Redundancy begets complexity

In 1902, two years after the rediscovery of Mendel’s laws of inheritance, British physician Sir Archibald Garrod made an historic discovery while studying several English families with an obscure disorder that caused their urine to turn brown, then black when exposed to the air.

A brown pigment discoloured the cartilage and connective tissues of individuals with alkaptonuria, and they developed arthritis. Their urine contained high levels of two amino acids, tyrosine and phenylalanine.

Garrod, a specialist in the chemistry of urine, observed that the disorder conformed to a Mendelian recessive pattern of inheritance in affected families, and postulated that it involved a mutation in a gene involved in tyrosine metabolism. He was the first person to link a biochemical abnormality to a genetic error.

In 2004, a research team led by Sydney University haematologist Professor John Rasko, head of the gene and stem cell therapy program at the Centenary Institute of Cancer Medicine and Cell Biology, published a paper in Nature Genetics describing mutations in a gene involved in Hartnup disease, another autosomal recessive disorder of amino-acid transport.

The study led to a longstanding collaboration known as the Australian Aminoaciduria Consortium, including the research groups of Professor Stefan Broer and Dr Juleen Cavanaugh at the Australian National University.

Rasko and his colleagues showed that Hartnup disorder, originally described in the Hartnup family of London in 1956, was caused by a recessive mutation in the SLC6A19 gene on chromosome 5.

SLC6A19 codes for a transporter protein for neutral amino acids like leucine, valine, isoleucine, methionine and tryptophan. It is expressed in the tubules of the kidney, and the lumen of the small intestine.

Hartnup disease’s symptoms include a light-sensitive rash on the skin, balance and coordination problems due to cerebellar ataxia, emotional instability due to a deficiency of tryptophan – the precursor for the calming neurotransmitter serotonin – and high urine concentrations of neutral amino acids. But for most people, the only symptom is the telltale urinary abnormalities.

---PB--- Metabolic disorders

After their success in identifying the gene and several point mutations involved in Hartnup disorder, Rasko and his colleagues turned their attention to iminoglycinuria (IG) and hyperglycinuria (HG), two related metabolic disorders in which affected individuals excrete 100 to 1000 times the normal level of glycine and proline in their urine.

Iminoglycinuria and hyperglycinuria were first described 30 years ago. Rasko describes their detection as a “no brainer” – the amino acids involved are present in gross excess in the urine. Once again, the disorders were expected to involve autosomal recessive point mutations in a transporter gene expressed in the kidney tubules.

“The kidney is an amazing organ,” Rasko says. “It does a superb job of washing the blood of unwanted waste products in the body.

“Millions of years ago, it evolved a system that dumps all wastes from plasma into a bucket, and then picks through them and pulls back what the body wants to retain.

“So every small molecule, including amino acids, gets dumped into the urine, but as it passes through the tubules of the kidney, transporter molecules in the apical membrane capture and pump them back into the bloodstream.”

Iminoglycinuria and hyperglycinuria are rare, and Rasko says it was a challenge to find multi-generational pedigrees whose families would agree to be involved in the search for the mutant gene involved.

Eventually, they were able to recruit seven affected families, identified by screening for iminoglycinuria in newborns in Australia and Canada, and conducted a candidate gene sequencing study.

“The rest should have been pretty straightforward, or so we thought,” Rasko says. “We believed the disorders would involve mutations in a specific pump. No such single pump exists, even though we believed we had identified all the kidney’s pumps from their genetic signatures.”

Only one family conformed to a classic, monogenic pattern – affected individuals inherited mutations in the SLC36A2 gene that rendered it non-functional. But Rasko says that in six of the seven pedigrees, iminoglycinuria and hyperglycinuria turned out to be multigenic in origin – and extraordinarily complex.

“Some individuals would waste glycine, but not proline or hydroxyproline – they would have 100 times the normal level of glycine in their urine, but their urinary proline levels were normal.

“Our study provides an insight into the amazing complexity of the genome. Normally when we think about a recessive gene disorder, we think about dead alleles of a single gene. But IG and HG involve the interaction of mutant alleles of two, three or even four different genes. The mutations converge to produce a phenotype, but they’re not all-or-nothing mutations.

“Some of the genes retain partial function. For the disorder to manifest, a specific membrane pump’s capacity to recover the amino acid from the urine and pump it back into the bloodstream has to fall below a threshold level.”

---PB--- Redundancy begets complexity

The Sydney-Canberra collaborators published their findings in the Journal of Clinical Investigation last December. The study zeroed in on the SLC36A2 transporter gene as the chief culprit in both disorders. It seemed to exhibit a semi-dominant pattern of expression: two mutant alleles produced the IG phenotype, one mutant allele caused HG.

As it turned out, mutant alleles in SLC36A2 that retained some activity caused IG when they were combined with mutations in the imino acid transporter gene SLC6A20.

Rasko and his colleagues identified further mutations in genes for a putative glycine transporter, SLC6A18, and the neutral amino acid transporter SLC6A19, in families affected by either IG or HG. The findings indicated that SLC36A2 mutations combined in various ways with mutations in three other genes that modulated its activity, to cause either IG or HG.

Rasko says the result is exciting, because it suggests that other inherited disorders that superficially exhibit recessive or semi-dominant patterns of inheritance may also involve complex interactions with one, two or three other genes.

He says their findings highlight the checks and balances that natural selection has built into some genetic networks that underpin vital bodily functions – redundancy begets complexity.