GA1) is a preventable cause of acute brain damage in early childhood, leading to a severe dystonic-dyskinetic disorder that is similar to cerebral palsy and ranges from extreme hypotonia to choreoathetosis to rigidity with spasticity. Degeneration of the putamen and caudate typically occurs between 6 and 18 months of age and is probably linked to changes in metabolic demand caused by normal maturational changes and superimposed catabolic stress. Recognition of this biochemical disorder before the brain has been injured is essential to outcome. Diagnosis depends upon the recognition of relatively non-specific physical findings such as hypotonia, irritability and macrocephaly, and on performance of urine organic acid quantification by gas chromatography–mass spectrometry or selective searches of urine or blood specimens by tandem mass spectrometry for glutarylcarnitine. The diagnosis may also be suggested by characteristic findings on neuroimaging. In selected patients diagnosis can only be reached by enzyme assay. Specific current management by the authors of this paper includes pharmacological doses of L-carnitine, as well as dietary protein restriction. Metabolic decompensation must be treated aggressively to avoid permanent brain damage. Multicentre studies are needed to establish best methods of diagnosis and optimal therapy of this disorder.

Isolated 3-methylcrotonyl coenzyme A carboxylase (MCC) deficiency was documented in four adult women from the Amish/Mennonite population of Lancaster County, Pennsylvania. Metabolic and enzymatic investigations in these individuals were instituted after the detection of abnormal acylcarnitine profiles in blood spots obtained from their newborn children, in whom MCC activity was normal.

Genetic studies of complex hereditary disorders require for their mapping the determination of genotypes at several hundred polymorphic loci in several hundred families. Because only a minority of markers are expected to show linkage and association in family data, a simple screen of genetic markers to identify those showing linkage in pooled DNA samples can greatly facilitate gene identification. All studies involving pooled DNA samples require the comparison of allele frequencies in appropriate family samples and subsamples. We have tested the accuracy of allele frequency estimates, in various DNA samples, by pooling DNA from multiple individuals prior to PCR amplification. We have used the ABI 377 automated DNA sequencer and GENESCAN software for quantifying total amplification using a 58 fluorescently labeled forward PCR primer and relative peak heights to estimate allele frequencies in pooled DNA samples. In these studies, we have genotyped 11 microsatellite markers in two separate DNA pools, and an additional four markers in a third DNA pool, and compared the estimated allele frequencies with those determined by direct genotyping. In addition, we have evaluated whether pooled DNA samples can be used to accurately assess allele frequencies on transmitted and untransmitted chromosomes, in a collection of families for fine-structure gene mapping using allelic association. Our studies show that accurate, quantitative data on allele frequencies, suitable for identifying markers for complex disorders, can be identified from pooled DNA samples. This approach, being independent of the number of samples comprising a pool, promises to drastically reduce the labor and cost of genotyping in the initial identification of disease loci. Additional applications of DNA pooling are discussed. These developments suggest that new statistical methods for analyzing pooled DNA data are required.