274

Southern blot of proband with severe haemophilia A and of family.

Arrow = aberrant band in proband and sister.

Mosaicism may be a common feature of mutagenesis and shouldbe taken into account during genetic counselling in sporadic cases ofhaemophilia and other diseases. There are four reports of possiblegermline mosaicism associated with Duchenne muscular

dystrophy,5-8 one example of which was discovered during prenataldiagnosis.’ Mosaicism was also found to be associated with oneother case of sporadic haemophilia A.9 Because mosaicism can onlybe observed in certain informative pedigrees, its frequency insporadic disease is difficult to estimate. For example, in our study ofFinnish pedigrees, only two of the six patients with sporadichaemophilia A and known mutations had the appropriate familymembers in which to look for germline mosaicism. Prenatal

diagnosis should be offered to women who have had a singlehaemophilic child, but have no genetic history of haemophilia. Forthose cases in which the haemophilia-causing mutation has beenidentified, fetal tissue can be tested for the mutation. For cases inwhich the mutation is unknown, haemophilia might be excluded byfetal sexing and analysis of polymorphisms. If haemophilia cannotbe excluded, diagnosis may be made by fetoscopy and fetal bloodsampling.l0,11Howard Hughes Medical Institute,University of California,San Francisco, California 94143, USA

JANE GITSCHIERBARBARA LEVINSON

Department of Medical Genetics,University of Helsinki,Helsinki, Finland

ANNA-ELINA LEHESJOKIALBERT DE LA CHAPELLE

1. Gitschier J, Wood WI, Goralka TM, et al. Detection and sequence of mutations in thefactor VIII gene of haemophiliacs. Nature 1985; 315: 427-30.

2. Youssoufian H, Kazazian HH Jr, Phillips DG, et al Recurrent mutations in

haemophilia A give evidence for CpG mutation hotspots. Nature 1986; 324:380-82.

3 Gitschier J, Wood WI, Goralka TM, et al Characterization of the human factor VIIIgene Nature 1984; 312: 326-30.

4. Janco RL, Phillips JA III, Orlando PJ, Woodward MJ, Wion KL, Lawn RM.Detection of hemophilia A carriers using mtragenic factor VIII·C DNA

polymorphisms. Blood 1987; 69: 1539-415. Monaco AP, Bertelson CJ, Colletti-Feener C, Kunkel LM. Localization and cloning

of Xp21 deletion breakpoints involved in muscular dystrophy Hum Genet 1987; 75:221-27.

6. Lanman JT Jr, Pencak-Vance MA, Bartlett RJ, et al. Familial inheritance of aDXS164 deletion mutation from a heterozygous female. Am J Hum Genet 1987, 41:138-44.

7. Bakker E, Van Broeckhoven C, Bonten EJ, et al Germline mosaicsim and Duchennemuscular dystrophy mutations. Nature 1987; 329: 554-56.

8 Darras BT, Francke U. A partial deletion of the muscular dystrophy gene transmittedtwice by an unaffected male. Nature 1987; 329: 556-58.

9. Gitschier J. Maternal duplication with gene deletion in sporadic hemophilia. Am JHum Genet 1988; 43: 274-79.

10 Firshein SI, Hoyer LW, Lazarchick J, et al. Prenatal diagnosis of classic hemophilia. NEngl J Med 1979; 300: 937-41

11. Mibashan RS, Rodeck CH, Thumpston JK, et al Plasma assay of fetal factors VIIICand IX for prenatal diagnosis of haemophilia. Lancet 1979, i: 1309-11

POINT MUTATION IN c-HA-ras ONCOGENE INNORMAL AND ABNORMAL CELLS

SIR,-The discovery that retrovirus oncogenes are homologousto cellular oncogenes led to the unveiling of several potentialcancer-causing genes capable of transforming NIH 3T3 cells.

Transforming ras genes differ from their normal cellular

counterpart by a single aminoacid substitution, and where suchmutations alter restriction enzyme sites Southern blot hybridisationcan be used to study them. The Ha-ras oncogene, first identified inthe Harvey sarcoma virus,’ is homologous to the human EJ bladdercarcinoma oncogene.2 Ha-ras can be activated by a single pointmutation: any aminoacid substitution at positions 12 or 61 activates

Fig 1-Southern blot analysis of DNA from lymphocytes (lanes 1and 3) and prostate (lane 2).

Lane 1 = control DNA; lanes 2 and 3 DNA from patient. Pattern inpatient’s leucocyte and prostate DNA is the same; he seems to be

heterozygous for mutation in codon 12 (ie, shows both 412 and 355 bpfragments).

the transforming capability of the ras gene product.3 The 12thaminoacid in normal c-Ha-ras gene is glycine and any mutation atthis position obliterates the MspI recognition site.DNA from a bladder tumour cell line with an altered aminoacid

at the 12th position transforms NIH 3T3 cells.’ Similar pointmutations in other ras gene families also have transformingcapability, and it has been hypothesised that these mutations lead tocarcinogenesis in man. A study of bladder, lung, and colon cancershowed mutation at position 12 in c-Ha-ras occurred infrequentlybut in a similar percentage of all three tumours.5 (Mutations at fourother "hot spots" in this gene cannot readily be detected byrestriction enzyme analysis.) We have looked for mutations in the12th aminoacid of c-Ha-ras in benign prostatic hyperplasia (BPH)and in the human metastatic prostate cell line (LNCaP).6 ControlDNA was obtained from the blood of five healthy men aged 20-25.High molecular weight DNA was digested with Mspl, separatedelectrophoretically on 2% agarose, and analysed by Southern blotanalysis.! †DNA from the LNCaP cell line had an extra copy of c-Ha-ras

which was rearranged but no point mutation at position 12 wasfound (data not shown). Normal prostate and lymphocyte DNAfrom a man with hyperplasia of the prostate and a history ofcarcinoma of prostate and bladder was heterozygous for the pointmutation. A rearrangement and an extra copy of Ha-ras was alsoevident (fig 1); neither abnormality was present in control DNA.G-banded karyotyping of this patient was normal. None of theother five BPH patients has the point mutation or a rearrangementof Ha-ras. The mutation found may then be associated with this

patient’s history of bladder and prostate cancer. The fact that thisabnormality was present in the germ line and not just in the tissue,may explain why diseases such as bladder cancer often recur evenafter complete resection.

Leucocyte DNA from one of the five controls showed him to behomozygous for the Ha-ras mutation at position 12 (fig 2). This isthe first report of a substitution of one of the hot spots of mutation ofc-Ha-ras in a healthy individual. Clearly this man should be

tTechnical details are available from 7he Lancet.

275

Fig 2-Southern blot analysis of DNA from lymphocytes of twonormal, healthy individuals.

Lane 1 shows 412 bp band consistent with mutation in codon 12 of Ha-ras.Lane 2 is normal, showing 355 bp fragment.

carefully followed up, and a larger number of "normal" individualsshould now be tested for this anomalous gene.A single dose of N-nitroso-N-methylurea induces mammary

carcinomas in rats and this agent also causes a transition at codon 12in Ha-ras.8 Thus a mutation at position 12, whether introducedgenetically or caused by alteration of the somatic gene, maypredispose to carcinogenesis. We now need to find out the

frequency of this restriction fragment length polymorphism in thenormal population and to see if its presence does indeed havepredictive value.

Supported by the National Institutes of Health (CA 36264) (A. N.,R. G. S.) and the Southern Medical Association (J. A. J.). We also thank DrDavid Ledbetter (at Baylor College of Medicine) for the karyotype analysis.

Scott Department of Urology,and Department of Cell Biology,

Baylor College of Medicine,Houston, Texas, USA

ABHIJIT NAGJEFFREY A. JONESROY G. SMITH*

*Present address: Department of Growth Biochemistry and Physiology,Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065,USA.

1. Ellis RW, DeFeo D, Shih TY, et al The p21 src genes of Harvey and Kirsten sarcomaviruses originate from divergent members of a family of normal vertebrate genes.Nature 1981; 292: 506-10.

2 Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EG bladder carcinomaoncogene is homologue of Harvey sarcoma virus ras gene. Nature 1982; 297:474-78.

3. Wigler M, Fasano O, Taparowsky E, et al. Structure and activation of ras genes. In:vande Woude GF, Levine AJ, Topp WC, Watson JD, eds. Cancer cells II ColdSpring Harbor Laboratories, NY, 1984 419-23

4. Taparowsky E, Suard Y, Fasano O, Shimizu K, Goldfarb M, Wigler M. Activation ofthe T24 bladder carcinoma transforming gene is linked to a single ammo acidchange. Nature 1982; 300: 762-65

5. Feinberg AP, Vogelstein B, Droller MJ, Baylin SB, Nelkin BD. Mutation affecting the12th amino acid of the c-Ha-ras oncogene product occurs infrequently in humancancer. Science 1983, 220. 1175-77

6. Horoszewicz JS, Leong SS, Kawinski E, et al LNCaP model of human prostaticcarcinoma. Cancer Res 1983; 43: 1809-18.

7. Sukumar S, Notario V, Martin-Zanca D Barbacid M. Induction of mammarycarcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1locus by single point mutations Nature 1983; 306: 658-61.

8. Zarbl H, Sukumar S, Arthur AV, Martin-Zanca D, Barbacid M. Direct mutagenesisof Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammarycarcinogenesis in rats. Nature 1985, 315: 382-85

RETINOPATHY OF PREMATURITY

SIR,-Dr Ng and colleagues (Nov 26, p 1235) report on theepidemiology of retinopathy of prematurity (ROP) in a definedarea. I would like to know the total number of infants with a

birthweight less than 1500 g and less than 1000 g, who were livebornin the study period. If one or more of the five neonatal units in theregion had a higher than expected mortality rate, especially amongthe smallest infants who are most at risk of severe retinopathy, thiswould tend to lower the rate of, in particular, cicatricial disease. Inaddition, what is the total number of births in all birthweightcategories in the region and hence the annual incidence, to comparewith other population-based studies of cicatricial ROP?The frequency of acute ROP in the study of Ng and colleagues is

high, which is attributed to the schedule of frequent examinations.However, at least five "vigorous" unit-based studies’-5 also beganfundal examinations at a few weeks of age followed by weeklyexaminations, and another by fortnightly examination 6 Only thestudy by Hittner et aP has a higher frequency of acute ROP in theone birthweight category less than 1500 g (69%, versus 60% in thestudy by Ng et al). Ng and colleagues report that 60% of acute ROPwas stage 1 at its maximum. There are interpretation difficultieswith stage 1 disease despite the international classification.’ Thustwo or three "clock hours" of avascular retina in the periphery couldbe interpreted as normal development or as stage 1; both aretransient and the outcome of a normal eye will be the same.

Screening programmes which rely on an examination at this timealso require a further examination if the retina is other than nearlyfully vascularised. What were the stage, location, and extent of theapproximately 40% of cases which Ng et al state would have beenmissed by a single examination at 6-9 weeks.

Department of Paediatrics,Christchurch Hospital,Christchurch, New Zealand BRIAN DARLOW

1. Johnson L, Schaffer D, Boggs TR. The premature infant, vitamin E deficiency andretrolental fibroplasia Am J Clin Nutr 1974, 27: 1158-73.

2. Yu VYH, Hookman DM, Nave JRM. Retrolental fibroplasia: controlled study of 4years experience in a neonatal intensive care unit Arch Dis Child 1982; 57: 274-52.

3. Hitmer HM, Speer ME, Rudolph AJ, et al Retrolental fibroplasia and vitamin E inthe preterm infant—comparison of oral versus intramuscular: oral administration.Paediatrics 1984; 73: 238-49.

4. Reisner SH, Amir J, Shohat M, Krikler R, Nissenkorn I, Ben-Sira I Retinopathy ofprematurity: incidence and treatment. Arch Dis Child 1985; 60: 698-701.

5. Prendiville A, Schulenberg WE. Clinical factors associated with retinopathy ofprematurity Arch Dis Child 1988; 63: 522-27

6 Flynn JT, Bancalari E, Bachynski BN, et al. Retinopathy of prematurity. diagnosis,seventy, and natural history. Ophthalmology 1987; 94: 620-29.

7. Committee for the classification of retinopathy of prematurity An internationalclassification of retinopathy of prematurity Br J Ophthalmol 1984; 68: 690-97

*** This letter has been shown to Professor Fielder and colleagues,whose reply follows.-ED. L.

SIR,-In fig 2 we showed the number of infants with

birthweights less than 1000 g and less than 1500 g who survived thefirst three weeks of life and therefore entered the study. The totalnumbers of liveborn infants with birthweight below 1000 g and1500 g born during the study period were 138 and 461, respectively(from fig 2 plus the total who died in first three weeks and those notexamined).The mortality rates at the five neonatal units were dissimilar,

because of social and ethnic differences and also cross-referral ofinfants with specific problems to the more specialised units.

However, infants were not transferred out of the study area and asthis was a population and not a hospital-based study this lack ofhomogeneity would have had no effect on the overall disease rate. Ifthe overall mortality in this area was different from that in otherareas, this would confound any comparisons of incidence.From birth notification data we ascertained that there were about

50 950 livebirths in the area over the study period. The incidence ofnew cases of acute and cicatricial ROP can therefore be calculated bymultiplying the number of cases by 1.96 to give 487 and 10,respectively, per 100 000 births. However, the value of this exerciseeludes us as, of these 50 950 births, nearly all were full-term andconsequently not at risk of ROP.