William Check, PhD

If it’s true that a good trial lawyer never asks aquestion without first knowing the answer, then

molecular geneticists would make bad trial lawyers.Several hundred times each day across North Amer-ica a molecular genetics laboratory sets up a clini-cal sample for whole-gene sequencing withoutknowing beforehand what the outcome will be.Of course, no laboratory knows beforehand whetherany sample will be positive or negative. However,sequencing entails a whole new dimension of un-certainty. Like a traditional clinical laboratory test,a sequence can be negative—no mutation or a be-nign polymorphism; or positive—a mutationknown to be associated with the hereditary condi-tion in question. But, like a lawyer questioning a wit-ness without benefit of a deposition, sequencingsometimes pops up a wild card—a mutation whosepossible association to the hereditary condition inquestion is a complete unknown.

Vicky Pratt, PhD, FACMG, director of moleculargenetics at Quest Diagnostics-Nichols Institute in

Chantilly, Va., calls such variations“mutations of unknown clinical sig-nificance.” Dr. Pratt co-organized aworkshop on this topic during lastyear’s meeting of the Association forMolecular Pathology. The session wascalled “Missense Mutations DetectedDuring Sequencing” because mostambiguous or uncertain variants aremissense mutations; frameshifts orstop codons (truncating mutations)would most likely be deleterious.“The job of the laboratorian is to try tofigure out the clinical impact of thatchange,” Dr. Pratt told CAP TODAY.“It is hard for a laboratory to make agood interpretation in these situa-tions and hard for clinicians to un-derstand these reports and to knowwhat to do next.” The workshop wasdesigned to present the thoughts of

laboratorians who have experience sequencing alarge number of genes or many samples of a fewgenes. “We asked them to tell us how they resolvethese unknown mutations,” she says.

Dr. Pratt predicts that, as more knowledge isgathered about uncertain variants, the problemwill resolve to some degree. But with large complexgenes like BRCA1 and BRCA2, mutations of un-known clinical significance will continue to be dis-covered. More important, Dr. Pratt and other expertsagree that an increasing number of genes will be se-quenced to aid clinical diagnosis. So the conun-drum posed by mutations of unknown clinical sig-nificance won’t go away, and it’s likely to grow.

“We are increasingly moving into the realm ofwhole-gene analysis rather than detection of a lim-ited panel of mutations,” Elaine Lyon, PhD, told CAPTODAY. “We can detect any mutation and anyvariant, and many times we find a variant that hasnot been described before and for which no clinicalstudies have been done,” says Dr. Lyon, who is as-sistant professor of clinical pathology at the Uni-versity of Utah and medical director of moleculargenetics at ARUP Laboratories, Salt Lake City. As aresult, “The clinician gets a report saying that wefound something that we are calling an uncertain

variant, which leaves the patient maybe with moreuncertainty than they started with.”

“I think this problem will only grow,” saysWayne Grody, MD, PhD, professor in the Divi-sions of Molecular Pathology and Medical Genet-ics in the Departments of Pathology and LaboratoryMedicine, Pediatrics, and Human Genetics, UCLASchool of Medicine. “As sequencing technologygets cheaper and easier, we will consider sequenc-ing more genes and even sequencing the wholegenome on some patients,” Dr. Grody says. Myri-ad Genetics’ work with the BRCA1 and BRCA2genes, the longest and most extensive experiencewith whole-gene sequencing for clinical purposes,foreshadows what can be expected, he says. In theBRCA genes, uncertain variants can arise at virtu-ally any position. “I don’t think the BRCAgenes aremore mutable than any other genes,” Dr. Grodysays. “The fact that Myriad has found thousands ofvariants is a foreboding that we will find the samething in any and every gene that we subject to se-quencing.”

Sequencing for clinical investigation of heredi-tary diseases, most often a hereditary predis-

position to cancer, is a relatively new practice. Myr-iad Genetics has been sequencing BRCA1 andBRCA2 genes for about eight years. Dr. Lyon notesthat sequencing of the beta globin gene for structuralanomalies that produce hemoglobinopathies (sick-le cell disease) and altered amounts of beta-globintranscripts (thalassemias) has been done clinicallyfor about five years. However, she says, “The betaglobin gene is so well studied that we have yet tofind a mutation that hasn’t already been charac-terized.” More recent additions to the sequencingmenu are the CFTR gene in cystic fibrosis and sev-eral genes that give rise to hereditary colon cancers,which are as problematic as the BRCA genes.

Interpreting a mutation of unknown clinical sig-nificance is difficult. To make the situation evenmore stressful, medical geneticists know that ma-jor clinical actions can ride on their decisions. In anasymptomatic woman with a family history ofbreast or ovarian cancer, for instance, finding amutation and calling it deleterious can have “seri-ous life impact,” Dr. Pratt says. A deleterious mu-

tation in one of the BRCA genes canlead to prophylactic bilateral mastec-tomy or oophorectomy.

Brian Ward, PhD, is senior vicepresident at Myriad Genetic Labora-tories, which sequences both BRCAgenes and several predispositiongenes for colon cancer. He lists manypossible implications of calling a mu-tation deleterious. First is a drasticrise in the individual’s lifetime riskof cancer. “For almost all of these can-cer predisposition genes,” he says, “ifyou are asymptomatic and get testedbecause of a family history, finding adeleterious mutation increases yourrisk from the population risk of 10percent to a lifetime risk of 80 per-cent [breast cancer] to almost 100 per-cent [colorectal cancer].” For ovariancancer, the risk goes from about onepercent to as high as 30 percent–40percent. (All of this information is atMyriad Genetics’ site: www.myri-adtests.com/inherited.htm.)

Calling a mutation deleterious alsohas implications for the person’s fam-ily. “If an affected individual carries adeleterious mutation, 50 percent ofthat person’s first-degree relatives—children, brothers, and sisters—are atrisk of the same mutation,” Dr. Ward

60 / CAP TODAY April 2006

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FILE—& PROOF— April Page 60

Annotated hand-curated alignments Novel mutation,

G1247R

CFTR

orthologs

Paralogs

(ABC trans)

ATP

G1247

Exon 20

Sec. Structure:

Walker A, P-loop

Same location as

G1302R in MRP6,

AR-PXE

G1381S in SUR1,

PHHI

100%

conserved

across

orthologs and

paralogs

Arlene B

uller, PhD

The Mutation Inference Scoring Tool being developed for the Molecular GeneticsLaboratory at Quest Diagnostics in San Juan Capistrano uses public databases to align thenucleotide sequence of a patient’s CFTR gene with an uncertain variant with homologousor related genes. This alignment shows the degree of evolutionary conservation of thepatient’s mutation and helps to arrive at a quantitative estimate of how likely it is that themutation is deleterious. In this case, the mutation in question, G1247R, corresponds to thesite of known mutations in two other human genes that code for transport proteins.Structural information shows that the mutation is found in the P loop of the CFTR protein.

Mutational wild cardscontinued from page <None>

Annotated hand-curated alignments