It started with a 10-month-old boy, who I’ll call Jake, who was feeding poorly. Between 6 and 10 months, a time when infants should be growing rapidly, he hadn’t gained a pound. His diapers were constantly wet – he was urinating at a high rate. He was irritable and fussy.
When a lab test found an extremely elevated blood calcium level, Jake was sent to the emergency room. Such an extreme elevation posed a risk of compromising his heart and kidney function. His blood pressure was sky-high. “He needed immediate IV fluid to bring his calcium down, and an immediate workup,” says Andrew Dauber, then a first-year fellow in endocrinology.
Jake was admitted to Children’s and Dauber took part in the consult with his mentor Joel Hirschhorn, a pediatric endocrinologist trained in genetics. “His kidneys were so calcified they were turning into stones,” Dauber recalls. The senior nephrologist on the team said Jake had the most severe case of nephrocalcinosis he’d seen.
The team put Jake on low-calcium formula and his calcium level slowly came down. His diagnosis was idiopathic infantile hypercalcemia — meaning they had no idea what was causing Jake’s calcium levels to be so high.
Dauber searched the medical literature and found many similar cases, but no explanation of the cause. He began emailing calcium experts all over the world. “They gave me all different advice,” he recalls.
One expert, assuming that the excess calcium was coming from Jake’s bones, suggested giving a bisphosphonate, an osteoporosis drug that inhibits the digestion of bone. But Jake’s bone X-rays were normal, as were blood tests measuring hormonal markers of bone loss. Other experts suggested steroids, to interfere with calcium absorption. But none of the other cases in the literature had definitively shown a problem with calcium absorption.
Did Jake? “We decided to figure out what was happening with this kid,” says Dauber. “We wrote a protocol for a one-family research study.”
Above, ultrasound of a normal kidney. Below, Jake’s kidney (click to enlarge). The white area shows calcium deposits – essentially turning Jake’s kidney into stone.
Dauber reached out to Steve Abrams at Baylor College of Medicine, a world expert in calcium metabolism. Abrams was enthused about the case and happened to be coming to Boston, where he met with Dauber and helped write up the study protocol. Most crucially, he gave Dauber thousands of dollars worth of calcium isotopes that could be used to trace the passage of calcium through the body. Since Dauber and Hirschhorn had no funding, this was a fortunate break.
To test Jake’s calcium absorption, they gave him one isotope,46Ca, in a single IV infusion, and put another, 48Ca, in Jake’s formula. They then collected Jake’s urine over the next five days and used mass spectrometry to measure amounts of each isotope. From these measurements, Dauber could deduce how much Jake was absorbing from each source – his formula and the IV infusion.
It turned out that Jake was absorbing 90 percent of the calcium he took in through his formula – twice the rate that’s considered normal for infants. “He had one of the highest intestinal calcium absorptions ever recorded in a human,” Dauber says, “completely off the charts. This cemented that putting him on a low-calcium diet was the right thing to do.”
But what was the cause? Dauber and Hirschhorn did a genetic study — made easier by the fact that the Jake’s parents are closely related (his father and maternal grandmother are cousins). This made it likely that the mutation was a recessive one, requiring two copies (one from each parent) for any trait to appear. It also made the gene easier to find.
With DNA samples in hand from both parents, Dauber and Hirschhorn used a technique called homozygosity mapping to look for blocks of DNA that were identical on both of Jake’s chromosomes – one from his mother, one from his father. A match would indicate a region where a recessive abnormality might lie.
They found 3 large chunks of DNA (on chromosomes 7, 16 and 20) that were identical, and knew their answer must lie in one of those chunks. These so-called homozygous regions, together, encompassed about 360 genes.
Hirschhorn took the samples to the Broad Institute, where he co-directs the Metabolism Initiative, and performed whole-exome sequencing of Jake’s DNA, including the homozygous regions. This uncovered more than 19,000 variant bits of DNA, as well as 367 short DNA insertions or deletions.
Analyzing just the homozygous regions, they found their smoking gun: a single base-pair deletion on the gene CYP24A1. Our DNA is made up of about 3 billion base pairs; Jake was missing a mere 3 pairs: a C, T, T was absent from each of his chromosomes.
Though tiny, this defect left Jake unable to properly make the enzyme that breaks down vitamin D. “He couldn’t inactivate his vitamin D, which promotes absorption of calcium in the intestines, and that was causing too much calcium absorption,” Dauber explains.
Little did Dauber and Hirschhorn know that a research team in Germany had just come to the same conclusion, through very different means. That team started with the idea that vitamin D must be involved, and found recessive mutations when they sequenced CYP24A1 alone in children with idiopathic infantile hypercalcemia.
The CYP24A1 defect seems to circulate at low levels in the population – Dauber and Hirschhorn found it in 7 of 1646 stored DNA samples. None of these subjects were affected, but Jake was, since he alone carried two copies of the defect – as Dauber and Hirschhorn reported in the Journal of Clinical Endocrinology & Metabolism.
Today at age 3 ½, Jake is growing and thriving. Moreover, he’s much happier. “His personality changed enormously by fixing this,” Dauber reports.
Now that Jake’s defect has been pinpointed, future siblings can be diagnosed early, either at birth or prenatally, and immediately placed on the diet to avoid any damage from high calcium levels.
“This case demonstrates how we can use modern genetic techniques to make new discoveries in biology from just one patient,” says Dauber. “The ability to sequence the entire genome is an extremely powerful tool that opens the door for discoveries in areas where little is understood.”
