National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

Overview of Rare Diseases Research Activities

As more is learned about disease mechanisms, through basic and clinical research, diseases that were once thought to be single entities have been demonstrated to be a collection of diseases with heterogeneous etiologies. These “new” diseases are often in a prevalence range that qualifies them as rare diseases (less than 200,000 in the U.S. population). This has been the case with almost all of the diseases and conditions for which NIDDK is responsible: diabetes and endocrine, metabolic, digestive, kidney, urologic and hematologic diseases. The Diabetes, Endocrinology, and Metabolic Diseases Division not only encompasses rarer subtypes of very common clinical entities but also, in its Inborn Errors of Metabolism Program, supports research into some of the several thousand genetic metabolic diseases. During the almost 50 years of its existence, NIDDK has been on the cutting edge of research on molecular and intracellular mechanisms of disease and has consistently found that basic knowledge obtained in the study of one disease will directly or indirectly assist and advance the study of other diseases. This is especially true with the rare disease because the genes responsible are isolated, leading to the elucidation of novel cellular mechanisms.

Recent Scientific Advances in Rare Diseases Research

Cystic Fibrosis

Improved therapy has transformed cystic fibrosis (CF) from a disease characterized by death in early childhood to a chronic illness, with most patients living to adulthood. In 1997, the FDA approved the use of an inhaled antibiotic that helps control lung infections and reduces the need for hospitalization in patients with CF. This year a randomized, placebo-controlled trial demonstrated the efficacy of this treatment in preserving lung function. Tobramycin is an antibiotic used to treat the chronic lung infections from Pseudomonas aeruginosa. CF patients with P. aeruginosa infection were divided into two groups. The treatment group received 300 mg of aerosolized tobramycin twice a day for 28 days and no treatment for 28 days for 3 cycles. The control group received a taste-matched placebo. The treated group showed a 10% improvement in FEV1, a measure of lung function, whereas the placebo group demonstrated a 2% decrease. This improvement was maintained throughout the 24-week study. In the treated group, however, density of P. aeruginosa in sputum decreased when patients were on tobramycin but returned almost to baseline during the 28-day period when tobramycin was not administered. Patients on the treatment also showed a small decrease in hospitalizations. This study suggests that this treatment, used in combination with other therapies, is beneficial in controlling infections, thus preserving lung function and reducing hospitalizations in CF.

CF is caused by mutations in CFTR, the gene for a chloride channel. The assembly of CFTR into a functional chloride channel at the cell surface has been further elucidated this year. When CFTR is assembled at the cell surface, there is uncertainty about how many molecules of CFTR compose a single chloride channel. To study this question, NIDDK-funded investigators linked two molecules. The fused CFTR dimers were transported to the cell surface and functioned normally. These findings have led investigators to conclude that two CFTR molecules form a single pore for chloride transport at the cell surface. Agents that can enhance cross-linking of two CFTR molecules could be useful in the treatment of CF.

Sandhoff Disease

One method for treating patients with genetic defects in metabolism is to restrict their diets to reduce the substance that cannot be metabolized. Another approach is to reduce the substrates that are used to synthesize these molecules; by limiting the synthesis, the accumulation that is so destructive in many storage diseases is prevented. An NIDDK intramural investigator has used mouse models to examine substrate deprivation therapy in the glycosphingolipid (GSL) pathway. The investigator had already created a mouse model of Sandhoff disease, which has a block in the GSL degradation pathway and accumulates GSLs; this mouse showed storage of GSL in neurons causing neurodegeneration and shortened lifespan. The investigator then introduced a block in the pathway for synthesis of GSL by mutating acetylgalactosaminyltransferase to see whether this had negative consequences. Overall, the mice appeared normal but demonstrated a complete absence of complex ganglioside and decreased myelination. These mice were crossed with the Sandhoff mice to obtain a double mutant homozygous mouse devoid of GSL storage. Compared to the Sandhoff mice, the double mutant mice had an increased lifespan and a delay in the appearance of symptoms of neurodegeneration such as ataxia. This genetic model mimics pharmacologic blocks of synthesis pathways and shows that this can be an effective treatment to ameliorate the symptoms of the disease.

Mucopolysaccharidosis

One of the challenges in gene therapy is to develop ways to deliver therapies to the brain, which is protected by the blood-brain barrier. Several groups have been developing gene therapy approaches using an animal model of mucopolysaccharidosis VII. This model has been used extensively because the mouse model is an accurate representation of the disease manifested in humans. The mouse model shows the classic clinical features, including liver and spleen enlargement, brain and corneal storage, and bone malformations. In addition, there is a dog model of this disease that could be used to scale up the treatment. Mucopolysaccharidosis type VII is caused by the absence of the enzyme ß-glucuronidase (GUSB). The most promising gene therapy vector is based on adenoassociated virus (AAV), which has never been shown to be pathogenic in humans. When AAV carrying the GUSB gene was administered intravenously, decreased storage of glycoaminoglycans could be demonstrated in both the liver and spleen; however, glycoaminoglycan storage in the brain was unaffected.

This year, several groups are experimenting with ways to treat the brain pathology. Most patients with Mucopolysaccharidosis are diagnosed after the newborn period. One approach was to see if stereotactic injection into the brain could deliver enough vector to reverse the pathology. In the mouse brain, this treatment could provide enough vector to clear most of the glycoaminoglycan storage from the brain for the 5 months of the study. Some of the cells were corrected as a result of cross-correction, a phenomenon whereby neighboring cells can endocytose the ß-glucuronidase that has been secreted by a cell. Again, this is a promising finding; however, numerous injections would be needed to scale this technique up to a human because the human brain is about 100 times the size of a mouse. In yet another approach, AAV-GUSB was injected into the cerebral spinal fluid, and brain pathology was studied. In these mice, the brain was also cleared of glycoaminoglycan storage. This technique holds promise for scaling up to a human because one of the limiting factors in the mouse was the volume of vector that could be delivered, which should be less problematic in humans.

Crigler-Najjar Syndrome Type I

The application of gene therapies to liver diseases has been limited by technical problems in the introduction of the “corrected gene” into the host’s genome. A novel approach relying on the normal cellular mechanism of DNA repair has been proposed by NIDDK investigators. This technique employs a novel chimeric RNA/DNA oligonucleotide (RNA/DNA ON) as the mediator of site-directed gene repair. Chimeric RNA/DNA ONs have now been used to correct site-specific mutations in vivo, circumventing many of the disadvantages associated with other strategies of gene therapies.

An attractive target for gene repair is the inherited deficiency of hepatic UDP-glucuronosyltransferase activity characteristic of Crigler-Najjar syndrome. Children born with this condition cannot conjugate bilirubin normally and rapidly develop marked elevations in serum-unconjugated bilirubin. If untreated, the condition leads to kernicterus and death within a few years. Current treatment requires 12 to 18 hours of light therapy daily and is only partially effective in preventing neurologic damage. Many patients undergo liver transplantation to correct the defect, even though the liver is normal in all respects except one. An excellent animal model of Crigler-Najjar syndrome is the “Gunn” rat, which has a nucleotide insertion in the UDP-glucuronosyltransferase gene. To correct the abnormal gene, RNA/DNA ONs have been developed specifically for the insertion of a single G in codon 1206 of the affected gene from cultured hepatocytes of Gunn rats. Most recently, these investigators have used the chimeric RNA/DNA ON to treat Gunn rats in vivo. Intravenous injection of the chimeric ON incorporated into liposomes or lactosylated protein leads to rapid distribution to the liver and incorporation into the nucleus. The rats were shown to have 15% to 20% correction of the abnormal gene and demonstrated a 50% decrease in serum bilirubin levels—a critically lifesaving amount in humans with this disease.

Hemochromatosis

Hereditary hemochromatosis is an autosomal recessive disorder characterized by progressive iron deposition and injury to the liver, heart, and endocrine organs caused by excessive dietary iron absorption. Approximately 85% of persons with hereditary hemochromatosis have a single gene mutation in the HFE gene on the short arm of chromosome 6 (C282Y). The function of the HFE gene is unknown, and the use of HFE gene screening for early detection of hemochromatosis is still being evaluated.

Insight has been gained into the role of the HFE gene in the regulation of absorption of dietary iron in the duodenum. Using a knockout mouse model of hemochromatosis (HFE-/-), investigators provide evidence that the mouse with deficient HFE expression accumulates excess iron in the liver and other tissues but nevertheless has a 7- to 8-fold increase in duodenal divalent metal transporter 1 (DMT1) mRNA, the major transporter of iron in the gastrointestinal tract. This lack of down-regulation of the iron transporter appears to result from the lack of colocalization of HFE with the transferrin receptor on the basolateral surface of intestinal crypt cells. The authors conclude that the HFE gene is the major regulator of the iron-sensing mechanism in the body that ordinarily decreases iron absorption when iron stores are replete.

Preliminary results on the clinical implications of HFE have (1) identified a large family with hereditary hemochromatosis who have a normal HFE gene and whose familial condition is not linked to HLA types and thus not present on the short arm of chromosome 6 (suggesting that the 15% of patients with hemochromatosis who have a normal HFE gene probably have another genetic cause of their iron overload); and (2) documented that 0.5% of the population are homozygous for the C282Y gene mutation in HFE, and 95% would have been identified by population screening using iron saturation as the screening test. The population-based study provides a simple and clinically useful algorithm for the evaluation and treatment of iron overload with or without HFE mutations. These studies were extensions of the research recommendations from the May 14–15, 1998, symposium on hemochromatosis sponsored by NIDDK, “Molecular Medicine and Hemochromatosis: At the Crossroads.”

Recessive Form of Polycystic Kidney Disease

Autosomal recessive polycystic kidney disease (ARPKD) is an important and often devastating form of polycystic kidney disease (PKD) that presents primarily in infancy and childhood. It involves the kidneys and the biliary tract, with an incidence of 1 in 20,000 live births. Thirty percent to 50% of affected neonates die within a few hours of birth. In affected children who survive the perinatal period, the principal causes of morbidity and mortality are progressive renal insufficiency and systemic and portal hypertension. The polycystic kidney and hepatic disease 1 (PKHD1) gene was initially mapped to 6p21.1-p12 in families primarily manifesting the less severe juvenile phenotype. A high-resolution sequence-ready contig map of the region has been developed for the positional cloning of PKHD1.

Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases. ADPKD is a disease of gradual onset, with major symptoms occurring in late middle life. The disease leads to cystic replacement of renal tissue and progressive renal failure in about half of the cases. ADPKD is caused by mutations in at least three genes. Two genes, polycystin-1 causing PKD1 and polycystin-2 causing PKD2, have been mapped to chromosomes 16 and 4, respectively. The function of the polycystins remains unknown. Mutations in these two genes have similar phenotypes; however, PKD1 patients have a more severe form of disease, with earlier onset and more rapid progression. Studies of the disease necessitate the investigation of the molecular and cellular pathology in both fetal and adult organs; animal models allow the sequence of molecular events that precede cyst formation to be studied in detail.

It was previously reported that targeted deletion of exon 34 in polycystic kidney disease 1 (PKD1), the mouse homologue of PKD1, results in renal cysts and perinatal death in homozygotes. It is now being reported that the null (Pkd1+/-) mice progressively develop scattered renal and hepatic cysts. Serial sections of 15 Pkd1+/- of mice 9–14 months of age, revealed cysts of more than 5 times the normal tubule diameter in 80% of the animals; after 16 months, 100% of the animals had developed innumerable bilateral cysts. Renal excretory function was normal in all, with the exception of one mouse with extensive disease. There were no cysts in age-matched control littermates. The phenotype of Pkd1+/- mice recapitulates the renal and hepatic phenotypes of human ADPKD. The gradual development of cysts and the absence of polycystin-1 in some cysts are consistent with clinical progression in humans and with the “two-hit hypothesis of cyst development,” providing a relevant model of ADPKD pathophysiology. This model provides an entry point into the pathways that lead to aberrant epithelial development. It should also be useful for the genetic testing of the intersection of various cystic pathways between the Pkd1 mutations and other mutations. These mice will likely be useful testing therapeutic approaches to autosomal dominant PKD as well as in the further design of therapeutic strategies.

Polycystins are membrane proteins that share significant sequence homology. In addition to the two members of the polycystin family (polycystin-1 and polycystin-2) that are mutated in ADPKD, there is polycystin-L, which is highly homologous to polycystin-2 and deleted in mice with renal and retinal defects. It has now been shown that polycystin-L is a calcium-modulated, calcium-permeable nonselective cation channel that is permeable to Na+, K+, and Ca++ ions. Patch-clamp experiments revealed single-channel activity. Channel activity was substantially increased when either the extracellular or intracellular ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signaling in vivo. Its ion selectivity, large single-channel conductance and long open time distinguish it from other structurally related channels of the transient receptor potential family, and voltage gated Ca++ and Na+ channels. It is hypothesized that alteration of these channels leads to the abnormal fluid secretion and cellular proliferation that are hallmarks of PKD. The elucidation of the channel properties of polycystins should provide new therapeutic strategies for PKD.

Friedreich’s Ataxia

Frataxin, a mitochondrial protein, is defective in Friedreich’s ataxia (FRDA), an autosomal recessive disease that is the most common form of inherited spinocerebellar ataxia. Recent studies have demonstrated that frataxin is required for mitochondrial export of iron and that mutations in the nuclear gene encoding the mitochondrial protein frataxin are responsible for FRDA. Yeast strains with a deletion in the frataxin gene homologue YFH1 accumulate excess iron in mitochondria and evidence mitochondrial damage.

New research shows that in the absence of YFH1, mitochondrial damage is proportional to the concentration and duration of exposure to extracellular iron, establishing mitochondrial iron accumulation as causal to mitochondrial damage. Reintroduction of YFH1 results in the rapid export of accumulated mitochondrial iron into the cytosol as free, non-heme bound iron, demonstrating that mitochondrial iron in the yeast FA model can be made bioavailable. These results demonstrate a mitochondrial iron cycle in which the gene product Yfh1p regulates mitochondrial iron efflux.

The observation that the YFH1 gene product Yfh1p affects iron efflux indicates that under normal conditions, iron can both enter and exit yeast mitochondria, suggesting the existence of a mitochondrial iron cycle. The identification of excess mitochondrial iron as the cause of FRDA may allow the development of a treatment regimen based on iron chelation. Factors regulating the expression of YFH1 need to be identified and their role under conditions of varying iron availability examined. The elements involved in the mitochondrial iron cycle need to be elucidated. A protocol for treatment of Friedreich’s ataxia patients using iron chelation is being examined in a pilot study.

Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an anemia with decreased or absent red blood cell precursor cells (erythroblasts) in the bone marrow, but otherwise normal cells. Until now, the basic genetic molecular defect in DBA has remained unclear, even though a gene responsible for DBA was genetically mapped. Because DBA represents a specific differentiation arrest of the erythroid lineage, it is reasonable to suggest that the mutation results in a deficient protein with a key regulatory role in erythropoiesis. Investigators have now cloned and characterized the chromosomal region associated with DBA and have found that the critical gene encodes the ribosomal protein S19. The ribosomal protein S19 was sequenced, and the DNA of 40 unrelated patients with DBA was screened for mutations. Ten of the patients (25%) were found to have mutations in S19. This is the first reported direct demonstration of a human disease resulting from a mutation in a ribosomal protein—although this had already been speculated on the basis of abnormal invertebrate development traced to mutations in homologous ribosomal protein genes. Ribosomes are cytoplasmic organelles that provide the structural backbone for the synthesis of proteins in response to genetic instructions.

Rare Diseases Research Initiatives

NIDDK issued an RFA (DK-97-010) for Gene Therapy Core Centers for Cystic Fibrosis and Other Genetic Diseases. The purpose of this initiative to provide shared resources to enhance the efficiency of research and foster collaborations within and among institutions with strong existing bases of research relevant to gene therapy of genetic diseases. This program is cosponsored by the Cystic Fibrosis Foundation, which will provide funds for up to 10 pilot and feasibility studies relevant to CF to each of these centers. NIDDK has funded two Core Centers in FY 1998 and two Core Centers in FY 1999 in response to this RFA.

NIDDK issued an RFA (DK-98-011) for Cystic Fibrosis Research Centers, which will foster multidisciplinary approaches to research ranging from elucidation of the molecular pathogenesis of CF to development of new therapies for CF. Two Centers were funded in FY 1999.

NIDDK issued an RFA (DK-00-013) for Cystic Fibrosis Core Centers. Applications are due June 16, 2000.

This year NIDDK has issued two RFAs to enhance research on polycystic kidney disease. PKD: Innovative Imaging to Assess Progression (DK-99-003) funded a Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease consisting of four Centers and a Coordinating Center. Interdisciplinary Centers for PKD Research (DK-99-012) requested applications for interdisciplinary research on PKD. Four Centers were funded in FY 1999.

Other RFA research solicitations in the rare diseases area include DK-99-015, Interstitial Cystitis Clinical Trials Group; DK-99-003, Biology of Iron Overload, and New Approach to Therapy; and HL97-013, Clinical Research on Cooley’s Anemia. Another current RFA (DK-00-008) is for Type 2 Diabetes in the Pediatric Population, a rare disease (compared with the incidence in adults) that is growing in importance.

Current program announcements on rare diseases include PA-99-040, Molecular and Genetic Mechanisms in Pancreatitis; PA-94-036, Characterization and Treatment of Genetic Metabolic Diseases; and PA-98-002, Polycystic Kidney Disease.

Rare Diseases-Related Program Activities

NIDDK collaborated with ORD to support a workshop on oxalosis (December 8–9, 1999), designed to report progress on developing treatments and to chart a course for research.

NIDDK collaborated with ORD to support a workshop on acute renal failure. Investigators met to design a pilot multicenter controlled trial. This meeting provided an opportunity to review the treatment for acute renal failure.

In collaboration with ORD, NIDDK has proposed funding a workshop on lipoatrophic diabetes and other lipodystrophic syndromes (scheduled for October 2000). These syndromes make up a collection of rare diseases associated with fat atrophy and insulin resistance, including polycystic ovarian syndrome.