Doctors In Pennsylvania Dutch Country Find New Genetic Links To Half Dozen Diseases In Past Year

Microarray-Based SNP Analysis Provides Researchers with Faster, Cheaper and More Definitive Method to Identify and Diagnose Genetic Disorders

In a corner of Pennsylvania Dutch country, doctors at the Clinic for Special Children are using Affymetrix GeneChip microarrays to discover the genetic causes of disease at an unprecedented pace -- more than a half dozen diseases in the past 12 months. In last month's early on-line issue of American Journal of Medical Genetics, the small group of physicians reported their most recent discovery: a mutated gene causing a mysterious developmental disorder in the Mennonite population.

The non-profit, community-supported Clinic -- part of their budget comes from their annual quilt sale -- is staffed by medical director Holmes Morton, MD, pediatrician Kevin Strauss, MD, and geneticist Erik Puffenberger, PhD. Morton and his wife Caroline started the clinic nearly 20 years ago to try to find treatments for a variety of rare genetic diseases that have long afflicted children in Plain sect communities, such as the Amish. In just the past year though, using a new GeneChip microarray technology from Affymetrix, Morton and colleagues have made breakthrough discoveries for genetic causes of at least six different diseases. While these scientists are studying diseases far more common in the Plain sects, their findings have worldwide ramifications, providing the basis for genetic tests of rare diseases in the general population.

The most recent discovery began when the parents of a 4 year-old girl suffering from slow growth and developmental delay informed the Clinic doctors of a cousin with similar symptoms, raising suspicion that the disease was hereditary. While recent estimates place about 25,000 genes in the human genome, the team narrowed their search to just 55 genes on chromosome 6 by using GeneChip Mapping 10K microarrays to scan 10,000 genetic variations from the two affected cousins and each of their parents. The team suspected two of the genes were involved in the disease and found that one of them -- SLC17A5 -- was mutated in the developmentally-delayed patients. The mutation turned out to be the identical one that causes Salla disease in the Finnish population; the Clinic physicians were then able to diagnose Salla disease in 4 additional individuals by testing for the SLC17A5 mutation directly in patients who had similar disease symptoms.

"Our most recent study using GeneChip arrays took less than one month to complete and cost less than $4,000 dollars to make a definitive diagnosis of Salla disease," said Strauss. "That same patient had undergone four years of standard medical exams, totaling nearly $20,000 dollars, but the diagnosis was elusive until we looked at her genome with the 10K microarray."

Affymetrix Microarray Technology
The key to these doctors' genetic discoveries is that they used the 10K microarray to look at the genome more quickly, more affordably, and in far greater detail than ever before. The experiments are not complicated. The scientists process only a handful of DNA samples and use little more than Microsoft(R) Excel for their statistical analyses. However, these physician-scientists work closely with their patients to understand disease symptoms; they then tease out causative mutations using high-density GeneChip microarrays as a powerful lens to view their patients' genomes, uncovering any hidden mutations.

To accelerate their discoveries, Affymetrix has donated a GeneChip system to the Clinic for Special Children; the donation comprises an annual number of GeneChip microarrays, a FS400 Fluidics Station to perform the microarray experiment, a GCS 3000 Scanner to image the microarrays, and all the software required to control the system and analyze array data.

"The goal is to identify the molecular lesions causing disease in our patient population. With this information, we can perform molecular genetic testing and identify affected individuals before they are symptomatic," said Puffenberger. "Currently, the most effective tool for us to map disease genes and identify the disease-causing mutations is the GeneChip mapping array."

Other Recent Findings

1. Cortical Dysplasia and Focal Epilepsy
   The Clinic team used the 10K to scan the genome of four Amish patients and mapped the disease mutation to a small part of chromosome 7 containing 83 genes. The scientists sequenced two of those genes in their patients and identified a single base deletion in all four patients. By screening all their seizure patients for this mutation, they were able to diagnose six other children with the same condition (research publication forthcoming).

2. "Pretzel" syndrome
   Studying just seven families, each with at least one affected child, the Clinic team discovered a small genetic deletion in a single gene located on chromosome 17 that had been previously missed. They made the discovery by noticing that one of the 10,000 SNPs on the microarray was missing in all seven patients; that missing SNP was part of the DNA deleted in the Pretzel syndrome patients (research publication forthcoming).

   Pretzel syndrome is a previously undescribed disorder characterized by skeletal deformity, malformation of the brain (with accompanying seizures), electrolyte imbalances, and variable malformations of the heart and other organs.

3. Down Syndrome, Patau Syndrome and other chromosomal disorders
   Using microarrays to genotype 10,000 SNPs from infants seen at the Clinic, the team quickly detected chromosomal copy number disorders like Trisomy 21 (Down syndrome) and Trisomy 13 (Patau syndrome) that are caused by having too many or too few chromosomes. With a basic 10K microarray scan, they found one patient with trisomy 13, confirmed two suspected cases of Down, identified a deletion on chromosome 13 in another patient, and discovered a 23 megabase duplication of chromosome 2 in yet another patient (research publication forthcoming).

   Trisomy occurs when a patient has three chromosomes instead of two, and is typically diagnosed by looking for extra chromosomes with a microscope. While SNP genotyping can confirm simple diagnoses of common chromosome duplications, they can also detect duplications or deletions of small parts of chromosomes that cause similar hereditary disease and are more difficult to diagnose than having an extra chromosome.

4. Sudden Infant Death with Dysgenesis of the Testes (SIDDT)
   The scientists identified a gene linked to a form of the largely mysterious sudden infant death syndrome (SIDS). While the genetic basis of SIDS has eluded scientists for decades, the group used the Affymetrix 10K array to analyze the DNA of just four infants, along with their family members, and within two weeks had found the mutation. The Clinic research team published these findings in the August 2004 issue of PNAS.

Upcoming studies
The researchers are currently planning to study a group of over 200 children with undiagnosed developmental delays to find the inherited mutations responsible for disease. Using the 10K array, the team will compare SNP genotypes between their patients with unexplained developmental delay. By grouping patients with the most similar clinical features, they hope to identify shared genomic regions, locate potential candidate genes, identify mutations, and ultimately make a genetic diagnosis. Other ongoing studies include combined variable immune deficiency, epilepsy, and cardiomyopathy.

"These Plain sect studies at the Clinic for Special children are a perfect example of how GeneChip microarrays are enabling scientists to explore the human genome and quickly discover the mutations underpinning disease -- it's a glimpse into the future of medicine," said Greg Yap, vice president of DNA Products at Affymetrix. "While 10,000 SNPs work well for studying small familial populations, 100,000 SNP products and 500,000 SNP products are doing the same thing for studies of complex diseases like diabetes and hypertension."

Broader Implications
Scientists elsewhere are discovering the genetics behind common complex diseases, like hypertension and diabetes, taking an approach similar to the Clinic for Special Children doctors, but using microarrays with far more processing power. Instead of 10,000 SNPs, scientists like Josephine Hoh at Yale University are able to hone in on disease associated mutations by using microarrays that genotype hundreds of thousands of SNPs; these microarrays provide enough genetic information to filter out the normal DNA differences between unrelated people that share the same disease. Hoh's recent study, published in the April 2005 issue of Science, scanned 100,000 SNPs from just 146 people to find a mutation associated with age related macular degeneration. Using previous technologies that looked at far fewer SNPs to generate results with the same scientific significance would have required Hoh to study thousands of people.

SOURCE: Affymetrix