Ghaleb Daouk

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.

Localization and genetic linkage of the human immunoglobulin heavy chain genes and the creatine kinase brain (CKB) gene: identification of a hot spot for recombination.

The immunoglobulin heavy chain (IGH) gene cluster and the gene coding for the brain form of the enzyme creatine kinase (CKB) have previously been localized to chromosome 14, at 14q32.3 and 14q32, respectively. Here we report more precise regional localization of these genes by dosage studies using DNA from a child hemizygous for the region from 14q32.32 to 14qter. CKB and IGH are present in a single dose in the proband. Dosage studies in a second patient with a similar but smaller deletion due to a ring chromosome 14 show that CKB is proximal to the IGH cluster. An EcoRI restriction site polymorphism was found with probes for the CKB gene. Linkage analysis of family data indicates that CKB is closely linked to IGH. Linkage analysis also revealed unusually high recombination (beta = 3.2%) between the C delta and C gamma 3 genes of the IGH constant region, which are only 60 kb apart. This finding, in combination with a previous observation of linkage equilibrium in the region, suggests that the C delta-C gamma 3 region contains a recombination hot spot.

Tuberous sclerosis complex and renal angiomyolipoma: case report and review of the literature

A 5-year-old boy with a known diagnosis of tuberous sclerosis complex was found to have an enlarging renal mass on routine ultrasound. He was diagnosed with an angiomyolipoma (AML) and scheduled for close observation. Follow-up magnetic resonance imaging demonstrated the AML to be significantly enlarged and hypervascular. Selective arterial embolization of the tumor was performed, which resulted in an appropriate decrease in tumor size. Angiomyolipoma is a known and well-described complication of the tuberous sclerosis complex that is usually found among patients in their adolescent and adult years. The case presented here illustrates the need for early and repeated renal imaging of younger pediatric patients with tuberous sclerosis. Our experience adds to the literature on young pediatric patients requiring embolization for treatment of large renal angiomyolipomas.

Exome Sequencing Discerns Syndromes in Patients from Consanguineous Families with Congenital Anomalies of the Kidneys and Urinary Tract

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the leading cause of CKD in children, featuring a broad variety of malformations. A monogenic cause can be detected in around 12% of patients. However, the morphologic clinical phenotype of CAKUT frequently does not indicate specific genes to be examined. To determine the likelihood of detecting causative recessive mutations by whole-exome sequencing (WES), we analyzed individuals with CAKUT from 33 different consanguineous families. Using homozygosity mapping and WES, we identified the causative mutations in nine of the 33 families studied (27%). We detected recessive mutations in nine known disease-causing genes: ZBTB24, WFS1, HPSE2, ATRX, ASPH, AGXT, AQP2, CTNS, and PKHD1 Notably, when mutated, these genes cause multiorgan syndromes that may include CAKUT as a feature (syndromic CAKUT) or cause renal diseases that may manifest as phenocopies of CAKUT. None of the above monogenic disease-causing genes were suspected on clinical grounds before this study. Follow-up clinical characterization of those patients allowed us to revise and detect relevant new clinical features in a more appropriate pathogenetic context. Thus, applying WES to the diagnostic approach in CAKUT provides opportunities for an accurate and early etiology-based diagnosis and improved clinical management.

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

Abstract The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP2−, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing NADPH NADP + concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the NADPH NADP + concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057) . It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.

Postnatal transient renal insufficiency in the feto-fetal transfusion syndrome

Abstract Twinning and higher-order multiple-gestation pregnancies have become relatively frequent in the current era of assisted reproductive techniques. Vascular interconnections are present in nearly all monochorionic twin placentae, yet hemodynamically significant arteriovenous anastomoses resulting in the feto-fetal transfusion syndrome occur in only 5%–18% of these. When arteriovenous connections through a shared placental cotyledon are present, variable amounts of blood may be transfused from one fetus to the other, and feto-fetal transfusion syndrome may result. While reports of renal failure due to a small non-functioning kidney in the donor infant pre- or postnatally have been published, recoverable renal insufficiency has not been previously delineated in feto-fetal transfusion syndrome. This article describes a case of postnatal transient renal insufficiency in a donor infant from a pair of monozygotic twins.

Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome

BACKGROUND AND OBJECTIVES Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. RESULTS In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. CONCLUSIONS Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.

Genetic variants in the LAMA5 gene in pediatric nephrotic syndrome

BACKGROUND Nephrotic syndrome (NS), a chronic kidney disease, is characterized by significant loss of protein in the urine causing hypoalbuminemia and edema. In general, ∼15% of childhood-onset cases do not respond to steroid therapy and are classified as steroid-resistant NS (SRNS). In ∼30% of cases with SRNS, a causative mutation can be detected in one of 44 monogenic SRNS genes. The gene LAMA5 encodes laminin-α5, an essential component of the glomerular basement membrane. Mice with a hypomorphic mutation in the orthologous gene Lama5 develop proteinuria and hematuria. METHODS To identify additional monogenic causes of NS, we performed whole exome sequencing in 300 families with pediatric NS. In consanguineous families we applied homozygosity mapping to identify genomic candidate loci for the underlying recessive mutation. RESULTS In three families, in whom mutations in known NS genes were excluded, but in whom a recessive, monogenic cause of NS was strongly suspected based on pedigree information, we identified homozygous variants of unknown significance (VUS) in the gene LAMA5. While all affected individuals had nonsyndromic NS with an early onset of disease, their clinical outcome and response to immunosuppressive therapy differed notably. CONCLUSION We here identify recessive VUS in the gene LAMA5 in patients with partially treatment-responsive NS. More data will be needed to determine the impact of these VUS in disease management. However, familial occurrence of disease, data from genetic mapping and a mouse model that recapitulates the NS phenotypes suggest that these genetic variants may be inherited factors that contribute to the development of NS in pediatric patients.

Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract

BACKGROUND Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patients CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.