Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.

Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

Steroid disorders in children: Congenital adrenal hyperplasia and apparent mineralocorticoid excess

Our research team and laboratories have concentrated on two inherited endocrine disorders, congenital adrenal hyperplasia (CAH) and apparent mineralocorticoid excess, in thier investigations of the pathophysiology of adrenal steroid hormone disorders in children. CAH refers to a family of inherited disorders in which defects occur in one of the enzymatic steps required to synthesize cortisol from cholesterol in the adrenal gland. Because of the impaired cortisol secretion, adrenocorticotropic hormone levels rise due to impairment of a negative feedback system, which results in hyperplasia of the adrenal cortex. The majority of cases is due to 21-hydroxylase deficiency (21-OHD). Owing to the blocked enzymatic step, cortisol precursors accumulate in excess and are converted to potent androgens, which are secreted and cause in utero virilization of the affected female fetus genitalia in the classical form of CAH. A mild form of the 21-OHD, termed nonclassical 21-OHD, is the most common autosomal recessive disorder in humans, and occurs in 1/27 Ashkenazic Jews. Mutations in the CYP21 gene have been identified that cause both classical and nonclassical CAH. Apparent mineralocorticoid excess is a potentially fatal genetic disorder causing severe juvenile hypertension, pre- and postnatal growth failure, and low to undetectable levels of potassium, renin, and aldosterone. It is caused by autosomal recessive mutations in the HSD11B2 gene, which result in a deficiency of 11β-hydroxysteroid dehydrogenase type 2. In 1998, we reported a mild form of this disease, which may represent an important cause of low-renin hypertension.

Evidence of evolutionary up-regulation of the single active X chromosome in mammals based on Clc4 expression levels in Mus spretus and Mus musculus

Previous studies have shown that the chloride channel gene Clc4 is X-linked and subject to X inactivation in Mus spretus, but that the same gene is autosomal in laboratory strains of mice. This exception to the conservation of linkage of the X chromosome in one of two interfertile mouse species was exploited to compare expression of Clc4 from the X chromosome to that from the autosome. Clc4 was found to be highly expressed in brain tissues of both mouse species. Quantitative analyses of species-specific expression of Clc4 in brain tissues from mice resulting from M. spretus × laboratory strain crosses, demonstrate that each autosomal locus has half the level of Clc4 expression as compared with the single active X-linked locus. In contrast expression of another chloride channel gene, Clc3, which is autosomal in both mouse species is equal between alleles in F1 animals. There is no evidence of imprinting of the Clc4 autosomal locus. These results are consistent with Ohno’s hypothesis of an evolutionary requirement for a higher expression of genes on the single active X chromosome to maintain balance with autosomal gene expression [Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes (Springer, Berlin)].

Sex-specific quantitative trait loci affecting longevity in Drosophila melanogaster

Senescence, the decline in survivorship and fertility with increasing age, is a near-universal property of organisms. Senescence and limited lifespan are thought to arise because weak natural selection late in life allows the accumulation of mutations with deleterious late-age effects that are either neutral (the mutation accumulation hypothesis) or beneficial (the antagonistic pleiotropy hypothesis) early in life. Analyses of Drosophila spontaneous mutations, patterns of segregating variation and covariation, and lines selected for late-age fertility have implicated both classes of mutation in the evolution of aging, but neither their relative contributions nor the properties of individual loci that cause aging in nature are known. To begin to dissect the multiple genetic causes of quantitative variation in lifespan, we have conducted a genome-wide screen for quantitative trait loci (QTLs) affecting lifespan that segregate among a panel of recombinant inbred lines using a dense molecular marker map. Five autosomal QTLs were mapped by composite interval mapping and by sequential multiple marker analysis. The QTLs had large sex-specific effects on lifespan and age-specific effects on survivorship and mortality and mapped to the same regions as candidate genes with fertility, cellular aging, stress resistance and male-specific effects. Late age-of-onset QTL effects are consistent with the mutation accumulation hypothesis for the evolution of senescence, and sex-specific QTL effects suggest a novel mechanism for maintaining genetic variation for lifespan.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Abnormal skeletal patterning in embryos lacking a single Cbp allele: A partial similarity with Rubinstein–Taybi syndrome

CBP is a transcriptional coactivator required by many transcription factors for transactivation. Rubinstein–Taybi syndrome, which is an autosomal dominant syndrome characterized by abnormal pattern formation, has been shown to be associated with mutations in the Cbp gene. Furthermore, Drosophila CBP is required in hedgehog signaling for the expression of decapentapleigic, the Drosophila homologue of bone morphogenetic protein. However, no direct evidence exists to indicate that loss of one copy of the mammalian Cbp gene affects pattern formation. Here, we show that various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice resulting from a C57BL/6-CBA × BALB/c cross. In support of a conserved signaling pathway for pattern formation in insects and mammals, the expression of Bmp7 was found to be reduced in the heterozygous mutants. The frequency of the different abnormalities was significantly lower in a C57BL/6-CBA background, suggesting that the genetic background is an important determinant of the variability and severity of the anomalies seen in Rubinstein–Taybi syndrome patients.

DNA typing in thirty seconds with a microfabricated device

We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microfluidic electrophoresis device. We have achieved baseline-resolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci CSF1PO, TPOX, THO1, and vWA (abbreviated as CTTv) were performed in less than 2 min. This constitutes a 10- to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 μm × 100 μm in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50°C. A fluorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 μl was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (α, β, and γ). In human, autosomal recessive mutations of α, β, or γENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of αENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of αENaC driven by a cytomegalovirus promoter in αENaC(−/−) knockout mice [αENaC(−/−)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na+ transport in renal, colonic, and pulmonary epithelia. At days 5–9, however, αENaC(−/−)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult αENaC(−/−)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na+ absorption in this transgenic mouse results in a mouse model for PHA-1.

The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway.

A deletion within the murine Werner syndrome helicase induces sensitivity to inhibitors of topoisomerase and loss of cellular proliferative capacity

Werner syndrome (WS) is an autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases. The gene responsible for WS encodes a member of the RecQ-like subfamily of DNA helicases. Here we show that its murine homologue maps to murine chromosome 8 in a region syntenic with the human WRN gene. We have deleted a segment of this gene and created Wrn-deficient embryonic stem (ES) cells and WS mice. While displaying reduced embryonic survival, live-born WS mice otherwise appear normal during their first year of life. Nonetheless, although several DNA repair systems are apparently intact in homozygous WS ES cells, such cells display a higher mutation rate and are significantly more sensitive to topoisomerase inhibitors (especially camptothecin) than are wild-type ES cells. Furthermore, mouse embryo fibroblasts derived from homozygous WS embryos show premature loss of proliferative capacity. At the molecular level, wild-type, but not mutant, WS protein copurifies through a series of centrifugation and chromatography steps with a multiprotein DNA replication complex.

Substantial narrowing of the Niemann–Pick C candidate interval by yeast artificial chromosome complementation

Niemann–Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11–12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

A CDKN2-like polymorphism in Xiphophorus LG V is associated with UV-B-induced melanoma formation in platyfish–swordtail hybrids

The genetic basis of spontaneous melanoma formation in spotted dorsal (Sd) Xiphophorus platyfish–swordtail hybrids has been studied for decades, and is adequately explained by a two-gene inheritance model involving a sex-linked oncogene, Xmrk, and an autosomal tumor suppressor, DIFF. The Xmrk oncogene encodes a receptor tyrosine kinase related to EGFR; the nature of the DIFF tumor suppressor gene is unknown. We analyzed the genetic basis of UV-B-induced melanoma formation in closely related, spotted side platyfish–swordtail hybrids, which carry a different sex-linked pigment pattern locus, Sp. We UV-irradiated spotted side Xiphophorus platyfish–swordtail backcross hybrids to induce melanomas at frequencies 6-fold higher than occur spontaneously in unirradiated control animals. To identify genetic determinants of melanoma susceptibility in this UV-inducible Xiphophorus model, we genotyped individual animals from control and UV-irradiated experimental regimes using allozyme and DNA restriction fragment length polymorphisms and tested for joint segregation of genetic markers with pigmentation phenotype and UV-induced melanoma formation. Joint segregation results show linkage of a CDKN2-like DNA polymorphism with UV-B-induced melanoma formation in these hybrids. The CDKN2-like polymorphism maps to Xiphophorus linkage group V and exhibits recombination fractions with ES1 and MDH2 allozyme markers consistent with previous localization of the DIFF tumor suppressor locus. Our results indicate that the CDKN2-like sequence we have cloned and mapped is a candidate for the DIFF tumor suppressor gene.

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.