HEPATOLOGY Elsewhere T. JAKE LIANG, EDITOR Gastrointestinal Unit

Massachusetts General Hospital 32 Fruit St Boston, MA 02114

GRJ-724

WALKING THE ETHICAL HIGHWIRE: GENETIC SCREENING AND HEREDITARY TYROSINEMIA

Grompe M, St,-Louis M, Demers SI, al-Dhalimy M, Leclerc B, Tanguay RM. A single mutation of the fu- marylacetoacetate hydrolase gene in French Canadi- ans with hereditary tyrosinemia type I. N Engl J Med 1994; 331:353-357.

ABSTRACT

Background. Hereditary tyrosinemia type I is an au- tosomal recessive inborn error of metabolism caused by a deficiency of the enzyme fumarylacetoacetate hy- drolase. The disorder clusters in the Saguenay-Lac-St.- Jean area of Quebec. In this region, 1 of 1846 newborns is affected and 1 of every 22 persons is thought to be a carrier. Recently, we identified a splice mutation and two nonsense mutations in the fumarylacetoacetate hy- drolase gene in two patients from Quebec with tyro- sinemia type I.

Methods: We used allele-specific-oligonucleotide hy- bridization to examine the frequency of these three can- didate mutations in patients with tyrosinemia type I and in the population of Quebec.

Results: The splice mutation was found in 100 percent of patients from the Saguenay-Lac-St.-Jean area and in 28 percent of patients from other regions of the world. Of 25 patients from the Saguenay-Lac-StJean region, 20 (80 percent) were homozygous for this mutation, a guanine-to-adenine change in the splice-donor sequence in intron 12 of the gene, indicating that it causes most cases of tyrosinemia type I in the region. The frequency of carrier status, based on screening of blood spots from newborns, was about 1 per 25 in the Saguenay-Lac-St.- Jean population and about 1 per 66 overall in Quebec.

Conclusions: This study identified the most prevalent mutation causing hereditary tyrosinemia in French Canada; it also showed the feasibility of DNA-based test- ing for carriers in the population at risk. (N Engl J Med 1994;331:353-7.)

COMMENTS

Progress in identifying the genetic basis of a disorder frequently catalyzes the rapid development of molecu- lar techniques that correctly identify affected individu- als andlor potential carriers of the disease. Realisti- cally, however, the process of developing therapeutic interventions based on newly described molecular mechanisms may lag behind diagnostic capabilities for

ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO

HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA

NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY

JOEL LAVINE, Boston, MA RICHARD H. MOSELEY, Ann Arbor, MI

many years. As a result, determining how to best use these new diagnostic aids becomes critically important. This decision process is best made in the context of the particular circumstances of the disease in question. How many individuals are affected by the disease? Is the disease curable? How onerous is the therapy? What kind of quality of life can be expected if treated? What kind of burden does the knowledge of disease carrier status place on individuals? Does this knowledge pro- vide access to different alternatives that would not oth- erwise be available? Physicians are asked to balance these difficult issues while policies are developed re- garding the use of new diagnostic advances for diseases with limited or costly therapies. As we attempt to step across this ethical highwire, we must recognize that we will be buffeted by social, religious, political, and economic forces, any one of which may make the cross- ing hazardous or nearly impossible. One recently cre- ated example of this situation exists with hereditary tyrosinemia (HT) and a newly developed genetic screening test described by Grompe et a1.l

Although HT is a rare autosomal recessive disorder having an estimated incidence of 1 in 100,000, it is much more common in certain populations. The inci- dence among French Canadians of the Saguenay-Lac- St.-Jean region of northern Quebec reaches 1 in 1,800 liveborn infant^.^,^ Researchers have established that HT results from deficient activity of fumarylacetoace- tate hydrolase (FAH), the last enzymatic step in tyro- sine metab~lism.~ Subsequent accumulation of toxic in- termediates is thought to cause liver, renal, and peripheral nerve injury (Fig. 1).

The clinical course of the disease, although variable, is frequently deva~tat ing.~ Liver disease can be dra- matic, presenting with marked coagulopathy shortly after birth. Cirrhosis develops almost invariably. Hepa- tocellular carcinoma often complicates HT early in childhood. Proximal renal tubular dysfunction can lead to the development of hypophosphatemic rickets. Addi- tionally, paroxysmal episodes of paralysis are postu- lated to result from a porphyrialike inhibition of delta- aminolevulinic acid dehydratase by succinylacetone (Fig. 1).

Interventions for HT are limited. Dietary restriction of protein intake with careful attention to phenylala- nine and tyrosine loads may ameliorate renal dysfunc- tion but do not halt the progression of liver disease.

1193

1194 HEPATOLOGY Elsewhere HEPATOLOGY April 1995

phenylalanine I

tyros i n e

delta aminolevulinic acid + 'A (**) succinylacetone +

2*) (**) maleylacetoacetate

(**) fumarylacetoacetate A 3

porp hobilinogen fumarylace t oace ta te hydrolase

fumarate + acetoacetate

FIG. 1. Abbreviated tyrosine metabolism pathway showing presumed toxic intermediates ('? and site of NTBC activity (*). Also illustrated is succinylacetone blockade of delta aminolevulinic acid dehydratase activity.

Liver transplantation, sometimes in combination with kidney transplantation, can be curative. Without transplantation, death usually occurs in childhood.

Establishing this fateful diagnosis has relied on a compatible clinical picture and the demonstration of elevated succinylacetone levels in the blood or urine.6 Previously, no reliable tests have been available to de- termine carrier status. By definition, parents of af- flicted children are heterozygous for the disorder and have a 1 in 4 chance of having a second afflicted child. Prenatal diagnosis has relied on invasive methods such as determination of succinylacetone levels in amniotic fluid or FAH assay using aminocytes or chorionic villus c e k 7

The cloning of the FAH complementary DNA and mapping to chromosome 15 q23-25 in 1991 ushered in a new era.*,' Several mutant FAH sequences responsi- ble for HT now have been described.lO,ll Among French Canadian patients, a missense mutation in the FAH structural gene has been detected in a single family. A second mutation with a guanine to adenine alteration in the splice donor consensus sequence of intron 12 region of the FAH gene has been reported. Addition- ally, a third genetic error resulting from two different nonsense mutations producing two translation stop sig- nals in the FAH sequence has been described. Using this information, Grompe et a1 postulated that one or more of the above-mentioned mutations would account for a majority of the FAH genotypes that result in HT in French Canada.

These investigators isolated genomic DNA from blood samples of healthy adult volunteers and 61 pa- tients with HT (25 of 29 patients from Quebec were from the Saguenay-Lac-St.-Jean region, and 32 pa-

tients were from outside French Canada). Because the previously mentioned mutations could be found within a 998-base pair region between intron 12 and 13 of the FAH gene, polymerase chain reaction amplification of specimen DNA from this region was performed using appropriate primers and subsequently spotted on dot blot apparatus. Oligonucleotide probes, which repre- sented the known mutations and wild-type FAH se- quences, were synthesized, radiolabeled, and each se- quentially hybridized to the blot.

The results demonstrated that the intron 12 splice donor mutation was present in all patients with HT from the Saguenay-Lac-%.-Jean region of Quebec (20 of 25 being homozygous for this disorder), while it was not present in the 4 French Canadians from outside this region. Outside of Quebec 9 of 32 or 28% of patients with HT had the mutation. DNA extracted from ran- dom infant blood spot samples obtained anonymously from the Quebec neonatal screening program showed a 1 in 25 carrier rate for the intron 12 FAH mutation in the Saguenay-Lac-St.-Jean region, and a 1 in 66 carrier rate in Quebec overall. This corresponds well to the observed incidence of HT in the region.

With this report these investigators showed the fea- sibility of genetic testing for HT in French Canada, particularly in the Saguenay-Lac-St.-Jean region. It is estimated that 81% of carriers in the region can be detected with the intron 12 probe. Discovery of other FAH mutations may improve this sensitivity and make it possible to genetically screen for HT in other locales.

However, the truly dominant question becomes how to use this new methodology. For example, where does one screen for HT: Saguenay-Lac-St.-Jean, Quebec, or all of Canada? When should screening take place: at

HEPATOLOGY Elsewhere 1195 HEPATOLOGY Vol. 21, No. 4, 1995

birth, in secondary school, during premarital testing? Although screening may make sense from a socioeco- nomic point of view, how would individual rights be protected? Will programs be developed to encourage identified individuals to look for mates outside the higher-risk region? Are identified at-risk couples en- couraged not to have children? Would identified couples be directed to undergo prenatal testing with future pregnancies and be encouraged to abort fetuses deter- mined to be affected? Importantly, how will public pol- icy be determined when the condition is found substan- tially within an ethnic minority of a larger population?

The availability of new therapies to treat HT makes the decision regarding disease screening a dynamic and fluid process. 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3- cyclohexadione (NTBC) is believed to interrupt the pro- duction of toxic tyrosine intermediates (Fig. 1). Promis- ing results have been reported.12 Yet, it is not clear whether NTBC will sufficiently prevent ongoing liver disease or the subsequent development of hepatocellu- lar carcinoma. Furthermore, the optimal timing of therapy with NTBC is not known. It is important to determine whether FAH genotypes can predict NTBC responsiveness in HT. If genetic screening is imple- mented, NTBC therapy offered either prenatally or postnatally may be an option for identified carriers with in utero-diagnosed HT not desiring termination of pregnancy.

As molecular diagnostic advances multiply, the med- ical profession will be called on more frequently to be the “Wallendas of the ethical highwire,” attempting to go forward while balancing the inequitable weights of diagnostic and therapeutic modalities. Careful consid- eration must precede our recommendations for genetic screening tests.

RENE ROMERO, MD Assistant Professor of Pediatrics Division of Gastroenterology Emory University School of Medicine Atlanta, GA

JOEL E. LAVINE, MD, PhD Assistant Professor of Pediatrics Center for Childhood Liver Diseases Children’s Hospital and Harvard Medical School Boston, MA

REFERENCES

1. Grompe M, St.-Louis M, Domers SI, al-Dhalimy M, Leclerc B, Tanguay RM. A single mutation of the fumarylacetoacetate hy- drolase gene in French Canadians with hereditary tyrosinemia type I. N Engl J Med 1994;331:353-357.

2. DeBraekeleer M, Larochelle J . Genetic epidemiology of heredi- tary tyrosinemia in Quebec and Saguenay-Lac-St.-Jean. Am J Hum Genet 1990;47:302-307.

3. Kvittingen EA. Hereditary tyrosinemia type I-an overview. Scand J Clin Invest 1986;46(suppl I):27-34.

4. Linblad B, Lindstedt S, Steen G. On the enzymatic defects in hereditary tyrosinemia. Proc Natl Acad Sci 1977; 74:4641-4645.

5. Paradis K, Mitchell G, Russo P. Tyrosinemia. In: Suchy FJ, ed. Liver disease in children. St. Louis: Mosby, 1994:803-818.

6. Grenier A, Lescault A, Laberge C, Mamer 0. Detection of suc- cylacetone and the use of measurement in mass screening for hereditary tyrosinemia. Clin Chim Acta 1982; 123:93-99.

7. Jakobs C, Stellard F, Kvittingen EA, Henderson M, Lilford R. First-trimester prenatal diagnosis of tyrosinemia type I by amni- otic fluid succinylacetone determination. Prenat Diag 1990; 10: 133-134.

8. Agsteribbe E, van Faassen H, Hartog M V , Reversma T, Taan- mam JW, Pannekoek H, Evers RF, et al. Nucleotide sequence of cDNA encoding human fumarylacetoacetase. Nucl Acids Res 1990; 18:1887.

9. Phanuef D, Labelle Y, Berube D, Arden K, Cavenee W, Gagne R, Tanguay RM. Cloning and expression of the cDNA encoding human fumarylacetoacetae hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15. Am J Hum Genet 1991;48:525-535.

10. Grompe M, al-Dhalimy M. Mutations of the fumarylacetoacetate hydrolase gene in four patients with tyrosinemia type I. Hum Mutat 1993; 2:85-93.

11. Phanuef D, Lambert M, Laframboise R, Mitchell G, Lettre F, Tanguay RM. Type I hereditary tyrosinemia: evidence for molec- ular heterogeneity and identification of a casual mutation in a French Canadian patient. J Clin Invest 1992; 90:1185-1192.

12. Lindstedt S, Holme E, Lock EA, Hjalmarson 0, Strandvik B. Treatment of hereditary tyrosinemia type I by inhibition of 4-hydroxyphenyl pyruvate dioxygenase. Lancet 1992; 340: 813-817.

GENETIC HEMOCHROMATOSIS AND WILSON’S DISEASE: ROLE FOR OXIDANT STRESS?

Young IS, Trouton TG, Torney JJ, McMaster D, Callender ME, Trimble ER. Antioxidant status and lipid peroxidation in hereditary haemochromatosis. Free Rad Biol Med 1994; 16:393-397.

ABSTRACT Hereditary haemochromatosis is characterised by

iron overload that may lead to tissue damage. Free iron is a potent promoter of hydroxyl radical formation that can cause increased lipid peroxidation and depletion of chain-breaking antioxidants. We have therefore as- sessed lipid peroxidation and antioxidant status in 15 subjects with hereditary haemochromatosis and agdsex matched controls. Subjects with haemochromatosis had increased serum iron (24.8 (19.1-30.5) vs. 17.8 (16.1-19.5) pmoVL, P = 0.021) and % saturation (51.8 (42.0-61.6) vs. 38.1 (32.8-44.01, P = 0.025). Thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were increased in haemochromatosis (0.59 (0.48-0.70) vs. 0.46 (0.21-0.71) @morn, P = 0.045), and there were de- creased levels of the chain-breaking antioxidants alpha- tocopherol (5.91 (5.17-6.60) vs. 7.24 (6.49-7.80) pmoYmmo1 cholesterol, P = 0.001), ascorbate (51.3 (33.7-69.0) vs. 89.1 (65.3-112.9), P = 0.013)’ and retinol(l.78 (1.46-2.10) vs. 2.46 (2.22-2.70) pmoY1, P = 0.001). Patients with hereditary haemochromatosis have reduced levels of antioxidant vitamins, and nutritional antioxidant supplementation may represent a novel approach to preventing tissue damage. However, the use of vitamin C may be deleteri- ous in this setting as ascorbate can have prooxidant ef- fects in the presence of iron overload.

von Herbay A, de Groot H, Hegi U, Stremmel W, Strohmeyer G, Sies H. Low vitamin E content in plasma of patients with alcoholic liver disease, hemo-