Just two years ago, the origins of the fatal childhood neurological disorders called Batten disease were shrouded in mystery, and there were few prospects for effective treatment. Now, for the first time, researchers can describe the genetic underpinnings of all major childhood forms of the disease.
The discovery of the gene and protein responsible for most cases of late infantile Batten disease is reported in the September 19, 1997, issue of Science(1). The finding allows the first reliable diagnosis and carrier testing for the disease and is the first step toward developing an effective treatment. Ultimately, the finding also may yield new insights into the aging process. The work was supported in part by the National Institute of Diabetes and Digestive and Kidney Disorders (NIDDK) and the National Institute of Neurological Disorders and Stroke (NINDS).
A research team led by Peter Lobel, Ph.D., and David E. Sleat, Ph.D., of the Center for Advanced Biotechnology and Medicine in Piscataway, New Jersey, located the gene using a newly developed biochemical approach to identify the enzyme missing in the disease. The center is a joint program of the University of Medicine and Dentistry of New Jersey (UMDNJ) and Rutgers University. The scientists then compared this enzyme to known proteins and gene segments that are described in databases available through the National Center for Biotechnology Information at the National Library of Medicine. This allowed them to determine the enzyme?s probable function.
The newly discovered gene, CLN2, codes for a type of enzyme that has never before been described in humans or other mammals. Nonetheless, it appears to be one of the most common protein-degrading enzymes (proteases) in the brain, says Raju K. Pullarkat, Ph.D., of the New York State Institute for Basic Research in Developmental Disabilities in Staten Island. The protein also is normally produced in the heart, lung, kidney, and many other organs. Dr. Pullarkat has studied Batten disease for nearly 20 years and collaborated on the study that led to identification of the CLN2 gene.
"Now that we know the protein exists in humans, we can study how it normally works and look for its potential role in more common neurological disorders," says Dr. Lobel, of UMDNJ-Robert Wood Johnson Medical School. "It?s a string, and we can start to pull on it."
"This discovery of a novel protease is exciting. It's the kind of basic science finding that can lead to treatment for devastating genetic disorders like Batten disease," said Catherine McKeon, Ph.D., of the National Institute of Diabetes and Digestive and Kidney Diseases.
Late infantile Batten disease, also called Jansky-Bielchowsky disease or late infantile neuronal ceroid lipofuscinosis (LINCL), is one of four closely related metabolic disorders that lead to neurodegeneration. The four diseases are often collectively called Batten disease, although their technical name is neuronal ceroid lipofuscinoses (NCLs). The other major childhood forms include infantile NCL (also called Santavuori-Haltia disease) and juvenile NCL (Batten disease or Spielmeyer-Vogt-Sjogren disease). Genes for both of these disorders were identified in 1995. An unusual adult form of NCL (Kufs disease) also has been described.
Late infantile Batten disease affects about 300 children and accounts for about 40 percent of the NCL seen in the United States. Children with late infantile Batten disease appear normal until age two or three. Symptoms, which worsen over time, may include mental retardation, seizures, impaired coordination, dementia, and loss of speech and vision. There is no treatment that can alter the course of the disease, and most affected children die by age fifteen.
To be affected with any form of NCL, a child must receive two defective copies of the gene, one from each parent. People with only one defective copy of the gene do not develop the disease, but can pass the gene on to their children.
In the NCLs, a fluorescent pigment accumulates in bubble-like compartments called lysosomes that are found within cells. Lysosomes act as the cell?s recycling centers, taking in waste products and complex molecules and breaking them down into simple components that can be reused or eliminated. In LINCL, one of the many lysosomal enzymes that work together to degrade proteins is missing and the normal breakdown process is interrupted. This results in a buildup of protein and abnormal deposits of the pigment called ceroid lipofuscin, which appears greenish-yellow when viewed through a microscope in ultraviolet light.
Defects in lysosomal function have been linked to many other disorders, including Tay-Sachs disease. "The identification of this protein in humans provides a new avenue for investigators to explore that may lead to a better understanding of other disorders in this family," says Judy A. Small, Ph.D., of the Division of Fundamental Neuroscience and Developmental Disorders at NINDS.
The new approach used to identify the gene is based on knowledge that, when lysosomal enzymes are produced, they almost always contain a special sugar called mannose 6-phosphate. This sugar helps the cell direct the enzymes to the lysosomes. The normal brain contains about 75 of these enzymes, which appear as individual spots when spread out on paper in one kind of laboratory test. However, the brains of patients with late infantile Batten disease are missing one of these spots. By isolating and sequencing the missing enzyme and comparing it to known genes and proteins, the scientists pieced together a nearly complete sequence for the gene and mapped it to chromosome 11.
This is the first time researchers have used the mannose-6-phosphate strategy to identify a defective gene. For LINCL, this strategy proved faster than the common approach, called positional cloning, which is used to identify which of the body?s 100,000 genes are defective. While powerful, positional cloning can be extremely laborious, as researchers must examine many potential genes. By looking only at lysosomal enzymes, the investigators simplified this problem and rapidly identified the disease gene. They think this approach may be useful in identifying other lysosomal enzymes and determining which ones are missing in human diseases.
The most critical question in LINCL is what causes the massive neuronal death seen in the disease. As many as 50 or 60 percent of the brain?s neurons may die as the disease progresses, says Dr. Pullarkat. One possibility is that the accumulation of undigested proteins may harm the neurons. Since neurons don?t divide as other cells do, they are unable to reduce their load of undigested proteins. A second possibility is that the normal enzyme is required to produce something neurons need to survive, such as a nerve growth factor.
The researchers are now developing DNA and protein-based tests that will improve diagnosis and carrier testing for LINCL. The finding also opens the door to gene therapy and enzyme therapy for the disease. Preliminary studies of enzyme therapy have been partially successful for Gaucher?s disease, another metabolic disease caused by a defective lysosomal enzyme. However, researchers must overcome many technological problems and find a way to circumvent the blood-brain barrier for gene therapy or enzyme therapy to be successful for Batten disease.
Discovery of this and other proteins involved in Batten disease may ultimately yield new insights about how humans age. Previous studies have suggested that buildup of substances within lysosomes may cause the termination of cell division that occurs during aging. The lipofuscin that accumulates within lysosomes in Batten disease is similar in some ways to the lipofuscin that accumulates in aged cells, says Dr. Pullarkat. This buildup of lipofuscin suggests that, in both aging and Batten disease, the cellular disposal system gets clogged and slows down.
The NINDS and the NIDDK are part of the National Institutes of Health located in Bethesda, Maryland. The NINDS is the nation's leading supporter of research on the brain and nervous system and a lead agency in the Congressionally designated Decade of the Brain. The NIDDK leads the federal research effort on metabolic diseases, including diabetes and genetic metabolic diseases.
(1) Sleat DE, Donnelly RJ, Lackland H, Liu C-G, Sohar I, Pullarkat RK, Lobel P. "Associations of mutations in a lysosomal protein with classical late infantile neuronal ceroid lipofuscinosis." Science, Vol. 277, No. 5333, September 19, 1997, pp. 1802-1805.
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Science