Like many of his fellow Anabaptist Protestants, young Jacob Hostetler fled the religious intolerance of Central Europe in 1738, and with his wife and two small children escaped aboard the ship Charming Nancy to freedom of worship in America. They ended up in and around Berks County, Pa., where their descendants live to this day.

They are Old Order Mennonites, a sect noted for simplicity of life, which shuns modern conveniences such as automobiles. Instead, they travel by horse-drawn buggy, as did their ancestors centuries ago. Moreover, Mennonite communities practice intermarriage to protect the purity of their heritage.

One inheritance is Hirschsprung disease (HD), more descriptively known as congenital aganglionic megacolon. It's primarily an inherited disorder of infants and children, but may not become manifest until later in life. In 1888, Harald Hirschsprung (1830-1916), a Danish physician, described the disease that now bears his name. Sixty years later, in 1948, several researchers demonstrated the aganglionic nature of the segment of colon that was not dilated. Shortly thereafter, two surgeons described an operation that included excision of the aganglionic distal segment of colon while preserving the anal sphincter - thereby curing what was previously an incurable disease.

This bowel obstruction begins in neonatal infants at birth, when during the first 24 hours they cannot pass meconium, the first intestinal discharges of the newborn. Among Mennonites, the incidence of HD is one in 500 births - 10 times greater than that in the general population, where it is estimated at one in 5,000. An estimated half-million to 1 million Mennonites are living from coast to coast in the U.S., and in a number of other countries. Despite this high incidence of HD among Mennonite families, multiple cases of the disease are rare, owing to the decreased penetrance of disease-causing gene mutations.

An article in the issue of Nature Genetics released online Sept. 23, 2002, is titled: "Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease." Its senior author is molecular geneticist Aravinda Chakravarti, director of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University School of Medicine in Baltimore.

"We conducted a nearly complete recruitment of individuals affected with HD in the Old Order Mennonite communities of Lancaster and Berks counties, Pa.," Chakravarti observed, "and a settlement founded in Ontario, Canada, by multiple migrations from Pennsylvania. We obtained blood samples from each consenting individual and extracted DNA from either whole blood or established lymphoblastoid cell lines. The population sample we studied," he continued, "comprises a total of 36 probands, six siblings (four of them affected), 69 parents (two affected) and eight other second- or third-degree relatives (two affected)."

A Tale Of Two Human Genes: RET, EDNRB

He and his co-authors carried out a field survey of 43 Mennonite trios (both parents plus one affected child), searching for association arising from common ancestry 12 generations ago. "We based this approach on a recent genetic bottleneck, which occurred with the founding of this Mennonite population in the 1700s. We identified susceptibility loci on the long arms of human chromosomes 10, 13 and 16. The gene at 13 is EDNRB, which stands for encoding endothelin receptor type B' - the primary HD susceptibility factor. Endothelin," Chakravarti explained, "is an extremely potent vasoconstrictor. Its three gene products are found in brain, kidney and intestine. RET, the gene on chromosome 10, encodes a receptor for tyrosine kinase.

"The mouse system," he went on, "allowed us to analyze single-gene effects, in isolation and in combination, on the development of specific cell types and tissues, and facilitated correlation with human disease. Heterozygous mice, lacking one of two murine Ret alleles, came down with abdominal distention or distress by weaning (three to four weeks postpartum)."

By cross-breeding transgenic mice between the murine equivalent of the two genes, the co-authors concluded that "genetic interaction [epistasis] between mutations in RET and EDNRB is an underlying mechanism for this complex disorder. These mutations account for up to 50 percent and 5 percent of HD cases, respectively, in the general population," Chakravarti pointed out. "Our data clearly indicate that the variation in expression of the HD phenotype and the sex bias observed in humans can be generated with greater accuracy in mice." (The male-to-female sex ratio in HD incidence is 4-to-1.)

Hirschsprung disease is caused by the lack of enteric ganglia - an aggregation of nerve cell bodies located in the peripheral nervous system - including variable segments of the intestine. Agangliosis is the commonest cause of congenital intestinal obstruction, with a heritability of roughly 100 percent. The absence of these ganglia, which normally course down along the lower third of the colon, causes digested food to back up behind the missing nerve cells. Thus, the colon cannot relax, to allow a peristaltic wave to pass. Peristalsis is the intestinal tract's continuous wave motion that nudges fecal material along to the rectum. This damming up of its fecal excretion causes the upstream colon tracts to dilate enormously.

One HD Variant, Toxic Enterocolitis, Can Be Lethal

Surgery - excision of the aganglionic segment - is virtually the only form of treatment for HD. Pending this colonostomy, growing infants are treated with cathartics and enemas to relieve their chronic constipation. A somewhat older child undergoing such care is typically anorexic and resists defecation. A definitive diagnosis is made by a full-thickness rectal biopsy.

A deadly variant of HD is toxic enterocolitis, owing to overgrowth of bacteria and bacterial toxins. It leads to severe diarrhea and rectal bleeding, followed by shock, anuria (absence of urine formation) and death.

"We carried out a whole-genome association scan," Chakravarti's paper recounted, "using a combination of microsatellite and single nucleotide polymorphism genetic markers to test for linkage disequilibrium in Mennonites with HD. Two advantages of linkage disequilibrium analysis for family trios are that marker genotypes from families with only one affected individual can be used, and that the analysis does not rely on a predefined model of inheritance. We analyzed a total of 2,083 loci spanning all autosomes and the X chromosome in the 43 families. Thus, our study constitutes one of the first instances of a genome-wide association scan showing genetic interaction between susceptibility variants in two genes, resulting in complex inheritance of a disease phenotype."

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