Letters to the Editor J Med Genet: First Published As 10.1136/Jmg.38.1.43 on 1 January 2001

Letters 43

Letters to the Editor J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

J Med Genet 2001;38:43–47 Sensitivity and predictive value of typifying LFS, but not selected on family history, to determine the proportion of gene carriers among them. The criteria for p53 germline mutation studies on patients with bone sarcoma or STS25–29 showed screening that up to a third of the group with early onset, an unusual family history, or multiple primary tumours may be carriers. Children with adrenocortical carcinoma were found to have the highest rate (50-80%).30 31 The frequency of EDITOR—The history of Li-Fraumeni syndrome (LFS) is a mutations among patients with multiple primary tumours good illustration of the delineation of dominantly inherited was estimated to be between 7 and 20%.32–34 Far lower rates family cancer syndromes. The identification of this were found for patients with brain tumours,35–39 or early syndrome is the result of the combination of two kinds of onset/familial breast cancer,40–43 although the breast cancer evidence, firstly, a number of reports on particular familial risk was clearly high in p53 mutation carriers. In some of aggregations12 and, secondly, systematic family studies of these studies, a selection bias on family history may be sus- childhood sarcomas.3–6 Among these studies, the decisive pected. Indeed, a significant proportion of mutations were contribution came from Li and Fraumeni3 who were the found among cases with a strong positive family history, the first to publish the results of a family study on 641 children frequency of which appeared to be unusually high. with rhabdomyosarcoma which led to the identification of None of these studies allowed an estimation of cancer four families in which a sib or a cousin was aVected by risk in mutation carriers, although unaVected carrier rela- rhabdomyosarcoma or another soft tissue sarcoma (STS). tives are found in family studies. Indeed, LFS selection cri- These families also had several members who were aVected teria are so stringent that it is impossible to correct for by diverse types of malignant tumours, in particular sarco- selection bias. Even looser criteria, such as Li-Fraumeni- mas and breast cancer at a very young age. This prompted like21 44 (LFL) or Li-Fraumeni incomplete20 (LFI) do not the authors to propose the existence of a new familial syn- allow correction for ascertainment bias. This was the drome.7 A prospective study on these families over 12 years reason that we undertook a study at the Institut Gustave provided evidence of a strong predisposition to cancer with Roussy with very loose criteria which oVered two a strikingly high frequency of multiple tumours.8 The term advantages: (1) they did not imply the existence of highly “Li-Fraumeni syndrome” was used for the first time in penetrant susceptibility genes and therefore potentially 19829 and the criteria, which subsequently became the allowed the detection of mutations associated with a low classical definition of the syndrome, were proposed by Li cancer risk; (2) correction for selection bias was possible and Fraumeni in 1988.10 These were a proband with a sar- for the estimation of cancer risks in individual subjects. coma before 45 years of age, a first degree relative with Our main conclusions are: (1) that cancer risks are very high, (2) although unaVected carriers may be observed, cancer before this age, and another close (first or second http://jmg.bmj.com/ degree) relative in the lineage with either cancer before this there is no evidence for the existence of mutations with age or a sarcoma at any age. These criteria led to the selec- particularly low penetrance, and (3) the proportion of de tion of 24 families which exhibited a wide variety of novo mutations is probably substantial.45 tumours including bone sarcomas, STS, breast cancer, While the above mentioned were gradually defining with brain tumours, leukaemia, adrenocortical carcinoma, ever greater accuracy the relationship between constitu- lymphoma, lung, stomach, pancreas, and prostate cancer, tional mutations and cancer types and risks, an inter- but only the first six types were significantly in excess of the national multidisciplinary group was trying to establish 44 46–50 expected proportion among subjects aVected by cancer recommendations for predictive testing. For such on September 26, 2021 by guest. Protected copyright. before 45 years in the American population. The follow up testing, it was essential, as a first step, to evaluate individual of these families confirmed an unusually high predisposi- and familial criteria to undertake the initial search in a tion to cancer.11 Other studies have indicated that a number family, in terms of sensitivity and predictive value. We of other cancers may occur in these families, the most report here the results obtained from our study on notable being melanoma, germ cell tumours, gastric carci- childhood cancer at the Institut Gustave Roussy and on a noma, and Wilms’ tumour.5 12–16 study of breast cancer in very young women performed at The definition of the syndrome shifted from clinical and the Institut Curie in France. familial criteria to molecular criteria after Malkin et al17 and The family history of cancer in children under 18 years Srivastava et al18 described the involvement of germline p53 treated for all types of solid malignant tumours in the mutations. The mutations initially found were all missense Department of Paediatric Oncology at the Institut Gustave mutations of exon 7, but further studies, extensively Roussy in Villejuif (France) was investigated between reported by Varley et al19 showed that other regions might January 1991 and May 1997. Information was collected also be involved. Studies on series of families with the clas- through a direct interview with a trained counsellor for sical LFS criteria showed that 50 to 70% of these families families of patients treated in the department during the displayed a p53 mutation,19–23 indicating that mutation study period. Information was obtained via a mailed ques- screening may have overlooked alterations that aVect regu- tionnaire and completed in most cases by a telephone latory regions and not p53 coding sequences or that germ- interview for patients treated before that period and no line mutation of other gene(s) may be responsible for LFS. longer followed up or who had died. To minimise possible Indeed, the study recently published by Bell et al24 showed biases owing to genetic and environmental heterogeneity, that heterozygous germline mutations in hCHK2 occur in only white children were included in the study. LFS. The proportions of p53 mutations are somewhat Family data were collected through the proband’s lower when less stringent criteria are applied.20 21 parents. They included information on each of the After ascribing LFS to germline p53 mutations, diVerent proband’s first and second degree relatives and first studies were conducted on series of patients with tumours cousins. When necessary, additional family members,

www.jmedgenet.com 44 Letters previously informed by the proband’s parents, were tive needs a high specificity, which is the probability that a J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from contacted for a telephone interview. Information on mutation will not be found given that the criteria are not relatives included general characteristics (sex, date of birth, fulfilled. The positive predictive value can be estimated by malformations, date and cause of death) and the the proportion of subjects carrying a germline p53 occurrence of any cancer. When cancers had occurred, mutation among those tested using given criteria. The esti- confirmation of the diagnosis and age at onset were sought mation of sensitivity and specificity requires reference cri- in medical and pathology records. Only invasive cancers teria that would allow the ascertainment of carriers and were considered, excluding non-melanoma skin cancer and non-carriers from an unselected population. These para- in situ carcinoma. meters therefore cannot really be estimated. However, it is A subgroup of children in whom the frequency of cancer possible to estimate the relative sensitivity by the ratio susceptibility genes would be potentially increased was between the number of mutations detected when given selected on the basis of the occurrence of either of the fol- criteria are applied and the number of mutations detected lowing criteria: (1) at least one cancer case aVecting a first in the whole sample. Besides, since a negative result is of no or second degree relative before the age of 46 (familial value at this point, specificity is not particularly interesting. cases) or (2) multiple primary cancers in the proband At this point, the importance of wording should be empha- regardless of his/her family history (multiple tumour cases). sised. The sentence “a mutation will be found” is used In the original protocol, the family was also included if can- instead of “a mutation exists”, because this would also cer had occurred only in first cousins. This criterion had to depend on the sensitivity of the method used to detect be removed since it dramatically increased the proportion of mutations, which is not the subject of the present study. chance aggregation in the selected sample. The positive predictive value and the relative sensitivity are p53 was genotyped in peripheral lymphocytes isolated estimated in relation to the whole sample when more and from fresh blood samples. Direct sequencing was used for more stringent criteria are applied on: (1) the number and the first set of 100 samples. Genomic DNA was amplified age of aVected relatives, (2) the tumour spectrum as three fragments including respectively exons 2-4, exons (probands and relatives), and (3) the existence of multiple 5-8, and exons 9-11 which were fully sequenced. Genotyp- primary tumours. ing was subsequently carried out with a functional assay in Of the 2691 children eligible for the family study on 1 yeast (FASAY), as described by Ishioka et al51 and modified January 1998, 239 fulfilled the selection criteria and by Flaman et al52 when this test became available. Vent consented to give a blood sample. Among these 239 DNA polymerase (New England Biolabs) was used to children, 211 had at least one first or second degree relative amplify p53 reverse transcripts before transfection in yeast. aVected by cancer before 46 years of age, 16 had at least two Yeast colonies carrying a p53 mutant allele were identified primary malignant tumours, and 12 fulfilled both familial either as His-auxotroph or as red colonies. p53 cDNA was and multiple tumour criteria. Among these cases, 15 muta- extracted from mutant colonies and sequenced. The tions were detected, nine in the first group (4.3%), one in FASAY has been reported to show over 90% of p53 mutant the second (a de novo mutation in a child with rhabdomy- alleles52 as does direct sequencing of amplified p53 exon osarcoma and adrenocortical carcinoma) (6.2%), and five scores in our hands. in the third group (4.2%). The complete descriptions of Women suVering from invasive breast cancer before 36 families with mutations are published elsewhere.45

years, which was diagnosed between January 1990 and Among the 223 children (211 + 12) fulfilling the famil- http://jmg.bmj.com/ August 1995 and followed up at the Institut Curie, were ial criteria, four levels of nested criteria were defined interviewed about their family history and were requested according to the number and tumour type in the aVected to give a blood sample for the study of genes involved in relative(s) and are listed in table 1: very loose criteria (223 breast cancer predisposition. Among the 275 women children), at least one first or second degree relative fulfilling these criteria, 119 were interviewed between aVected by any cancer; loose criteria (141 children), the January 1993 and August 1995 and 116 gave their tumour type in the aVected relative(s) is restricted to informed consent for DNA analysis. sarcoma, brain tumours, breast cancer, adrenocortical car-

The pedigrees were constructed by taking into account cinoma, haematological malignancies, stomach cancer, on September 26, 2021 by guest. Protected copyright. first to third degree relatives of the proband on both paren- melanoma, and germ cell tumours, which are the most tal sides. Information concerning the family history of prevalent tumours described in LFS; stringent criteria (81 tumours and age at onset of the tumours was verified when children), the tumour spectrum in relative(s) is restricted possible in medical and pathology records. to unquestioned tumours, that is, sarcomas, brain tumours, Screening for the presence of mutations was performed breast cancer, and adrenocortical carcinoma (narrow spec- by analysis of PCR products from genomic DNA with trum); very stringent criteria (21 children), a new criterion denaturing gradient gel electrophoresis (DGGE). Exons 4, is added to the previous ones, at least another first or sec- 5, 6, 7, 8, 9, 10, and 11 and respective flanking regions were studied53 (unpublished data). PCR products exhibiting a Table 1 Definition of the four levels of nested criteria according to the number and tumour type in the aVected relative(s) in the study on variant electrophoretic pattern were directly sequenced on childhood cancer both strands. In order to confirm the loss of biological function of missense mutations detected, a functional assay Criteria on relatives Definition in yeast was performed according to Flaman et al.52 Any Very loose At least one first or second degree relative aVected mutation identified was confirmed on a second independ- by any cancer ent blood sample. Loose Tumour type in the aVected relative(s) restricted to The objectives of defining criteria for recommending p53 sarcoma, brain tumours, breast cancer, adrenocortical carcinoma, haematological mutation screening are triple: (1) to look for a mutation in malignancies, stomach cancer, melanoma, and situations in which it is likely to be found; (2) to miss as few germ cell tumours mutations as possible; (3) not to select subjects who are not Stringent Tumour spectrum in relative(s) restricted to unquestioned tumours, ie sarcomas, brain carriers. The first objective needs a high positive predictive tumours, breast cancer, and adrenocortical value, which is the probability that a mutation will be found carcinoma (narrow spectrum) for given criteria. The second objective needs a high sensi- Very stringent New criterion added to the previous ones: at least another first or second degree relative aVected by tivity, which is the probability that the criteria will be cancer before 46 years or a sarcoma at any age fulfilled, given that the mutation is found. The third objec-

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Table 2 Positive predictive value and relative sensitivity of criteria on probands and relatives for predictive p53 screening in childhood cancer J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

Criteria on relatives

Very loose Loose Stringent Very stringent

Predictive Relative Predictive Relative Predictive Relative Predictive Relative Criteria on proband value sensitivity value sensitivity value sensitivity value sensitivity

Any tumour 6% (14/223) 93% (14/15) 9% (13/141) 87% (13/15) 15% (12/81) 80% (12/15) 43% (9/21) 60% (9/15) Narrow spectrum 12% (12/102) 80% (12/15) 16% (11/67) 73% (11/15) 23% (11/47) 73% (11/15) 53% (8/15) 53% (8/15) ond degree relative aVected by cancer before 46 years or a cerned a case of bilateral breast carcinoma at 29 and 30 sarcoma at any age. Criteria were also defined by stratifica- years whose family history was clearly indicative of tion on the tumour type in the proband with two levels, a Li-Fraumeni syndrome, including chondrosarcoma at 16 narrow spectrum tumour (102 children) or any tumour. years, leukaemia at 26 years, breast cancer at 20 years, and The results of the combination of criteria for relatives and renal tumour at 36 years (unconfirmed) in the sibship, and probands among the 223 familial cases are given in table 2. the father aVected with a soft tissue tumour of an unknown They show that the positive predictive values for the histological nature at 64 years. The third mutation criterion “any tumour” in the proband are quite low (less (Arg175Gly) was detected in a woman suVering from than 15%) except in the category “very stringent” criteria in osteosarcoma at 18 years and bilateral breast cancer at 27 relatives. It is significantly higher when the tumour type in and 29 years. Her father had developed a rectal carcinoma the proband is restricted to the narrow spectrum and attains at 39 years, meningioma at 54 years, and pancreatic carci- 23% when stringent criteria are fulfilled among relatives. noma at 55 years, and her paternal uncle had developed a The parameters estimated among the 28 (16 + 12) mul- germ cell tumour at 45 years. tiple tumour cases (excluding bilateral tumours of paired Because of the small number of mutations found, we had organs) are shown in table 3. The positive predictive values to consider a smaller number of criteria than in the previ- are much higher than in the group selected on a familial ous section, and only two levels of nested criteria were basis. However, this criterion overlaps markedly with the defined: loose criteria, at least one first or second degree previous ones; in the six carriers of a germline mutation, relative aVected by any cancer before 46 years of age or a five fulfil the “narrow spectrum” for the first tumour of the proband with multiple primary malignant tumours; proband, of which four also fulfil the “stringent” criteria on stringent criteria, the tumour spectrum in relative(s) (or in relatives. Thus, adding the criterion “multiple tumours” in the proband in case of multiple tumours) is restricted to the proband to the combination of narrow spectrum in the the narrow spectrum. However, since breast cancer is proband’s tumour and stringent criteria for relatives yields common in the general population, familial aggregation of 24 new cases, two of which have a germline mutation. This breast cancers may be either because of chance or germline results in an increase in relative sensitivity from 73% BRCA1/2 mutations. Therefore, two situations were (11/15) to 87% (13/15) and a decrease in the predictive considered, the narrow spectrum tumour is breast cancer value from 23% (11/47) to 18% (13/71). If both tumours (situation A) or another tumour (situation B). (or at least two tumours) are restricted to the narrow spec- Thirty three cases fulfilled the loose criteria (two muta- trum, then only six families are added, one of which carries tions), 21 cases the stringent criteria A (no mutation), and http://jmg.bmj.com/ a mutation, resulting in a relative sensitivity of 80% (12/15) two the stringent criteria B (two mutations). The positive and a predictive value of 23% (12/53). There are very few predictive values are presented in table 4, but not the rela- multiple tumours among relatives (excluding bilateral tive sensitivities which would be meaningless with only tumours of paired organs): six families among which three three mutations. fulfil the stringent criteria in relatives and four fulfil the Most of the studies on germline p53 mutations narrow spectrum in the proband. There are two p53 muta- conducted to date and quoted in the introduction did not

tions, two of which belong to the group defined by narrow permit evaluation of diVerent selection criteria. Some of on September 26, 2021 by guest. Protected copyright. spectrum and multiple tumours in the proband and strin- them concerned families ascertained on the basis of strong gent criteria in relatives. Consequently, the predictive value familial aggregation (corresponding roughly to our very and the relative sensitivity are not modified when multiple stringent criteria) and the relevance of looser criteria could tumours are added to the narrow LFS spectrum in relatives not be assessed. Other studies concerned series of tumours (data not shown). with very limited information on family history, so that it Among the 116 breast cancer cases fully analysed, a total was impossible to evaluate criteria. The most well of three germline p53 mutations (2.5%) were detected. documented studies are by the group of Jillian Birch, based Two of them are missense mutations (Leu130Phe, on the Manchester Children’s Tumour Registry.16 21 These Arg175Gly) and one is an in frame deletion authors showed the relatively high predictive value (GluAla346del3Asp). The deleterious eVects of both mis- (4/18=22%) of the so called Li-Fraumeni-like criteria, that sense mutations have been confirmed with FASAY. One is, a proband with any childhood cancer or sarcoma, brain mutation (Leu130Phe) was found in a woman who was tumour, or adrenal cortical tumour diagnosed under the aVected at 31 years and had no family history of cancer and age of 45 years with one first or second degree relative with in particular five unaVected sisters aged from 34 to 49 a typical LFS cancer at any age, plus a first or second years. The second mutation (GluAla346del3Asp) con- degree relative in the same lineage with any cancer under the age of 60 years. There is some degree of overlap Table 3 Positive predictive value and relative sensitivity in families ascertained on the existence of multiple tumours in the proband child Table 4 Positive predictive value in families of women with breast cancer before 36 years according to criteria Criteria on relatives Predictive value Relative sensitivity Criteria Positive predictive value None 21% (6/28) 40% (6/15) Very loose 42% (5/12) 33% (5/15) None 3% (3/116) Loose 63% (5/8) 33% (5/15) Loose 7% (2/29) Stringent 71% (5/7) 33% (5/15) Stringent A 0% (0/21) 2 9% (2/23) Very stringent 100% (3/3) 20% (3/15) B 100% (2/2) 3

www.jmedgenet.com 46 Letters between these criteria and ours; 18 families conform to this recommend a search for p53 mutation in families fulfilling J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from definition in our sample, four of which exhibit a p53 muta- the following criteria: (1) a proband aVected by a narrow tion, which is exactly the same number and proportions as spectrum cancer before 36 years and at least one first or those found by these authors. Note that all positive families second degree relative aVected by a narrow spectrum fulfil our stringent criteria in relatives and narrow spectrum tumour (other than breast cancer if the proband is aVected tumour in the proband. by breast cancer) before 46 years or multiple primary In the present study, we have quantified various criteria tumours; (2) a proband with multiple primary tumours two which can be a very useful basis for establishing of which belong to the narrow spectrum and the first of recommendations for conducting p53 screening. which occurred before 36 years, whatever the family history; The functional assay used in the study on childhood (3) a proband with adrenocortical carcinoma whatever the cancer detects about 90% of mutations in p53.52 Mutations age of onset and family history. Using such criteria, we in exons 2 and 3 are very rare and were not studied in the expect to find a mutation in about 20% of cases and to miss study on breast cancer A small number of mutations may about 20% (3/15) of the mutations which would be detected thus have been missed and the positive predictive value of using the loosest criteria. These criteria could of course be criteria may thus be slightly higher, which should not modified in the near future with more eYcient laboratory modify our conclusions. detection methods and re-evaluated prospectively. At present, searching for p53 germline mutations is still a cumbersome task and laboratories which perform such We are indebted to Thierry Frébourg for functional assays and helpful sugges- tions and to Céline Delouis for her help in p53 testing. We are grateful to Marion screening in France are in favour of criteria yielding a posi- Gauthier-Villars for her help in data management and to Lorna Saint-Ange for tive predictive value of at least 20%. In our study on child- editing the manuscript. We acknowledge the financial support of the Fondation de France, Fondation A et P Sommer, the Association pour la Recherche sur le hood cancer, this value is achieved when the following cri- Cancer, la Ligue Nationale Contre le Cancer, la Caisse Nationale d’Assurance teria are used: a proband with a narrow spectrum tumour Maladie, l’Institut Electricité Santé, l’Association ISIS, l’Institut Gustave V Roussy, Le Ministère de la Recherche et de l’Enseignement Supérieur, and at least one first or second degree relative a ected SNECMA, INSERM, and CNRS. before 46 years by a narrow spectrum cancer or multiple AGNÈS CHOMPRET*† primary tumours or a proband aVected by multiple ANNE ABEL*† primary tumours whatever the family history. It should be DOMINIQUE STOPPA-LYONNET‡ noted that when very stringent criteria are used, that is, that LAURENCE BRUGIÈRES† SABINE PAGÈS‡ are very close to those used in previous studies to define JEAN FEUNTEUN§ LFS, very similar results are obtained with a positive CATHERINE BONAÏTI-PELLIÉ* predictive value of 53%. *Unité de Recherche en Epidémiologie des Cancers (U521 INSERM), For cancer occurring in adulthood, our data concern Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France only very early onset breast cancer, which is the most fre- †Département de Pédiatrie, Institut Gustave Roussy, 39 rue Camille quent tumour in p53 carriers (80% of tumours occurring Desmoulins, 94805 Villejuif Cedex, France 45 among female carriers after 16 years of age ). As it is also ‡Service de Génétique Oncologique, Institut Curie, 26 rue d’Ulm, 75248 common in the general population, we have presented the Paris Cedex 05, France results separately when breast cancer is the narrow §Laboratoire de Génétique Oncologique (UMR 1599 CNRS), Institut spectrum tumour in the relative who was the determinant Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France Correspondence to: Dr Bonaïti-Pellié, [email protected] factor in the inclusion of the family (criterion A) and when http://jmg.bmj.com/ it is another cancer (criterion B). Although this distinction results in loss of precision of estimations owing to a 1 Bottomley RH, Condit PT. Cancer families. Cancer Bull 1968;20:22-4. 2 Lynch HT, Mulcahy GM, Harris RE, Guirgis HA, Lynch JF. Genetic and decrease in sample size, it should be noted that no pathologic findings in a kindred with hereditary sarcoma, breast cancer, mutation among 21 cases was detected when criterion A brain tumors, leukemia, lung, laryngeal, and adrenal cortical carcinoma. Cancer 1978;41:2055-64. was found, whereas both cases with criterion B were p53 3 Li FP, Fraumenii JF. Rhabdomyosarcoma in children: epidemiologic study carriers (p=0.02, exact test). Consequently, as in childhood and identification of a cancer family syndrome. J Natl Cancer Inst 1969;43: 1365-73. cancer, it seems reasonable to use stringent criteria and to 4 Birch JM, Hartley AL, Marsden HB, Harris M, Swindell R. Excess risk of add this restriction on tumour type, that is, exclude breast breast cancer in the mothers of children with soft tissue sarcomas. Br J on September 26, 2021 by guest. Protected copyright. Cancer 1984;49:325-31. cancer from the tumour spectrum in relatives. 5 Strong LC, Stine M, Norsted TL. Cancer in survivors of childhood soft tis- Although the results obtained by ascertaining early onset sue sarcoma and their relatives. J Natl Cancer Inst 1987;79:1213-20. 6 Birch JM, Hartley AL, Blair V, Kelsey AH, Harris M, Teare MD, Morris breast cancer probands cannot necessarily be extrapolated Jones PH. Cancer in families of children with soft tissue sarcomas. to all tumours of the narrow spectrum, we expect that the Cancer 1990;66:2237-48. 7 Li FP, Fraumeni JF. Soft tissue sarcomas, breast cancer and other predictive value for the other tumours will be higher than neoplasms: a familial syndrome? Ann Int Med 1969;71:747-52. that obtained for breast cancer, given the high frequency of 8 Li FP, Fraumeni JF. Prospective study of a cancer family syndrome. JAMA 1982;247:2692-4. this tumour in the population. 9 Pearson ADJ, Craft AW, RatcliVe JM, Birch JM, Morris-Jones PH, Roberts In addition, although we have very few data on adreno- DF. Two families with the Li-Fraumeni cancer family syndrome. JMed Genet 1982;19:362-5. cortical carcinoma not selected on family history, it seems 10 Li FP, Fraumeni JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA, logical to add this criterion considering the results of pub- Miller RW. A cancer family syndrome in twenty-four kindreds. Cancer Res 1988;48:5358-62. lished studies quoted in the introduction. 11 Garber JE, Goldstein AM, Kantor AF, Dreyfus MG, Fraumeni JF, Li FP. Finally, it should be noted that although leukaemia is Follow-up study of twenty-four families with Li-Fraumeni syndrome. Can- cer Res 1991;51:6094-7. usually reported as belonging to the LFS, this was not 12 Hartley AL, Birch JM, Marsden HB, Harris M. Malignant melanoma in included among narrow spectrum tumours in the present families of children with osteosarcoma, chondrosarcoma and adrenal cortical carcinoma. J Med Genet 1987;24:664-8. study for two reasons. (1) It is absent in our childhood 13 Hartley AL, Birch JM, Kelsey AM, Marsden HB, Harris M, Teare MD. Are probands because the Department of Paediatrics at the germ cell tumours part of the Li-Fraumeni syndrome? Cancer Genet Cytogenet 1989;42:221-6. Institut Gustave Roussy treats only solid tumours. (2) 14 Hartley AL, Birch JM, Tricker K, Wallace SA, Kelsey AM, Harris M, Mor- Leukaemia is frequent in the general population and the risJonesPH.Wilms’ tumor in the Li-Fraumeni cancer family syndrome. Cancer Genet Cytogenet 1993;67:133-5. inclusion of families with one relative aVected by this can- 15 Varley JM, McGown G, Thorncroft M, Tricker KJ, Teare MD, Santibanez- cer would dramatically increase the probability of chance Koref MF, Houlston RS, Martin J, Birch JM, Evans DGR. An extended Li-Fraumeni kindred with gastric carcinoma and a codon 175 mutation in aggregation. TP53. J Med Genet 1995;32:946-50. With our knowledge of the interest of the criteria 16 Varley JM, McGown G, Thorncroft M, Santibanez-Koref MF, Kelsey AM, Tricker KJ, Evans DGR, Birch JM. Germline mutations of TP53 in evaluated in this study and considering the state of the art for Li-Fraumeni families: an extended study of 39 families. Cancer Res p53 mutation screening, the French LFS group decided to 1997;57:3245-52.

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Li-Fraumeni syndrome - a molecular 37 Chen P, Iavarone A, Fick J, Edwards M, Prados M, Israel MA. and clinical review. Br J Cancer 1997;76:1-14. Constitutional p53 mutations associated with brain tumors in young 20 Brugières L, Gardes M, Moutou C, Chompret A, Meresse V, Martin A, adults. Cancer Genet Cytogenet 1995;82:106-15. Poisson N, Flamant F, Bonaïti-Pellié C, Lemerle J, Feunteun J. Screening 38 Felix CA, Slavc I, Dunn M, Strauss EA, Phillips PC, Rorke LB, Sutton L, for germ line p53 mutations in children with malignant tumors and a fam- Bunin GR, Biegel JA. p53 gene mutations in pediatric brain tumors. Med ily history of cancer. Cancer Res 1993;53:452-55. Pediatr Oncol 1995;25:431-6. 21 Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM, Harris 39 Li YJ, Sanson M, Hoan-Xuang K, Delattre JY, Poisson M, Thomas G, M, Morris Jones PH, Binchy A, Crowther D, Craft AW, Eden OB, Evans Hamelin R. Incidence of germ-line p53 mutations in patients with gliomas. DGR, Thompson E, Mann JR, Martin J, Mitchell ELD, Santibanez-Koref Int J Cancer 1995;64:383-7. MF. Prevalence and diversity of constitutional mutations in the p53 gene 40 Børresen AL, Andersen TI, Garber J, Barbier-Piraux N, Thorlacius S, among 21 Li-Fraumeni families. Cancer Res 1994;54:1298-304. Eyfjörd J, Ottestad L, Smith-Sorensen B, Hovig E, Malkin D, Friend SH. 22 Birch JM, Heighway J, Teare MD, Kelsey AM, Hartley AL, Tricker KJ, Screening for germ line TP53 mutations in breast cancer patients. Cancer Crowther D, Lane DP, Santibanez-Koref MF. Linkage studies in a Res 1992;52:3234-6. Li-Fraumeni family with increased expression of p53 protein but no germ- 41 Prosser J, Porter D, Coles C, Condie A, Thompson AM, Chetty U, Steel Br J Cancer 70 line mutation in p53. 1994; :1176-81. CM, Evans HJ. Constitutional p53 mutations in a non Li-Fraumeni cancer 23 Frebourg T, Barbier N, Yan YX, Garber JE, Dreyfus M, Fraumeni JF, Li family. Br J Cancer 1992;65:527-8. FP, Friend SH. Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome. Am J Hum Genet 1995;56:608-15. 42 Sidransky D, Tokino T, Helzlsouer K, Zehnbauer B, Rausch G, Shelton B, Prestigiacomo L, Vogelstein B, Davidson N. Inherited p53 gene mutations 24 Bell DW, Varley JM, Szydlo TE, Kang DH, Wahrer DCR, Shannon KE, Cancer Res Lubratovich M, Verselis SJ, Isselbacher KJ, Fraumeni JF, Birch JM, Li FP, in breast cancer. 1992;52:2984-6. Garber JE, Haber DA. Heterozygous germ line hCHK2 mutations in 43 Warren W, Eeles RA, Ponder BA Easton DF, Averill D, Ponder MA, Ander- Li-Fraumeni syndrome. Science 1999;286:2528-31. son K, Evans AM, DeMars R, Love R, Dundas S, Stratton MR, 25 Toguchida J, Yamaguchi T, Dayton SH, Beauchamp RL, Herrera GE, Trowbridge P, Cooper CS, Peto J. No evidence for germline mutations in Ishizaki K, Yamamuro T, Meyers PA, Little JB, Sasaki MS, Weichselbaum exons 5-9 of the p53 gene in 25 breast cancer families. Oncogene RR, Yandell DW. Prevalence and spectrum of germline mutations of the 1992;7:1043-6. p53 gene among patients with sarcoma. N Engl J Med 1992;326:1301-8. 44 Eeles RA. Germline mutations in the TP53 gene. Cancer Surv 1995;25:101- 26 Porter DE, Holden ST, Steel CM, Cohen BB, Wallace MR, Reid R. A sig- 24. niﬁcant proportion of patient with osteosarcoma may belong to 45 Chompret A, Brugières L, Ronsin M, Gardes M, Dessarps-Freichey F, Abel Li-Fraumeni cancer families. JBoneJointSurgBr1992;74:883-6. A, Hua D, Ligot L, Dondon MG, Bressac-de Paillerets B, Frebourg T, 27 Iavarone A, Matthay KK, Steinkirchner TM, Israel MA. Germ-line and Lemerle J, Bonaïti-Pellié C, Feunteun J. Prevalence of p53 germline muta- somatic p53 mutations in multifocal osteosarcoma. Proc Natl Acad Sci USA tions in childhood cancer and estimation of cancer risk for carrier individu- 1992;89:4207-9. als. Br J Cancer 2000;82:1932–7. 28 McIntyre JF, Smith-Sorensen B, Friend SH, Kassell J, Børresen AL, Yan 46 Li FP, Correa P, Fraumeni JF. Testing for germline p53 mutations in cancer YX, Russo C, Sato J, Barbier N, Miser J, Malkin D, Gebhardt MC. Germ- families. Cancer Epidemiol Biomarkers Prev 1991;1:91-4. line mutations of the p53 tumor suppressor gene in children with 47 Li FP, Garber JE, Friend SH Strong LC, Patenaude AF, Juengst ET, Reilly osteosarcoma. J Clin Oncol 1994;12:925-30. PR, Correa P, Fraumeni JF. Recommendations on predictive testing for 29 Diller L, Sexsmith E, Gottlieb A, Li FP, Malkin D. Germline p53 mutations germ line p53 mutations among cancer-prone individuals. JNatlCancer are frequently detected in young children with rhabdomyosarcoma. J Clin Inst 1993;84:1156-60. Invest 1995;95:1606-11. 48 Eeles RA. Predictive testing for germline mutations in the p53 gene: are all 30 Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA, Malkin D. High fre- the questions answered? Eur J Cancer 1993;29:1361-5. quency of germline p53 mutations in childhood adrenocortical cancer. J 49 Li FP, Fraumeni JF. Collaborative interdisciplinary studies of p53 and other Natl Cancer Inst 1994;86:1707-10. predisposing mutations in Li-Fraumeni syndrome. Cancer Epidemiol 31 Varley JM, McGown G, Thorncroft M, James LA, Margison GP, Forster G, Biomarkers Prev 1994;3:715-17. Evans DG, Harris M, Kelsey AM, Birch JM. Are there low-penetrance 50 Eng C, Schneider K, Fraumeni JF, Li FP. Third international workshop on TP53 alleles? Evidence from childhood adrenocortical tumors. Am J Hum collaborative interdisciplinary studies of p53 and other predisposing muta- Genet 1999;65:995-1006. tions in Li-Fraumeni syndrome. Cancer Epidemiol Biomarkers Prev 1997;6: 32 Malkin D, Jolly KW, Barbier N, Look AT, Friend SH, Gebhardt MC, 379-83. Andersen TI, Børresen AL, Li FP, Garber J, Strong L. Germline mutations 51 Ishioka C, Frebourg T, Yan YX, Vidal M, Friend SH, Schmidt S, Iggo R. of the p53 tumor-suppressor gene in children and young adults with second Screening patients for heterozygous p53 mutations using a functional assay

malignant neoplasms. N Engl J Med 1992;326:1309-15. in yeast. Nat Genet 1993;5:124-9. http://jmg.bmj.com/ 33 Shiseki M, Nishikawa R, Yamamoto H, Ochiai A, Sugimura H, Shitara N, 52 Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sameshima Y, Mizoguchi H, Sugimura T, Yokota J. Germ-line p53 Sappino AP, Limacher JM, Bron L, Benhattar J, Tada M, Van Meir EG, mutation is uncommon in patients with triple primary cancers. Cancer Lett Estreicher A, Iggo RD. A simple p53 functional assay for screening cell 1993;73:51-7. lines, blood, and tumors. Proc Natl Acad Sci USA 1995;92:3963-7. 34 Russo CL, McIntyre J, Goorin AM, Link MP, Gebhardt MC, Friend SH. 53 Hamelin R, Jego N, Laurent-Puig P, Vidaud M, Thomas G. EYcient Secondary breast cancer in patients presenting with osteosarcoma: possible screening of p53 mutations by denaturing gradient gel electrophoresis in involvement of germline p53 mutations. Med Pediatr Oncol 1994;23:354-8. colorectal tumors. Oncogene 1993;8:2213-20. on September 26, 2021 by guest. Protected copyright. J Med Genet 2001;38:47–49 Identification of a transcriptionally forming element associated with c-MYC.3 Besides genetic lesions, other epigenetic factors such as activation of compromised allele of c-MYC in a growth factor receptors may also lead to constitutive North American family expression of c-MYC. Thus, considerable eVorts have been made systematically to identify the c-MYC transcriptional apparatus (promoters and enhancers) in an eVort to control c-MYC expression. While c-MYC transcription EDITOR—Chromosomal translocations that target c-MYC potentially initiates from one of three promoters, P0, P1, at 8q24 are found in all Burkitt’s lymphomas (BL), AIDS and P2 which reside in the exon 1 region, the P2 promoter related non-Hodgkin’s lymphoma (AIDS-NHL), mouse normally accounts for 75-90% of cytoplasmic c-MYC plasmacytomas (PCTs), in many examples of diVuse large RNAs. To date, more than 20 transcription factors have cell lymphoma (DLCL), and in multiple myeloma (MM). been found to reside in the proximity of exon 1 of c-MYC.1 Indications are that c-MYC is under strict control and Actually, c-MYC was one of the first genes to exhibit tran- when deregulated results in unchecked cellular prolifera- scriptional blockage. RNA polymerase II initiation com- tion and hyperplasia. Non-random chromosomal translo- plexes were shown to pause on P2 before activation.4–6 cations found such as t(8;14), t(8;22), or t(2;8) in these Upon chromosomal translocation, the insertion of IG lymphoid neoplasias places c-MYC under the control of enhancer elements renders a shift in promoter usage from strong immunoglobulin enhancers, which leads to overex- P2 towards P1 and loss of transcriptional blockage.7 pression.12 In addition, c-MYC is amplified in many In concert with the intensely regulated transcriptional tumours including breast, prostate, gastrointestinal, ovar- machinery, the sequences surrounding c-MYC appear to ian, MM, myeloid leukaemia, and melanoma suggesting be strictly conserved across species boundaries and little in that the overall transcriptional level is probably a key trans- the way of sequence variation or allelic polymorphisms has

www.jmedgenet.com 48 Letters been identified. In fact, attempts to position c-MYC in the panels of AIDS-NHL (around 200), BL (around 40), MM J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from mouse by genetic recombination techniques were ultimately (around 20), small cell lung carcinoma (around 25), or achieved only when wild mice were backcrossed to inbred neuroblastoma/neurocytoma (around 60). Thus, S288K mice.8 Recently, we and others9 have identified several alleles appears to be the lowest frequency MYC variant allele of human c-MYC (S11N, CAA-33) through single nucle- identified to date in the North American population. To otide polymorphism (SNP) analysis of the coding region learn more about the origin of the S288K allele, we among a large random panel of normal healthy subjects of obtained peripheral blood samples from the North Ameri- African-American and white descent.10 Comparisons of the can family of the proband and we have concluded that the S11N and CAA-33 alleles to wild type alleles at the RNA father (No 557) and a daughter (No 554) are heterozygous level showed that the CAA-33 allele is transcribed less carriers and present with no apparent phenotypic abnor- eYciently in peripheral blood leucocytes. Although the malities. The family is white with a largely western nature of this diVerence remains to be elucidated, the finding European background and no apparent predisposition to that CAA-33 is transcriptionally compromised and is found the development of cancer or other metabolic diseases. We almost exclusively in people of African descent underlines have compared expression of the S288K allele to the wild the importance of the genetic background in studies on the type allele by SSCP and RT-PCR amplification of RNA control of c-MYC expression. Thus, studies of allelic diVer- made from peripheral blood (fig 1). We find that S288K is ences and transcription of human c-MYC will provide useful expressed at extremely low levels or not at all in either sub- paradigms in the attempt to control or regulate c-MYC ject and this result has been confirmed by sequencing indi- expression in normal and disease conditions. vidual subclones (13 in total) of RT-PCR amplified RNA We have considered the possibility that sequence diVer- from No 554 (a ratio of 12:1 subclones for codon 288 K:S). ences that exist in the coding region of c-MYC could result We present two hypotheses to explain the compromised in feedback inhibition of transcription. For example, expression of S288K in comparison to wild type. The somatic point mutations have been found in both BL11 12 conformational change associated with a serine to lysine and AIDS-NHL13 14 to be clustered in c-MYC exon 2 in the change at codon 288 could abrogate binding of a transcrip- region responsible for binding of P107 or other factors to tion factor and lead to repression of c-MYC. In fact, the the transactivation domain (TAD).15 16 This suggests that transcription factor YY1 which acts to down regulate disruption of binding in this region might lead to a c-MYC expression through both direct18 and indirect functional inactivation of c-MYC. Indeed, a substitution at eVects19 is known to bind in the proximity of this region.20 An residue Thr-58 in the TAD of v-Myc in the avian alternative hypothesis is that S288K carries additional retroviruses MC29, MH2, and OK10 is known to contrib- sequence diVerences in the 5' untranslated region which ute to the transformation of fibroblasts.17 While it is aVect transcription. We cannot distinguish between these believed that a major consequence of somatic mutation in alternatives until more detailed cloning and sequencing is c-MYC could be loss of function, it is not clear which resi- accomplished. Nevertheless, S288K represents the second dues are critical. instance of an allele of c-MYC (in addition to CAA-33) in We wish to report the discovery of a unique sequence which transcription is less robust in comparison to wild type. (S288K AGC→AAC) in the coding region of c-MYC Numerous reports of an L-MYC polymorphism have which we have recently found in a North American family been linked to disease susceptibility in soft tissue sarcomas,

(fig 1). The S288K substitution resides just distal to the oral cancers, colorectal cancers, NHL, breast carcinoma, http://jmg.bmj.com/ acidic domain and proximal to the nuclear localisation sig- and non-SCLC, whereas the same alleles seem to be asso- nal and was detected by PCR amplification and sequencing ciated with resistance to hepatocellular carcinoma.21 22 The of the exon 3 region of human c-MYC. Subsequently, we reason for this apparent paradox can be attributed to a developed a single stranded conformational polymorphism basic lack of quantitative expression data for L-MYC alle- (SSCP) assay for S288K which we have used to survey les at the RNA or protein level in tumour versus normal panels of normal, healthy, white (around 200), African- tissue.3 Understanding gene expression today has pro- American (around 200), Hispanic (two) and Asian-Pacific gressed from studies of the 5' untranslated/promoter

(two) subjects. We were unable to ﬁnd further evidence of regions to include large constructs of enhancers, matrix on September 26, 2021 by guest. Protected copyright. the S288K allele among these subjects or among disease attachment regions, locus control regions, and methylation

12 34

556 557

554 563 566

554 556 557 554 Figure 1 Family pedigree for the S288K variant. (Left) Pedigree shows the distribution of the wild type allele (grey) and the S288K allele (black). Subjects 554 (proband) and 557 correspond to the aVected daughter and father, respectively. (Right) The SSCP assay of genomic DNA (lane 1, 554, lane 2, 556, and lane 3, 557) and peripheral blood RNA from 554 (lane 4). Arrows depict wild type alleles (grey) and S288K speciﬁc alleles (black).

www.jmedgenet.com Letters 49 sites. Even through the use of large YAC constructs, we 7 Strobl LJ, Kohlhuber F, Mautner J, Polack A, Eick D. Absence of a paused transcription complex from the c-MYC P2 promoter of the translocation J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from know that not all the components necessary for regulation chromosome in Burkitt’s lymphoma cells: implication for the c-MYC P1/P2 of transcription have been identified, nor will they be found promoter shift. Oncogene 1993;8:1437-47. 8 Huppi K, Duncan R, Potter M. MYC-1 is centromeric to the linkage group in proximity to the coding region for the gene of interest. Ly-Sis-Gdc-1 on mouse chromosome 15. Immunogenetics 1988;27:215-19. c-MYC is no exception in that deregulation can occur in 9 Rothberg PG, Otto YM. A polymorphic variant of human c-Myc: conjunction with chromosomal translocations located as Asn11-Ser. Mamm Genome 1995;6:209-11. 10 Huppi K, Siwarski D, Kim J, Diaw L, Mainhart C, Rabkin C, Leitman S, far downstream as the PVT locus (260 kb distant to Chanock S. The frequency of variant alleles of the human c-MYC gene in c-MYC). Thus, we have attempted to identify alleles of healthy control and cancer populations. (Submitted.) 11 Bhatia K, Huppi K, Spangler G, Siwarski D, Iyer R, Magrath I. Point muta- c-MYC and to compare rates of transcription in a search for tions in the c-MYC transactivation domain are common in Burkitt’s controlling regulatory elements in c-MYC. Through the lymphoma and mouse plasmacytomas. Nat Genet 1993;5:56-61. 12 Yano T, Sander CA, Clark HM, Dolezal MV, JaVe ES, RaVeld M. Clustered identification of CAA-33, S11N, and S288K alleles, we can mutations in the second exon of the MYC gene in sporadic Burkitt’s begin the process of systematic classification of c-MYC lymphoma. Oncogene 1993;8:2741-8. 13 Clark HM, Yano T, Otsuki T, JaVe ES, Shibata D, RaVeld M. Mutations in expression and predisposition to disease. the coding region of c-MYC in AIDS-associated and other aggressive lym- DAVID SIWARSKI phomas. Cancer Res 1994;54:3383-6. JENNIFER KIM 14 Bhatia K, Spangler G, Gaidano G, Hamdy N, Dalla-Favera R, Magrath I. Mutations in the coding region of c-MYC occur frequently in acquired LENA DIAW immunodeficiency syndrome-associated lymphomas. Blood 1994;84:883-8. KONRAD HUPPI 15 Gu W, Bhatia K, Magrath IT, Dang CV, Dalla-Favera R. Binding and sup- Laboratory of Genetics, National Cancer Institute/NIH, Building 37, pression of the Myc transcriptional activation domain by p107. Science Room 2B-21, Bethesda, Maryland 20892, USA 1994;264:251-4. 16 Beijersbergen RL, Hijmans EM, Zhu L, Bernards R. Interaction of c-Myc Correspondence to: Dr Huppi, [email protected] with the pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation. EMBO J 1994;13:4080-6. 1 Marcu KB, Patel AJ, Yang Y.DiVerential regulation of the c-MYC P1 and P2 17 Frykberg L., Graf T, Vennström B. The transforming activity of the chicken promoters in the absence of functional tumor suppressors: implications for c-MYC gene can be potentiated by mutations. Oncogene 1987;1:415-21. mechanisms of deregulated MYC transcription. Curr Topics Micro Immunol 18 Riggs KJ, Saleque S, Wong KK, Merrell KT, Lee JS, Shi Y, Calame K. Yin- 1997;224:47-66. Yang-1 activates the c-Myc promoter. Mol Cell Biol 1993;13:7487-95. 2 Obaya AJ, Mateyak MK, Sedivy JM. Mysterious liaisons: the relationship 19 Shrivastava A, Yu J, Artandi S, Calame K. YY1 and c-Myc associate in vivo between c-MYC and the cell cycle. Oncogene 1999;18:2934-41. in a manner that depends on c-Myc levels. Proc Natl Acad Sci USA 1996; 3 Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human 93:10638-41. neoplastic disease. Oncogene 1999;18:3004-16. 20 Shrivastava A, Saleque S, Kalpana GV, Artandi S, Golf SP, Calame K. Inhi- 4 Bentley DL, Groudine M. A block to elongation is largely responsible for bition of transcriptional regulator Yin-Yang-1 by association with c-Myc. decreased transcription of c-MYC in diVerentiated HL-60 cells. Nature Science 1993;262:1889-92. 1986;321:702-6. 21 Crossen PE, Morrison MJ, Colls BM. Increased frequency of the S allele of 5 Eick D, Bornkamm GW. Transcriptional arrest within the first exon is a fast the L-MYC oncogene in non-Hodgkin’s lymphoma. Br J Cancer 1994;69: control mechanism in c-MYC gene expression. Nucleic Acids Res 759-61. 1986;14:8331-46. 22 Fernandez T, Saranth D, Advani SH, Deo MG, Soman CS, Restriction 6 Nepveu A, Marcu, KB. Intragenic pausing and anti-sense transcription fragment length polymorphism of the L-MYC oncogene in non-Hogkin’s within the murine c-MYC locus. EMBO J 1986;5:2859-65. lymphoma patients from India. Cancer Lett 1998;125:165-9.

J Med Genet 2001;38:49–52 E-cadherin is not frequently mutated in following criteria were introduced: (1) two or more docu-

mented cases of diVuse gastric cancer in first/second http://jmg.bmj.com/ hereditary gastric cancer degree relatives, with at least one diagnosed before the age of 50 or (2) three or more cases of documented diVuse gastric cancer in first/second degree relatives, independently of age of onset. In addition, criteria for familial intes- EDITOR—Inherited mutations in the E-cadherin gene 8 (CDH1) were first described in three Maori kindreds with tinal gastric cancer (FIGC) were defined. early onset, diVuse, familial gastric cancer.1 More recently, In the present study, we analysed 11 Finnish gastric can- 2–7 cer patients with a family history of disease and two

this finding has been confirmed in other populations and on September 26, 2021 by guest. Protected copyright. this dominantly inherited familial cancer syndrome has sporadic cases with germline E-cadherin gene mutations been designated a hereditary diVuse gastric cancer (table 1, fig 1). None of these families completely fulfilled (HDGC).4 So far, no germline mutations have been iden- the criteria for other inherited cancer syndromes with tified in site specific intestinal type gastric cancer. Based on predisposition to gastric cancer, for example, hereditary the guidelines of the First Workshop of the International non-polyposis colorectal syndrome (HNPCC), familial Gastric Cancer Linkage Consortium (IGCLC), the adenomatous polyposis (FAP), Peutz-Jeghers syndrome, or

Table 1 Features of the families studied

No of gastric Age of onset and histological subtype Family cancer cases (ddiVuse, iintestinal, uunknown) Other cancer types

17 33d,39u,40d,55d,66d,68d,75d Br*, Bla + Ov, Pro+Bas, Un 22 40d,42d Ski + Pan, Lu 33 37d,55d,57i Br, Mel, Sar, Un 42 36d,63u — 52 39d,67d Bas, CRC 66 41u,42d,50u,71u,71i,75u Br, Br, Leu† , Pan, Sar, Liv 71 56u Thy, Pan, Br, CRC 81 42d Lu, Kid 92 67d,68d Kid, Ut, Bas + Pro, Lu, Ov, Pan+Lu, Leu, Skin, Liv 10 2 27d,54d — 11 3 32u,64d,Unu CRC‡, Lip 12 3 61u,70u,76d CRC, Pan 13 7 45 i,53i,53i,55i,56u,65d,68u Bas, Lip§, Lu, Un, Un

*Gastric and breast cancer; †gastric cancer and leukaemia; ‡gastric and colon cancer; §gastric and lip cancer. Bas, basalioma; Bla, bladder cancer; Br, breast cancer; CRC, colorectal cancer; Kid, kidney cancer; Leu, leukaemia; Lip, lip cancer; Liv, liver cancer; Lu, lung cancer; Mel, melanoma; Ov, ovarian cancer; Pan, pancreatic cancer; Pro, prostate cancer; Sar, sarcoma; Ski, skin cancer; Thy, thyroid cancer; Un, unknown; Ut, uterine cancer.

www.jmedgenet.com 50 Letters

FAM2 FAM1 J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from Un (66)

4 6 Ga (68) Lu Ski (79) Pan (81) ** 236 * Ga (39) Ga (75) Bla (44) Ga (66) Ga (55) Ga (40) Bas (66) 3 Br (63) Ov (52) Pro (69) Ga (42) Ga (40) * 2 342524 Ga (33) 2

FAM3 FAM4 FAM5

Un (48) Ga (63) CRC (86)

4 3 3 Ga (55) Sar (77) Bas (69) Ga (67)

* * 6 * 3 3 5 Ga (36) Ga (39) Br (38)Mel (46) Ga (57) Ga (37) 3 2

FAM7 FAM6 CRC (91) Br (71) Liv (75)

* 2 3 6 Ga (71) Ga (50) Ga (71) Ga (75) Sar Ga (56) Pan (48) Leu * 8 3 21 9 6 2 Br (40) Ga (41) Br (50) Ga (42) Pan Thy (41) (40) 3 22 5

FAM8 FAM9 FAM10

Kid Liv (68)

* 3 5 8 Lu Ga (68) Bas (91) Lu (81) Ov (56) Pan + Lu Ga (54) Pro (94) (45)

* * 2 2 Ga (42) 8 2 Ut (63) Ga (67) Leu (62) Skin (62) Ga (27) http://jmg.bmj.com/ 5 Kid (41)

FAM11 FAM12 FAM13

Un Lip (52) Pan (75) Lu Un

5 3 3 Ga (32) Ga (61) CRC (73) Ga (70) Ga (68) on September 26, 2021 by guest. Protected copyright. * * * 21 4 2 6 Ga (64) Ga (76) Ga (65) Un (76) Lu (59) Ga (56) Ga (53) Ga (55) Ga (53) Bas (50) CRC (59) Lip (52) 22 Gastric cancer 3 5 Ga (45) Other/unknown type of cancer 2 Figure 1 Pedigrees of gastric cancer families. Patients analysed in this study are marked by an asterisk. An arrow depicts the person carrying the P172R change (family 1). Bas, basalioma; Bla, bladder cancer; Br, breast cancer; CRC, colorectal cancer; Ga, gastric cancer; Kid, kidney cancer; Leu, leukaemia; Lip, lip cancer; Liv, liver cancer; Lu, lung cancer; Mel, melanoma; Ov, ovarian cancer; Pan, pancreas cancer; Pro, prostate cancer; Sar, sarcoma; Ski, skin cancer; Thy, thyroid cancer; Un, unknown; Ut, uterine cancer. The age at diagnosis, when known, is shown in parentheses.

Li-Fraumeni syndrome (LFS).89Five of the families stud- from each of the families was isolated according to stand- ied fulfilled the criteria for HDGC syndrome (table 1, fig 1, ard procedures. Exons were amplified using primers Nos 1-3, 5, and 10) and five families included two or more described by Berx et al,10 except exons 4 and 5, which were cases of gastric cancer (one of which was confirmed to be amplified as described in Gayther et al.2 The reactions were of diVuse type) (table 1, fig 1, Nos 4, 6, 9, 11, and 12). carried out in a 50 µl reaction volume containing 100 ng of Family 13 included four intestinal type gastric cancer cases genomic DNA, PCR buVer (PE/ABI, Foster City, CA), and therefore seems to belong to FIGC. However, one of 200 µmol/l each dNTP (Finnzymes, Espoo, Finland), 0.6 the patients in this family had diVuse type carcinoma. In µmol/l each primer, and 1 unit AmpliTa q GOLD polymer- addition to gastric cancer, 11 families also displayed other ase (PE/ABI). The concentrations of MgCl2 in the reaction cancer types. mixture were as described by Berx et al,10 except that for

E-cadherin mutation analysis was performed by genomic exon 6 the concentration of MgCl2 was 1.5 mmol/l and for sequencing of the 16 coding exons including exon/intron exon 1 DMSO (5%) was included in the reaction mixture. boundaries. DNA from one patient with gastric cancer PCR reactions were carried out as described in Berx et al10

www.jmedgenet.com Letters 51 with the following changes: annealing temperature for exon

A J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from 2 was 54°C, for exon 6 56°C, for exon 8 54°C, and for exon 13 57°C. Sequencing reactions containing 40 ng of the PCR product with 3.2 pmol of the sequencing primer in a ATTTCCTAAA volume of 12 µl were performed using ABI Prism Dye Ter- minator Cycle Sequencing Ready Reaction Kit (Perkin- 140 Elmer, Foster City, CA) or ABI Big Dye Terminator Kit (Perkin-Elmer, Foster City, CA) according to the manufac- turer’s instructions. Sequencing reactions were electro- phoresed either on 6% Long Ranger gels, containing 8 mol/l urea, or 5% Long Ranger gels, containing 6 mol/l urea, and analysed on an Applied Biosystems model 373A or 377 automated DNA sequencers, respectively. Exons 1 and 4 were amplified as described above and analysed using SSCP from 84 and 212 cancer free controls, respectively. After PCR, 5 µl of each sample was mixed with 5 µl of denaturing loading buVer (95% formamide, 20 mmol/l EDTA, 0.05% bromphenol blue, 0.05% xylene ° B cyanole FF), denaturated for five minutes at 94 C and 12 345 loaded into a 0.4 mm × 30 cm × 45 cm gel. Electrophore- sis was performed for exon 1 using gels containing 0.5 × MDE solution (AT Biochem, Malvern, PA) and 0.6 × TBE buVer and were run at 4 W for 20 hours. Exon 4 was analysed using 1 × MDE solution and 2.5 mol/l urea at 6 W for 14.5 hours. The gels were silver stained according to standard procedures. We detected one potential missense mutation in the coding E-cadherin gene sequence (table 1, fig 1, No 1). A C to G Figure 2 (A) Direct sequencing shows a heterozygous C→G change (P172R, see change occurred in codon 172 in exon 4 resulting in substi- arrow). (B) SSCP analysis of the P172R tution of proline by arginine (P172R) (fig 2A). This family change. A positive control (lane 4) was included contains seven gastric cancer cases in three diVerent genera- in all SSCP runs. tions. Three of the aVected subjects had gastric cancer under 50 years of age (33, 39, and 40 years). One of them also had tion of the mutation in aVected cases in this particular fam- ductal breast cancer. In addition, one patient with both ily, it seems that this change is not a pathogenic mutation. It bladder and ovarian cancer and another with prostate cancer seems to be a very rare polymorphism because none of the and basalioma were found in this family. To investigate the 212 cancer free controls carries this change. This finding is segregation of this missense type change in the family, we interesting because altogether seven gastric cancer cases 8 screened two additional family members with gastric cancer were found in this family. Caldas et al have suggested that http://jmg.bmj.com/ (fig 1). DNA from paraYn embedded tissues was isolated E-cadherin should account for 25% of the families fulfilling according to standard procedures and mutation analysis was the established criteria for HDGC. However, PCR based performed as described above. However, neither of them screening methods used in this study do not allow detection carried the P172R change. One of the patients studied was of all mutation types, for example, large deletions. the mother of the mutation carrier. The father of this patient Our results support the notion that germline mutations died at the age of 94 years and was cancer free. This change in the E-cadherin gene are responsible for only a subset of was also absent in 212 control samples from cancer free gastric cancer patients with a family history of the disease. subjects, as screened by SSCP analysis (fig 2B). The change In our study, no mutations were found in 13 gastric cancer on September 26, 2021 by guest. Protected copyright. appears to be a rare polymorphism. probands. Five of the families studied fulfil the criteria for Four additional polymorphisms of the E-cadherin gene HDGC and one for FIGC. Our data suggest that for the were found in this series of gastric cancer patients. A C to purpose of eYcient E-cadherin mutation detection, there T silent change in codon 692 (from alanine to alanine) may be a need for more stringent criteria for HDGC, such occurred in eight of 13 (61.5%) gastric cancer patients. A as requiring three aVected subjects as is common in C to T change in codon 751, resulting in aspartate substi- research on familial breast and colon cancer. However, our tution by asparagine, was detected in three of 13 (23%) data set is limited. Loose inclusion criteria should encour- patients. These two polymorphisms have been previously age collection of gastric cancer families. This is important, reported.10 11 A C to G change was found before the start because further work is necessary to identify predisposing codon (−71 bp) in the non-coding region in one of 13 gene(s) for a subset of HDGC families, as well as families (7.7%) gastric cancer patients and in two of 51 (3.9%) segregating intestinal gastric cancer. cancer free controls. A T to C change at position +6 in intron 1 occurred in five of 13 (38%) gastric cancer We thank Inga-Lill Svedberg, Tuula Lehtinen, Sinikka Lindh, Annika Lahti, and Kirsi Laukkanen for technical assistance. patients and in 18 of 51 (35%) cancer free controls. EGLE AVIZIENYTE* So far, altogether 14 truncating E-cadherin germline VIRPI LAUNONEN* 8 mutations have been detected in gastric cancer patients. A REIJO SALOVAARA† few putative missense germline mutations have been TUULA KIVILUOTO‡ reported but their functional significance has not been LAURI A AALTONEN* 167 *Department of Medical Genetics, Haartman Institute, PO Box 21, tested. In the sporadic type of cancer there seems to be a FIN-00014 University of Helsinki, Finland cluster of mutations between exons 7 and 9 whereas germ- †Department of Pathology, Haartman Institute, PO Box 21, FIN–00014 line mutations are more evenly distributed.811A novel mis- University of Helsinki, Finland sense type change, P172R, found in this study is located in ‡Second Department of Surgery, FIN-00029 HUCH, Helsinki University exon 4 which encodes a large extracellular domain with Central Hospital, Helsinki, Finland Ca2+ binding motifs (exons 4-13).10 Based on the segrega- Correspondence to: Dr Aaltonen, lauri.aaltonen@helsinki.fi

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1 Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P, 6 Yoon KA, Ku JL, Yang HK, Kim WH, Park SY, Park JG. Germline Taite H, Scoular R, Miller A, Reeve AE. E-cadherin germline mutations in mutations of E-cadherin gene in Korean familial gastric cancer patients. J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from familial gastric cancer. Nature 1998;392:402-5. Am J Hum Genet 1999;44:177-80. 2 Gayther SA, Gorringe KL, Ramus SJ, Huntsman D, Roviello F, Grehan N, 7 Shinmura K, Kohno T, Takahashi M, Sasaki A, Ochiai A, Guilford P, Machado JC, Pinto E, Seruca R, Halling K, MacLeod P, Powell SM, Jack- Hunter A, Reeve AE, Sugimura H, Yamaguchi N, Yokota J. Familial gastric son CE, Ponder BAJ, Caldas C. Identification of germline E-cadherin cancer: clinicopathological characteristics, RER phenotype and germline mutations in gastric cancer families of European origin. Cancer Res p53 and E-cadherin mutations. Carcinogenesis 1999;20:1127-31. 1998;58:4086-9. 8 Caldas C, Carneiro F, Lynch HT, Yokota J, Wiesner GL, Powell SM, Lewis 3 Richards FM, McKee SA, Rajpar MH, Cole TRP, Evans DGR, Jankowski FR, Huntsman DG, Pharoah PDP, Jankowski JA, MacLeod P, Vogelsang JA, McKeown C, Sanders SA, Maher ER. Germline E-cadherin gene H, Keller G, Park KGM, Richards FM, Maher ER, Gayther SA, Oliveira C, (CDH1) mutations predispose to familial gastric cancer and colorectal Hum Mol Genet 8 Grehan N, Wight D, Seruca R, Roviello F, Ponder BAJ, Jacson CE. Famil- cancer. 1999; :607-10. J Med Genet 4 Guilford PJ, Hopkins JB, Grady WM, Markowitz SD, Willis J, Lynch H, ial gastric cancer: overview and guidelines for management. Rajput A, Wiesner GL, Lindor NM, Burgart LJ, Toro TT, Lee D, Limacher 1999;36:873-80. JM, Shaw DW, Findlay MPN, Reeve AE. E-cadherin germline mutations 9 Vogelstein B, Kinzler KW. The genetic basis of human cancer. New York: define an inherited cancer syndrome dominated by diVuse gastric cancer. McGraw-Hill, 1998. Hum Mutat 1999;14:249-55. 10 Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJF, van de Vijver MJ, 5 Keller G, Vogelsang H, Becker I, Hutter J, Ott K, Candidus S, Grundei T, Cornelisse C, van Roy F. E-cadherin is a tumour/invasion suppressor gene Becker K-F, Mueller J, Siewert JR, Höfler H. DiVuse type gastric and lobu- mutated in human lobular breast cancers. EMBO J 1995;14:6107-15. lar breast carcinoma in a familial gastric cancer patient with an E-cadherin 11 Berx G, Becker K-F, Höfler H, van Roy F. Mutations of the human germline mutation. Am J Pathol 1999;155:337-42. E-cadherin (CDH1) gene. Hum Mutat 1998;12:226-37.

J Med Genet 2001;38:52–58 The spectrum and evolution of exhibited autistic behaviour consisting of arm flapping, head banging, and repetitive and self-stimulatory manner- phenotypic findings in PTEN mutation isms. Hyperpigmented macules of the penile shaft were positive cases of first detected at the age of 7 years 3 months. They had not been present at 41⁄2 years during a previous evaluation Bannayan-Riley-Ruvalcaba syndrome when the diagnosis of BRRS had been considered based on the presence of macrocephaly, developmental delay, and a lipoma on the back. Laboratory evaluations included frag- ile X testing and karyotype analysis, both of which were EDITOR—Bannayan-Riley-Ruvalcaba syndrome (BRRS) is normal. At his most recent evaluation, at the age of 8 years an autosomal dominant condition which includes the fea- 10 months, he had height and weight measurements on the tures of macrocephaly, hyperpigmented penile macules, 95th centile. His OFC was 58.8 cm, on the 98th centile for and hamartomatous tumours, including lipomas, haeman- 1–4 an adult male. He had obvious mental retardation, with giomas, and gastrointestinal polyps. In 1996, it was rec- very few words and markedly autistic behaviour. His ognised that BRRS shared features with Cowden syn- palpebral fissures were downward slanting. drome, another autosomal dominant condition with 5 The proband’s older sister, III.1 in family 1, was 5 years multiple hamartomas. Cowden syndrome is characterised 9 months old at the time this family was first seen. She was by trichilemmomas (small, benign hair follicle tumours), born at ∼37 weeks’ gestation after an uncomplicated preg- oral papillomas, intestinal polyps, and increased frequency nancy with birth weight 4000 g (>97th centile) and length http://jmg.bmj.com/ V 6 of breast and thyroid cancers in a ected subjects. Germ- 50.8 cm (97th centile). She had an isolated, small, left PTEN line mutations in the gene (phosphatase and tensin groin lipoma at the initial evaluation. She sat at 7 months, homologue deleted on chromosome 10), a gene associated walked at 15 months, and exhibited delayed speech with somatic deletion in a number of cancer cell lines and although to a much lesser degree than her brother. At her some primary tumours, were identified in families with most recent evaluation at the age of 9 years 10 months, her Cowden disease the following year.7–10 At the same time, height was between the 75th and 90th centiles, her weight mutations in the PTEN gene were identified in several 11 was on the 75th centile, and her OFC was 57 cm, just less BRRS families, providing evidence that these conditions on September 26, 2021 by guest. Protected copyright. are allelic. Identical mutations have been identified in some than the 98th centile for an adult female. She had families with Cowden syndrome and in others with downward slanting palpebral fissures, joint hypermobility, BRRS.12 In addition, families whose members have and a high arched palate. overlapping features of both conditions have been III.3 in family 1 was first evaluated at 3 years. Her birth identified.13 14 However, publications on BRRS provide lit- history was unavailable. She walked at 18 months and used tle clinical information on the natural history and progres- a few single words at 3 years. When last evaluated, at the sion of this condition. Here we review our experience fol- age of 7 years 1 month, she had height on the 95th centile, lowing 10 subjects in three families with BRRS and PTEN weight greater than the 95th centile, and head circumfer- mutations. The criteria for ascertainment were at least one ence of 58 cm, greater than the 98th centile for an adult aVected person in a family with at least two of the three female. She exhibited mild mental retardation, most nota- features of macrocephaly, hamartomas (including at least bly speech delay, and had joint hypermobility and a high one lipoma, haemangioma, or intestinal polyp), and penile arched palate, but did not have any cutaneous manifesta- macules in males. AVected subjects were found to have tions of BRRS. germline PTEN mutations by DNA analysis.12 13 15 The III.4 in family 1 (fig 2A) was born at 37 weeks’ gestation pedigrees of the families are presented in fig 1 and their after an uncomplicated pregnancy with a birth weight of clinical features are summarised in table 1. 3490 g. Other birth parameters were unavailable. He Family 1 is of Native American descent and has been walked at 21 months and had three to four words at his first followed for six years (fig 1A). The father and four of his genetic evaluation at 22 months of age. At his next evalua- five children are aVected. The proband, III.2, was born at tion, aged 4 years 3 months, he had two small hyperpig- 37 weeks’ gestation after an uneventful pregnancy with mented macules involving the penis. He developed seizures birth length and OFC between the 90th and 97th centiles. related to hypoglycaemia at the age of 12 months, and had He has been a healthy child but had markedly delayed cog- an extensive metabolic workup that was not informative. nitive development. He sat at 8 months, walked at 16 Normal laboratory studies have included electrolytes, months, and had only one word at 4 years of age. He has thyroid function tests, insulin, growth hormone, cortisol,

www.jmedgenet.com Letters 53 lactate, carnitine (urine and plasma), urine organic acids, 64.2 cm, much greater than the 98th centile. He was J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from and plasma amino acids. He has a history of ketonuria hirsute and had a high arched palate. associated with hypoglycaemia. A muscle biopsy was III.5, the youngest child in family 1, is normocephalic, normal, without evidence of lipid myopathy, although the without hamartomas or penile macules. When last muscle carnitine levels were somewhat low and he is being evaluated at the age of 3 years 9 months, his height and treated with oral carnitine. A cranial MRI performed at 3 weight were between the 75th and 90th centiles and his years 8 months and repeated two years later showed OFC was 52 cm (80th centile). He had mild joint normal ventricles and patchy increased T2 signal in the hypermobility and a normal palate. He has had normal deep and subcortical white matter of both occipital lobes development. In this family, there has not been any docu- with prominent perivascular spaces. His EEG was mented breast or thyroid cancer, nor gastrointestinal abnormal, with diVuse slowing and epileptiform discharges polyps, although formal endoscopy has not been per- over both occipital lobes. When last evaluated at the age of formed. The father’s sibs and parents were unavailable for 7 years 2 months, he had height and weight both above the evaluation. 95th centile and an OFC of 58.2 cm (98th centile for an Family 2 is of Dutch and other European extraction (fig adult male). He had four visible macules on the shaft of the 1B). The proband, III.1, has been followed for three years. penis. His back was hirsute. He continued to exhibit global He was initially evaluated for macrocephaly, speech delay, developmental delay and had joint hypermobility and a and a family history of Cowden syndrome. Born at term, high arched palate. his birth weight was on the 90th centile and birth length The proband’s father, II.5 in family 1, has had learning was between the 10th and 50th centiles. Because of a large diYculties and macrocephaly (fig 2B). He was reportedly a head and a birthmark along the spine, he had a head CT large infant. He has multiple hyperpigmented macules on scan which showed megalencephaly without hydrocepha- his penis. At the age of 28, he had a thyroidectomy for goi- lus, and an MRI of the lumbar spine was normal. When tre. Pathological examination showed adenomatous nodu- evaluated at the age of 10 months, he had normal develop- lar hyperplasia without evidence of carcinoma. At the last ment, macrocephaly, and a lipomatous vascular malforma- evaluation aged 29 years, his height was on the 50th centile, tion in the lumbar spine region. When evaluated at 3 years weight was just greater than the 95th centile, and OFC was 2 months, he had a broad forehead, a fleshy vascular mal-

A Family 1 Native American B Family 2 European

I I

1 2 1 2 GI polyps

II II

22 3 http://jmg.bmj.com/ 1, 2 3, 4 5 6 1 2, 3, 4 5 6 29 y 30 y 26 y Thyroid Thyroid Ca E– E+ GI polyps III III

12345 1 10 y 9 y 7 y 6 y 4 y 3 y on September 26, 2021 by guest. Protected copyright. E+ E+ E+ E+ E– E+

C Family 3 European

1 2 3 IDDM 51 y d 36 y Seizures MVA II African American

26= Macrocephaly 1, 2 3 4 5 6 7 23 y 21 y 22 y = Lipoma/haemangioma Hydrocephalus Seizures Arnold-Chiari = MR/DD III = Penile macules 1 2 5 y 1 3 2 y E = PTEN mutation analysis E+ E+ Figure 1 Pedigree of (A) family 1, (B) family 2, and (C) family 3. MR/DD indicates mental retardation/developmental delay. MVA = motor vehicle accident. IDDM = insulin dependent diabetes mellitus.

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Table 1 Clinical features of BRRS patients J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

Family 1 Family 2 Family 3 Total (%)

Male:female ratio 3:2 2:0 2:1 7:3 Primary selection criteria for BRRS Macrocephaly 5/5 2/2 3/3 10/10 (100) Penile macules* 3/3 males 0/1 males 1/2 males 4/6 (67) Hamartomata* 2/5 2/2 0/2 4/9 (44) Lipomas 2103 Haemangiomas0101 Known features of BRRS/Cowden MR/DD 5/5 2/2 3/3 10/10 (100) Motordelay*4127 Speech delay* 4116 Thyroid abnormalities* 1/5 1/2 0/2 2/9 (22) GI polyps* 0/5 1/2 0/2 1/9 (11) Facial papules* 0/5 1/2 0/2 1/9 (11) Other ﬁndings High arched palate* 5/5 1/1 2/2 8/8 (100) Overgrowth 5/5 1/2 2/2 8/9 (89) Prenatal*† 2125 Postnatal3104 Joint hypermobility* 5/5 0/1 2/2 7/8 (88) Hypotonia* 2/5 1/1 2/2 5/8 (63) Frontal bossing* 0/5 1/1 2/2 3/8 (38) Hypoglycaemic episodes* 1/5 0/1 2/3 3/9 (33) Seizures 1/5 0/2 2/3 3/10 (30) Downsl palp ﬁssures* 2/5 0/1 0/2 2/8 (25) Broad thumbs/big toes* 0/5 0/1 2/2 2/8 (25) Café au lait macules* 1/5 1/1 1/2 3/8 (38)

MR/DD = mental retardation/developmental delay. Downsl palp fissures = downward slanting palpebral fissures. *An aVected parent(s) from families 1, 2, and/or 3 was unavailable for assessment of these features and is not included in the total. †Birth weight or length greater than 95th centile; birth parameters not available for several members of family 1. formation measuring ∼3 × 4 cm, located above the gluteal cleft (fig 3), and several small café au lait macules. He had delayed motor development, and had a vocabulary of only five to ten words. There were no hyperpigmented penile Figure 2 Features of BRRS in family 1. (A) Macrocephaly but otherwise normal cranial configuration of III.4. (B) The father (II.5) also macules. Normal laboratory studies included thyroid pro- has macrocephaly. file, growth hormone levels, and karyotype. A cranial MRI at 3 years 9 months showed a small left frontal venous mal- disorder, with normal EEG and MRI as an infant (except formation and evidence of mild prominence of the perivas- for macrocephaly), episodic ketotic hypoglycaemia, and a history of hypotonia. Motor milestones were moderately cular spaces. At 3 years 10 months, his height and weight http://jmg.bmj.com/ were above the 95th centiles, with an OFC of 59 cm (>98th delayed with crawling at 17 months and walking at 18 centile for an adult male). There were no penile macules. months, and he had language impairment with more severe II.5 in family 2, the proband’s father, has had expressive than receptive delays. When first evaluated in macrocephaly, multiple lipomas, and learning problems. the genetics clinic aged 2 years 4 months, he was noted to His birth weight was between the 10th and 50th centiles have macrocephaly with mild frontal bossing but no and the delivery was complicated owing to large head size. dysmorphic facial features. His skin examination was nota- At the age of 11 years 11 months, he was documented to ble for three café au lait macules on his trunk, only one have an OFC of 60.2 cm (>98th centile for an adult male), measuring greater than 0.5 cm in diameter, as well as a left on September 26, 2021 by guest. Protected copyright. with weight on the 75th and height on the 90th centiles. He preauricular telangiectasia from an involuted capillary had undergone several surgical resections of subcutaneous haemangioma. No lipomas or penile macules were identi- lipomas. A head CT scan was normal. He exhibited fied. At his most recent evaluation, aged 5 years 2 months, delayed motor and cognitive skills with intelligence in the his height was between the 50th and 75th centiles, weight borderline range (IQ 77-79). He developed a goitre in his between the 90th and 95th centiles, and OFC measured teenage years and underwent thyroidectomy at the age of 60.2 cm (>98th centile for an adult male). Other findings 26 for papillary carcinoma. He was later found to have included a high arched palate, joint hypermobility, and benign polyposis during evaluation for gastrointestinal broad thumbs and big toes, but no penile hyperpigmenta- complaints. Based on the presence of facial skin papules, he tion. He had speech articulation problems. A karyotype was tentatively given the diagnosis of Cowden syndrome, study was normal. although the biopsy was not confirmatory for trichilemmo- The proband’s maternal half brother, III.2 in family 3 mas. We have no information regarding penile macules (fig 4A, B), whose father is of probable African-American because he was not examined for this finding and is extraction, was born at 37.5 weeks’ gestation by repeat unavailable for evaluation. caesarean section. Prenatally detected macrocephaly Other contributory history for family 2 is a report of prompted amniocentesis, which showed a normal karyo- gastrointestinal polyps in I.1, the proband’s paternal type. Birth weight was 3940 g (97th centile) and length was grandfather. He did not have thyroid or learning problems. 53.5 cm (just greater than the 97th centile). OFC was not Family 3 is of predominantly European extraction and available. He had a normal newborn course but was evalu- has been followed in the genetics clinic for three years (fig ated at 6 months for hypotonia and motor delay. At 71⁄2 1C). The proband, III.1 (fig 4A, B), was born at 36 weeks’ months, he was noted to have height and weight on the gestation by caesarean section for fetal macrocephaly with 50th centile but OFC measuring 51 cm (>98th centile). birth weight 3630 g (97th centile). At the age of 4 months, During genetic evaluation at 10 months, he had macro- his OFC measured 45 cm (>98th centile) with weight and cephaly, frontal bossing, midface hypoplasia, hypotonia, height on the 25th centile. He had a generalised seizure and joint laxity. DiVuse hyperpigmentation of the penis was

www.jmedgenet.com Letters 55 J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

Figure 3 Lipomatous vascular malformation on the back of proband (III.1) in family 2. noted. At his most recent evaluation, at 3 years 7 months, Molecular analysis was performed on genomic DNA his growth parameters included height on the 50th-75th extracted from peripheral blood leucocytes as previously centile, weight on the 90th centile, and OFC of 59.8 cm described.12 15 In family 1, a germline heterozygous (>98th for adult male). Four discrete hyperpigmented nonsense mutation in the PTEN gene was identified at macules were identified on the penile shaft and two on the codon 130 leading to premature termination of the protein scrotum. He had mild hypotonia and joint hypermobility, (R130X) within the highly conserved phosphatase domain. as well as a high arched palate. In spite of mild motor delay, The father and four aVected children carried the R130X he has had normal language acquisition and has not quali- mutation, which was not present in the youngest fied for special education programmes. unaVected child, III.5. In family 2, blood was obtained for The biological mother of the two boys (II.6 in family 3) PTEN mutational analysis on the proband and his has not been available for evaluation and has not been their unaVected mother, and a mutation was identified in the caregiver owing to concerns of neglect. She is reported to proband at position 5 in intron 6 (IVS6+5G→T). His have an OFC of 63 cm (much greater than the 98th centile mother did not share this allele. In family 3, only the for an adult female) and a history of childhood seizures, proband and his half brother were available for testing. A hypoglycaemic episodes, and developmental disabilities. Of mutation in the PTEN gene at position 5 in intron 6 her four full brothers, two are reported to have macro- (IVS6+5G→A) was detected in one allele from each of the cephaly, one with hydrocephalus and an Arnold-Chiari two boys. This specific mutation has not been previously http://jmg.bmj.com/ malformation (II.3). The proband’s maternal grandfather described. The mutations in families 2 and 3 are likely to (I.1) reportedly had macrocephaly and diabetes and died lead to aberrant RNA splicing and a truncated protein aged 36 in a car accident. There are no reports of breast or product. thyroid cancer or gastrointestinal polyps in this family, In identifying our families with BRRS, we focused on although endoscopy has not been performed. subjects possessing at least two of the three features of on September 26, 2021 by guest. Protected copyright.

Figure 4 Features of BRRS in family 3. (A) Body habitus of aVected boys III.2 (left) and III.1 (right). (B) Macrocephaly in the same two boys.

www.jmedgenet.com 56 Letters macrocephaly, hamartomas, and penile macules, criteria variable, with the father exhibiting the mildest learning J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from which have been used to ascertain BRRS patients for problems. All of his aVected children were in special edu- molecular studies.11 13 It has been suggested that the mere cation programmes, and the two aVected sons had the presence of macrocephaly/macrosomia and developmental greatest impairment, with the oldest son at the age of 9 delay, without hamartomata or penile macules, should years showing autistic behaviour and minimal expressive warrant further evaluation for PTEN mutations.16 In fact, language. To our knowledge, autism has not been macrocephaly has been the most consistent finding in described previously in this condition. The second family BRRS in numerous reviews17–19 and was present in all of our had two aVected males with learning problems; the father aVected subjects. The macrocephaly consists of megalen- had borderline intelligence on repeated testing and his son cephaly without ventricular dilatation and is generally had significant global delay and behavioural problems. The present at birth.20 We were surprised by the degree of mac- aVected children in both of these families appeared to have rocephaly in all aVected members of family 1, with head more severe cognitive impairment than their parents, a circumference measurements greater than the 98th centile phenomenon which has been described in other families for age, in the absence of facial dysmorphism or other dis- with BRRS or Cowden syndrome.14 23 24 In family 3, both tinguishing cranial features such as frontal bossing. children exhibited motor and speech delay, although the Presumably, the overall large body size masked the dispro- oldest son was more severely aVected; their mother was portionately large head size in these subjects. Families 2 reported to have developmental disabilities as well. The and 3 also exhibited marked macrocephaly, and the true prevalence of mental retardation in this disorder aVected children had a broad, prominent forehead that has remains to be established, but for those children suspected been previously described in BRRS.21 of carrying this diagnosis, developmental assessments and The broad spectrum of hamartomata is illustrated by appropriate therapeutic interventions are important as- these families. Although family 1 was first brought to medi- pects of care. cal attention because of familial macrocephaly and develop- Based on the clinical features in these aVected subjects, mental delay, the diagnosis of BRRS was not suspected until summarised in table 1, we suggest that some “soft” clinical single lipomas were identified in two of the four aVected signs may aid in diagnosis when a young child exhibits children. As the children have grown, these lipomas have macrocephaly and learning delay but may not have devel- become less visible. None of the members in family 1 has oped hamartomata or, if male, penile macules. One of these been evaluated for gastrointestinal polyposis and symptoms features is a high arched palate, which has been identified such as intestinal bleeding or chronic abdominal pain have in 56-70% of BRRS patients in other surveys17 20 25 and was not been reported. The father in family 2 had several classi- identified in all of our patients. Overgrowth, of either pre- cal tumours of BRRS, including multiple lipomas evident by natal or postnatal onset, is a feature exhibited by almost his teens and intestinal polyps. His young son had only a 90% of our cohort. Approximately 50% of newborns with vascular malformation along the lumbar spine. In contrast, BRRS have been reported to have large birth weight and the aVected boys in family 3 have had no obvious length, with subsequent postnatal growth deceleration hamartomata. These cases illustrate the importance of a resulting in normal growth parameters by adulthood.18 20 thorough skin examination at each evaluation of a child with As newborns, five of the patients were macrosomic, with macrocephaly, although the absence of dermatological either birth weight or length greater than the 95th centile. V

abnormalities does not exclude the diagnosis. Three of the a ected children in family 1 have exhibited http://jmg.bmj.com/ Penile macules are perhaps the most distinctive and postnatal overgrowth, as has the aVected boy in family 2. potentially valuable diagnostic feature of this syndrome. The father and oldest daughter in family 1 now have nor- This is shown by family 1, in which penile macules were mal height and weight, suggesting that the overgrowth important in establishing the diagnosis and were not iden- resolves by adulthood and perhaps puberty. Since the dis- tified in the oldest son (III.2) at the age of 41⁄2 years but turbance in growth velocity appears to be age dependent, it were seen at 7 years 3 months. His younger brother had two may not be appreciated without following growth curves very small hyperpigmented macules identified at the age of over time. Other findings with greater than 50% incidence 1 4 ⁄2 years and four small penile macules by 7 years 2 in our cohort include joint hypermobility and hypotonia. on September 26, 2021 by guest. Protected copyright. months. Only one of the two aVected half brothers in fam- Hyperextensibility of joints has been reported in approxi- ily 3 had penile macules, which were first visualised mately 50% of patients with BRRS,17 20 25 and hypotonia in 17 20 between the ages of 21⁄2 and 31⁄2 years, although approximately 20%. These reports also identified generalised penile hyperpigmentation was seen in infancy. downward slanting palpebral fissures in a majority of their He is of mixed racial heritage, with an African-American patients, a finding which was less common in our cohort. father, which may increase the likelihood of pigmentation, Frontal bossing, hypoglycaemic episodes, seizures, and since his fully white older brother with the identical muta- café au lait macules were all identified in approximately 1/3 tion did not have penile macules. The penile macules of our patients. The presence of these less specific findings detected in all of the children were very small pigmentary may support the diagnosis of BRRS. changes that might be missed during a cursory examina- It has been suggested that BRRS shows a male prepon- tion and were far less obvious than many published photo- derance, and that this reflects the overall increased graphs.518We conclude that speckling of the penis is more incidence of macrocephaly in males.19 26 In previous likely to occur in later childhood, and its absence in infants surveys, even before the recognition of penile macules as a and toddlers should not exclude consideration of the diag- clinical feature of the disorder, ∼70% of identified patients nosis of BRRS. were male.17 20 In our three families, seven out of 10 Mental retardation has been a feature in case reports of subjects at risk for BRRS were male, with six of these males BRRS22 and has been reported in 15-20% of aVected sub- mutation positive. However, both at risk females in family jects in two previous surveys.17 20 More common are motor 1 were also aVected, so we do not have adequate numbers and speech delays occurring in childhood in approximately to draw conclusions regarding this male preponderance. 50% of patients. These delays are reported to improve with Since our inclusion of penile macules as a diagnostic crite- age in many cases,18 and adults are often described as hav- rion may lead to bias in recognising males with this disor- ing motor dysfunction with normal IQ.20 In our families, all der, further studies incorporating mutation analysis are aVected subjects had some degree of learning impairment. warranted to confirm the observation that more males are In family 1, the degree of cognitive disability was highly aVected with BRRS. The opposite sex ratio may exist for

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Cowden syndrome, which has been reported to exhibit a ily2beoVered to these brothers and their other at risk J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from female preponderance.24 27 However, breast cancer is much family members. more prevalent in females in general, and women may be In summary, our three families with PTEN mutation more likely to report facial papules because of cosmetic confirmed BRRS illustrate the phenotypic variability concerns, so females with Cowden syndrome may be more within family members with this condition and the time readily identified than aVected males. As a consequence of course for the development of some of the manifestations. these sex specific clinical features, the reported ratios in Macrocephaly appears to be the most consistent feature in each disorder may merely reflect ascertainment bias. BRRS, but may not be obvious and requires measurement PTEN mutations have now been identified in up to 80% and documentation. The natural history of this disorder of patients with Cowden syndrome and in up to 60% of suggests that the distinctive finding of penile macules in those with BRRS, indicating that they are allelic disor- males may not appear until mid childhood, and that cogni- ders.7121315In several cases, the same PTEN mutation has tive impairment, in addition to macrocephaly, may be a been identified in families with a diagnosis of either Cow- prominent feature of BRRS in many families. We suggest den syndrome or BRRS, and in other cases family that postnatal overgrowth during childhood may be members carrying the same PTEN mutation have diVerent common in this condition, and other features such as high diagnoses.13 14 23 There is significant overlap between Cow- arched palate, joint hypermobility, and hypotonia may aid den syndrome and BRRS, and two of the features found in in diagnosis. With the availability of PTEN molecular both conditions are macrocephaly and thyroid abnormali- analysis, genotype-phenotype correlations may be feasible. ties.5618 The father in family 1 had thyroidectomy for Our cases confirm the observation that the clinical features adenomatous nodular changes although he and his of BRRS and Cowden syndrome show significant overlap, children fit the description of BRRS better than Cowden and suggest that until these conditions are better syndrome. Family 2 also illustrates the overlap in understood, genetic counselling should include infor- phenotypic features of these conditions. The son had the mation about the risk of developing thyroid and breast BRRS findings of macrocephaly, a haemangioma, and cancers and gastrointestinal polyps for anyone with a documented PTEN mutation. developmental delay, while his father had macrocephaly, borderline intelligence, and features more consistent with We thank the families who participated in this study and genetic counsellors Cowden syndrome, including GI polyposis, thyroid cancer, Susie Ball and Roger Fick, who helped coordinate the evaluations and testing in and facial papules. In contrast, family 3 has no distinctive these families. We also thank Heather Hampel, genetic counsellor and research coordinator for the PTEN studies, and X P Zhou for technical assistance. This features usually associated with Cowden syndrome, research was funded in part by the National Institutes of Health, grant number although assessment of all at risk subjects has not been 5T32GM07454 (MAP), by the American Cancer Society, grant numbers RPG-97-064-01-VM and RPG 98-211-01-CCE (CE), and by the US Army performed and the boys are still quite young. Cowden syn- Research Medical and Material Command Breast Cancer Research Program, drome may be a more likely diagnosis in adolescents or grant number DAMD17-98-1-8058 (CE). adults because the cardinal features of GI polyposis and MELISSA A PARISI*† MARY BETH DINULOS* thyroid and breast carcinomas are of later onset than the KATHLEEN A LEPPIG* findings of macrocephaly and developmental delay identi- VIRGINIA P SYBERT‡ fied in children diagnosed with BRRS. As we accumulate CHARIS ENG§ PTEN LOUANNE HUDGINS¶ more data on these children with mutations and the *Division of Genetics and Development, Department of Pediatrics, http://jmg.bmj.com/ diagnosis of BRRS, they may develop features classically Children’s Hospital and Regional Medical Center, PO Box 5371/CH-25, associated with Cowden syndrome. Seattle, WA 98105-0371, USA Correlations between genotype and phenotype are †Division of Medical Genetics, Department of Medicine, University of beginning to be elucidated for Cowden syndrome and Washington,Seattle, WA,USA BRRS, with resulting implications for genetic counselling ‡Division of Dermatology, Department of Medicine, University of Washington,Seattle, WA,USA for cancer and related health risks. An association between §Clinical Cancer Genetics Program and Human Cancer Genetics the presence of a PTEN mutation and the development of Program, Ohio State University Comprehensive Cancer Center,

cancer or breast fibroadenomas has been observed in both Columbus, OH, USA on September 26, 2021 by guest. Protected copyright. BRRS and Cowden syndrome.13 Thus, aVected females in ¶Division of Medical Genetics, Department of Pediatrics, Stanford particular may have an increased risk of breast cancer, and University, Stanford, CA, USA we have recommended breast cancer surveillance begin- Correspondence to: Dr Parisi, [email protected] ning at puberty for the daughters in family 1. Mutations in the core phosphatase domain are common, and this 1 Riley HD, Smith WR. Macrocephaly, pseudopapilledema and multiple hemangiomata. Pediatrics 1960;26:293-300. domain appears to be a crucial region for the function of 2 Bannayan GA. Lipomatosis, angiomatosis, and macrencephalia. Arch Pathol the tumour suppressor.12 13 The PTEN mutation identified 1971;92:1-5. 3 Zonana J, Rimoin DL, Davis DC. Macrocephaly with multiple lipomas and in family 1, R130X, is present in this core motif, and has hemangiomas. J Pediatr 1976;89:600-3. been described in other families with either Cowden 4 Ruvalcaba RHA, Myhre S, Smith DW. Sotos syndrome with intestinal polyposis and pigmentary changes of the genitalia. Clin Genet 1980;18:413-16. syndrome, BRRS, or features that overlap both of these 5 Fargnoli MC, Orlow SJ, Semel-Concepcion J, Bolognia JL. Clinicopatho- conditions.12 13 23 The PTEN mutation in family 2 has also logic findings in the Bannayan-Riley-Ruvalcaba syndrome. Arch Dermatol 1996;132:1214-18. been identified as a somatic mutation in a tumour (C Eng, 6 Eng C. Cowden syndrome. J Genet Couns 1997;6:181-92. unpublished data) and probably results in abnormal RNA 7 Liaw D, Marsh DJ, Li J, Dahia PLM, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M, Eng C, Parsons R. Germline mutations of the processing with a truncated protein product. Since the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syn- father with overlapping BRRS/Cowden syndrome features drome. Nat Genet 1997;16:64-7. 8 Lynch ED, Ostermeyer EA, Lee MK, Arena JF, Ji H, Dann J, Swisshelm K, had GI polyposis as well as thyroid cancer in his mid-20s, Suchard D, MacLeod PM, Kvinnsland S, Gjertsen BT, Heimdal K, Lubs H, Moller P, King MC. Inherited mutations in PTEN that are associated we have recommended that the son has an annual manual with breast cancer, Cowden disease, and juvenile polyposis. Am J Hum thyroid examination as well as thyroid function studies, and Genet 1997;61:1254-60. 9 Nelen MR, van Staveren WCG, Peeters EAJ, Hassel MB, Gorlin RJ, Hamm that he seek medical attention for the development of any H, Lindboe CF, Fryns JP, Sijmons RH, Woods DG, Mariman ECM, Pad- breast or neck masses or rectal bleeding. The mutation in berg GW, Kremer H. Germline mutations in the PTEN/MMAC1 gene in patients with Cowden disease. Hum Mol Genet 1997;6:1383-7. family 3 is at the same intronic position as that in family 2, 10 Tsou HC, Teng DHF, Ping XL, Broncolini V, Davis T, Hu R, Xie XX, although the symptoms appear to be more subtle in the two Gruener AC, Schrager CA, Christiano AM, Eng C, Steck P, Ott J, Tavtigian SV, Peacocke M. The role of MMAC1 mutations in early-onset breast half brothers, who are still less than 6 years old. We have cancer: causative in association with Cowden syndrome and excluded in recommended that the same evaluations as those for fam- BRCA1-negative cases. Am J Hum Genet 1997;61:1036-43.

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11 Marsh DJ, Dahia PLM, Zheng Z, Liaw D, Parsons R, Gorlin RJ, Eng C. 17 Moretti-Ferreira D, KoiVmann CP, Souza DH, Diament AJ, Wajntal A. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Macrocephaly, multiple lipomas, and hemangiomata (Bannayan-Zonana J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from Nat Genet 1997;16:333-4. syndrome): genetic heterogeneity or autosomal dominant locus with at 12 Marsh DJ, Coulon V, Lunetta KL, Rocca-Serra P, Dahia PLM, Zheng Z, least two diVerent allelic forms? Am J Med Genet 1989;34:548-51. Liaw D, Caron S, Duboue B, Lin AY, Richardson AL, Bonnetblanc JM, 18 Gorlin RJ, Cohen MM, Condon LM, Burke BA. Bannayan-Riley-Ruvalcaba Bressieux JM, Cabarrot-Moreau A, Chompret A, Demange L, Eeles RA, syndrome. Am J Med Genet 1992;44:307-14. Yahanda AM, Fearon ER, Fricker JP, Gorlin RJ, Hodgson SV, Huson S, 19 DiLiberti JH. Inherited macrocephaly-hamartoma syndromes. Am J Med Lacombe D, LePrat F, Odent S, Toulouse C, Olopade OI, Sobol H, Tishler Genet 79 S, Woods CG, Robinson BG, Weber HC, Parsons R, Peacocke M, Longy 1998; :284-90. M, Eng C. Mutation spectrum and genotype-phenotype analyses in Cow- 20 Miles JH, Zonana J, Mcfarlane J, Aleck KA, Bawle E. Macrocephaly with den disease and Bannayan-Zonana syndrome, two hamartoma syndromes hamartomas: Bannayan-Zonana syndrome. Am J Med Genet 1984;19:225- with germline PTEN mutation. Hum Mol Genet 1998;7:507-15. 34. 13 Marsh DJ, Kum JB, Lunetta KL, Bennett MJ, Gorlin RJ, Ahmed SF, Bod- 21 Higginbottom MC, Schultz P. The Bannayan syndrome: an autosomal urtha J, Crowe C, Curtis MA, Dasouki M, Dunn T, Feit H, Geraghty MT, dominant disorder consisting of macrocephaly, lipomas, hemangiomas, and Graham JM Jr, Hodgson SV, Hunter A, Korf BR, Manchester D, Miesfeldt risk for intracranial tumors. Pediatrics 1982;69:632-4. S, Murday VA, Nathanson KA, Parisi M, Pober B, Romano C, Tolmie JL, 22 Saul RA, Bley R. Mental retardation in the Bannayan syndrome. Pediatrics Trembath R, Winter RM, Zackai EH, Zori RT, Weng LP, Dahia PLM, Eng 1982;69:642-4. C. PTEN mutation spectra and genotype-phenotype correlations in 23 Zori RT, Marsh DJ, Graham GE, Marliss EB, Eng C. Germline PTEN Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden mutation in a family with Cowden syndrome and Bannayan-Riley- syndrome. Hum Mol Genet 1999;8:1461-72. Ruvalcaba syndrome. Am J Med Genet 1998;80:399-402. 14 Celebi JT, Tsou HC, Chen FF, Zhang H, Ping XL, Kezis J, Peacocke M. 24 Hanssen AMN, Werquin H, Suys E, Fryns JP. Cowden syndrome: report of Phenotypic ﬁndings of Cowden syndrome and Bannayan-Zonana syn- a large family with macrocephaly and increased severity of signs in subse- PTEN J drome in a family associated with a single germline mutation in . quent generations. Clin Genet 1993;44:281-6. Med Genet 1999;36:360-4. 15 Marsh DJ, Dahia PLM, Caron S, Kum JB, Frayling IM, Tomlinson IPM, 25 Hayashi Y, Ohi R, Tomita Y, Chiba T, Matsumoto Y, Chiba T. Hughes KS, Eeles RA, Hodgson SV, Murday VA, Houlston R, Eng C. Bannayan-Zonana syndrome associated with lipomas, hemangiomas, and Germline PTEN mutations in Cowden syndrome-like families. J Med Genet lymphangiomas. J Pediatr Surg 1992;27:722-3. 1998;35:881-5. 26 Asch AJ, Myers GJ. Benign familial macrocephaly: report of a family and 16 Longy M, Coulon V, Duboue B, David A, Larregue M, Eng C, Amati P, review of the literature. Pediatrics 1976;57:535-9. Kraimps JL, Bottani A, Lacombe D, Bonneau D. Mutations of PTEN in 27 Starink TM, van der Veen JPW, Arwert F, de Waal LP, de Lange GG, Gille patients with Bannayan-Riley-Ruvalcaba phenotype. J Med Genet 1998;35: JJP, Eriksson AW. The Cowden syndrome: a clinical genetic study in 21 886-9. patients. Clin Genet 1986;29:222-33.

J Med Genet 2001;38:58–61 A frameshift mitochondrial complex I parkinsonism-MELAS overlap syndrome6 and somatic mutations including frameshift mutations have been found gene mutation in a patient with in human cancers.78 In contrast, inherited frameshift dystonia and cataracts: is the mutation mutations in mtDNA have not previously been reported. We now report the identification of an inherited frameshift pathogenic? mutation in a patient with dystonia and maternally inherited cataracts. The normal base pair (T) is replaced by AC at np 3308 (T3308AC) in the mitochondrial gene encoding the ND1 subunit of complex I. Dystonia910and EDITOR—Mitochondrial DNA (MtDNA) is highly poly- 11–13 morphic. Each person is estimated to diVer from another cataracts have each been linked previously to complex I on average at about 25 base pairs among the 16 569 that dysfunction and to mtDNA mutations but, for the reasons http://jmg.bmj.com/ comprise the mitochondrial genome.1 Thus, only a small outlined above, the pathogenicity of the T3308AC fraction of mtDNA variants are likely to be of pathogenic mutation remains uncertain. significance. Criteria currently used for determining the DNA was isolated by standard proteinase K and SDS likelihood that a missense mutation is pathogenic include digestion followed by phenol and chloroform extractions. heteroplasmy (the percentage of mtDNA molecules within DNA was isolated from muscle (III.4), fibroblasts (II.8), or a cell or tissue harbouring a mutation), evolutionary blood (I.1, III.1, IV.1, IV.5, and IV.6). Each of these conservation of the altered amino acid, a maternal inherit- subjects (except IV.1) underwent neurological and oph-

ance pattern, absence of the mutation in controls, clinical thalmological examinations. Clinical and molecular data on September 26, 2021 by guest. Protected copyright. features commonly linked to known pathogenic mtDNA were unavailable from other family members. Polymerase mutations, and defects in mitochondrial morphologies and chain reaction (PCR) amplification of mtDNA and enzyme activities.1 However, these criteria are inadequate sequencing on an ABI 377 automated sequencer (Perkin- 14 for several reasons. Many mitochondrial missense muta- Elmer) were performed as previously described. PCR tions are homoplasmic. Pathogenic mtDNA mutations are reactions for restriction digests were performed with prim- typically characterised by incomplete penetrance, even ers at np 3207-3223 (upper) and 3414-3401 (lower). when homoplasmic, presumably reflecting interactions Restriction digests were performed with MsiI (New with environmental and genetic factors.2 As a result, inher- England Biolabs) and analysed by ultraviolet illumination ited mtDNA mutations may manifest as “sporadic” disor- of a 2% agarose gel permeated with ethidium bromide. The ders rather than with the classical maternal inheritance mutation eliminates the single restriction site for this pattern. Biochemical assays may also be inconclusive, as enzyme. The normal 208 base pair (bp) PCR product is the expression of a defect in mitochondrial function cut into two 104 bp fragments, but in the presence of the depends on the nuclear background and tissue type in T3308AC mutation, a single 208 bp fragment remains. A which the mutation is studied.34 As a result, mtDNA normal control DNA sample was included in each assay to mutations identified in rare families or subjects with a confirm complete digestion by the enzyme. Other PCR and putative mitochondrial genetic disorder are often of uncer- sequencing primers have been published previously.14 tain pathogenic significance. Immunoblotting of the ND1 subunit of complex I was Over 100 point mutations have been identified in mito- performed using lysates of fibroblasts obtained from three chondrial genes in association with human disease, at least aVected family members and one control. Samples (10 µg) 45 of which are missense mutations in protein encoding were loaded and run on a 12% acrylamide minigel, rinsed, genes.5 However, frameshift mtDNA mutations are ex- transferred to a polyvinylidene fluoride membrane (Milli- ceedingly rare. An acquired frameshift 4 bp deletion muta- pore, Bedford, MD), and incubated in blocker containing tion was identified in the cytochrome b gene at nucleotide primary antibody (1:500), as described previously.15 position (np) 14 787-14 790 in a patient with Membranes were thoroughly rinsed, then incubated with

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I J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

12 34

123945678

III

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1 23 4 5 6 Figure 1 Pedigree. The darkened circle (III.4) represents the proband with early onset cataracts, focal dystonia, and episodes of paraesthesias. Cross hatched circles represent maternal relatives with early onset cataracts. Subject IV.1died in infancy of multiple congenital defects. Clinical examinations and DNA analyses were available for I.1, II.8, III.1, III.4, IV.1,IV.5,and IV.6.See text for further clinical details. horseradish peroxidase conjugated secondary antibody. was available for analysis (I.1, II.8, III.1, IV.1, IV.5, and Secondary antibodies were detected by chemilumines- IV.6). The mutation was absent in 108 control subjects cence (Amersham ECL, UK). including 29 with Parkinson’s disease and 23 with adult The proband (III.4, fig 1) developed a unilateral (right) onset focal dystonia. An initiating methionine at this site is cataract in her late teens. At the age of 30 she experienced highly conserved evolutionarily.16 several episodes lasting 30 minutes each of bilateral Sequencing of the entire mitochondrial genome in sub- paraesthesias in her arms and legs with right hemifacial ject III.4 showed 17 known errors or consensus changes in paraesthesias and paresis. Neurological examination the Cambridge sequence.17 Additional changes were showed sensory loss and weakness in the right face, as well observed as follows. Synonymous base pair changes: as right arm weakness. The following year, examination T6620C, C7028T, G11719A, G12007A, C12705T, and showed a right facial dystonia and diVuse hyperreflexia A14470G. Known polymorphisms in the non-coding D with a positive HoVman’s sign on the left, but full strength loop: C16223T, C16290T, G16319A, T16325C, and normal sensation. Plantar responses were flexor. Her T16362C, C64T, A73G, T146C, A153G, A235G, http://jmg.bmj.com/ sister (III.1) developed cataracts in her early teens with a T310C, and C514CAC. Known polymorphisms in rRNA severe left and mild right cataract. This sister’s daughter (IV.1) died a few months after birth with a hypoplastic left 3308 heart, polycystic kidneys, and an ectopic pancreas. The maternal grandmother (I.1) developed a right cataract by the age of 40 years. There was no history of ocular trauma T3308AC TTAAC AACC AACCC A A GG in any of the family members with cataracts. insertion- deletion An extensive evaluation of III.4 included normal brain on September 26, 2021 by guest. Protected copyright. (III·4) computed tomography and magnetic resonance imaging. Electromyography and nerve conduction studies were normal. Muscle biopsy (right vastus lateralis) showed normal light and electron microscopic results. Cytochrome oxidase c staining was normal. No ragged red fibres were seen. Complex I activity, measured as rotenone sensitive NADH cytochrome c reductase activity normalised to citrate synthase, was normal. Cytochrome oxidase activity was normal. Citrate synthase activity was raised (11.08 µmol/min/g compared to 3.35 ± 1.1 for controls). Serum and cerebrospinal fluid lactates were normal. Serum Normal TTTAAC AACCCC A A A GG ammonia was raised at 45 µmol/l (compared to normal of 9-33 µmol/l). Cerebrospinal fluid glucose and protein were normal with no cells or oligoclonal bands. Serum creatine kinase levels were normal. Sequencing both the H and L strands of PCR amplified muscle derived DNA in III.4 showedaTtoACinsertion/ deletion at np 3308 (T3308AC) (fig 2). This converts an ATA (methionine) codon to AACA, creating a frameshift in the initiating methionine codon of the mtDNA gene encoding the ND1 subunit of complex I. The presence of a homoplasmic mutation was confirmed by the elimination Figure 2 Electrophoretograms of subject III.4 (top) and a of an MsiI (New England Biolabs) restriction site in III.4 as control showing a frameshift mutation at position 3308 well as in each of her maternal relatives from whom DNA whereaTisreplaced by an AC in subject III.4.

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unlikely given the relatively mild clinical and biochemical J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from 1234 features associated with the mutation. Alternatively, translation may begin in frame at the next methionine codon, which occurs at np 3313-5, resulting in a truncation of the first two amino acids (methionine and proline) at the amino terminal end of the ND1 subunit, with preservation of a methionine at the amino terminal end. Except for the Figure 3 Western blot analysis of ND1 expression using a initiating methionine, amino acids at the amino terminal polyclonal ND1 specific antibody. Lanes 1, 2, and 4 are 26 from subjects with homoplasmic mutations (lane 4 is the end are not highly conserved evolutionarily. proband); lane 3 is a control subject. ND1 Mutations involving np 3308 have been reported in sev- immunoreactivity (arrow) was clearly detected in all eral patients with neurological abnormalities including two subjects. A minor cross reacting band was also seen (upper 16 band). with dystonia. Campos et al reported a woman with tran- sient ataxia and later seizures and marked generalised dys- genes: A663G, A1736G, and A2706G. Known polymor- tonia who harboured a missense point mutation at np phisms in protein coding genes associated with an altered 3308. Two other unrelated patients, each with multiple amino acid: A4824G (ND1), G8027A (COX II), and neurological deficits, also were found to harbour a C8794T (ATP6). Mutations at non-conserved sites within missense point mutation at 3308.27 One additional family the non-coding D loop (but not known polymorphisms): with maternally inherited hearing loss attributed to the C461T, C505T, and a TT insertion at np 311. The TT T7511C mtDNA mutation also harboured a T3308C insertion at 311 is not a known polymorphism, but a CC point mutation28 but it was not thought likely to be patho- insertion at this site is a known polymorphism. An genic as it was homoplasmic and had been reported previ- insertion ofaCat956occurs in a non-conserved region of ously in controls.27 MtDNA mutations at np 3308 were not the 12S rRNA gene. A mutation identified at 14 280 (A to present in any of our 108 controls. Campos et al16 also G) in the ND6 gene alters a non-conserved amino acid. found no mutations at this site in 130 controls. In contrast, Immunoblotting detected the ND1 protein (apparent Vilarinho et al27 reported that four of 150 controls MW ∼33 kDa) in all fibroblast samples (fig 3). Thus, the harboured a point mutation at 3308. This may reflect the ND1 protein is expressed in subjects with the frameshift ethnic diVerences between these control groups. None of mutation. An additional, minor, cross reacting band of the combined 388 controls in these studies carried a unknown identity and significance was also seen. frameshift mutation as found in the family reported here. Suspicion of a mitochondrial complex I gene mutation in However, the molecular consequences of a point mutation this family was raised by the presence of dystonia and cata- at this site may be identical to that of the T3308AC racts, each of which has been associated with complex I frameshift mutation, resulting in a truncation of the first dysfunction and mtDNA mutations.9–13 The origin of the two amino acids. The cellular impact of the T3308AC complex I deficiency in dystonia is unknown. In one study, mutation is uncertain. A missense mutation in the lung carcinoma derived cell lines expressing mtDNA from initiating codon of the cytochrome c oxidase subunit II nine patients with focal dystonia did not manifest complex gene has been shown to result in lower levels of protein I deficiency.18 However, the biochemical expression of synthesis for this subunit.29 In contrast, we found no

mtDNA mutations is influenced by tissue type as well as evidence of altered ND1 expression in association with the http://jmg.bmj.com/ nuclear background.34 Thus, the role of mtDNA muta- T3308AC mutation, and muscle complex I activity was tions in the complex I defect of patients with focal dystonia normal. The possibility remains that complex I activity remains uncertain. Dystonia clearly can be a prominent could be altered in the basal ganglia, the primary site of manifestation of mtDNA mutations, as shown by missense pathology in many dystonia patients.30 Furthermore, our mutations in a mitochondrial complex I gene in several complex I assay measures maximal enzyme activity families with dystonia plus Leber’s hereditary optic (“Vmax”), and thus an eVect of the mutation on enzyme neuropathy (LHON).19–22 Dystonia is commonly a promi- kinetics cannot be excluded. nent component of Leigh’s syndrome, which can be asso- This is the first identification of an inherited frameshift on September 26, 2021 by guest. Protected copyright. ciated with mtDNA point mutations or deletions.23 24 Dys- mutation in a human mitochondrial protein coding gene. tonia, along with other neurological deficits, was reported However, the pathogenic significance of this mutation in a subset of aVected members of a family harbouring the remains uncertain. This case highlights the diYculties often LHON associated 11 778 complex I mutation,14 and can encountered when interpreting associations between rare be the presenting feature of the 3243 “MELAS” mtDNA variants and putative mitochondrial disorders. mutation.25 Cataracts have also been reported as a clinical 11–13 correlate of mitochondrial complex I dysfunction and This work was supported by grants from the National Institutes of Neurological in patients with mtDNA mutations, including large Disorders and Stroke (K08NS01971, DKS), the National Eye Institute (RO1 13 EY10864, DRJ), and the National Institute on Deafness and Other Communi- deletions. The occurrence of both dystonia and mater- cation Disorders (R01 DC03401, DRJ). nally inherited early onset cataracts in our patient DAVID K SIMON* suggested the possibility of a mtDNA mutation. MARK A TARNOPOLSKY† Though a mtDNA mutation was suspected in this fam- J TIMOTHY GREENAMYRE‡ ily, the identification of a frameshift mutation was surpris- DONALD R JOHNS*§ *Department of Neurology, Beth Israel Deaconess Medical Center and ing given the mild phenotype and the lack of any previous Harvard Medical School, 77 Avenue Louis Pasteur, Room HIM847, reports of inherited frameshift mutations in human Boston, MA 02115, USA mitochondrial genes. Translation of the ND1 subunit nor- †Department of Neurology and Rehabilitation, McMaster University, mally begins at the initiating methionine codon at np Hamilton, Ontario, Canada 3307-9. An initiating methionine is highly conserved ‡Department of Neurology, Emory University, Atlanta, GA, USA 16 evolutionarily. If translation begins at the 3307-9 codon §Department of Ophthalmology, Beth Israel Deaconess Medical Center for the mutant sequence, then the frameshift at 3308 would and Harvard Medical School, Boston, MA, USA result in an asparagine (rather than methionine) as the ini- Correspondence to: Dr Simon, [email protected] tial amino acid and a premature stop codon after the 28th amino acid, whereas the full length ND1 subunit normally 1 Chinnery PF, Howell N, Andrews RM, Turnbull DM. Mitochondrial DNA consists of 318 amino acids. Such an abnormality seems analysis: polymorphisms and pathogenicity. J Med Genet 1999;36:505-10.

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2 Simon DK, Johns DR. Mitochondrial disorders: clinical and genetic 17 Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, features. Annu Rev Med 1999;50:111-27. Howell N. Reanalysis and revision of the Cambridge reference sequence for J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from 3 Hao H, Morrison LE, Moraes CT. Suppression of a mitochondrial tRNA human mitochondrial DNA. Nat Genet 1999;23:147. gene mutation phenotype associated with changes in the nuclear 18 Tabrizi SJ, Cooper JM, Schapira AH. Mitochondrial DNA in focal dystonia: background. Hum Mol Genet 1999;8:1117-24. a cybrid analysis. Ann Neurol 1998;44:258-61. 4 Cock HR, Tabrizi SJ, Cooper JM, Schapira AH. The inﬂuence of nuclear 19 Jun AS, Trounce IA, Brown MD, ShoVner JM, Wallace DC. Use of transmi- background on the biochemical expression of 3460 Leber’s hereditary optic tochondrial cybrids to assign a complex I defect to the mitochondrial neuropathy. Ann Neurol 1998;44:187-93. DNA-encoded NADH dehydrogenase subunit 6 gene mutation at 5 MITOMAP. Human Mitochondrial Genome Database. Center for Molecu- nucleotide pair 14459 that causes Leber hereditary optic neuropathy and lar Medicine, Emory University, Atlanta, GA, USA 2000;http:// dystonia. Mol Cell Biol 1996;16:771-7. www.gen.emory.edu/mitomap.html 20 ShoVner JM, Brown MD, Stugard C, Jun AS, Pollock S, Haas RH, Kaufman 6 De Coo IF, Renier WO, Ruitenbeek W,Ter Laak HJ, Bakker M, Schagger H, A, Koontz D, Kim Y, Graham JR. Leber’s hereditary optic neuropathy plus Van Oost BA, Smeets HJ. A 4-base pair deletion in the mitochondrial cyto- dystonia is caused by a mitochondrial DNA point mutation. Ann Neurol chrome b gene associated with parkinsonism/MELAS overlap syndrome. 1995;38:163-9. Ann Neurol 1999;45:130-3. 21 Nikoskelainen EK, Marttila RJ, Huoponen K, Juvonen V, Lamminen T, 7 Habano W, Sugai T, Yoshida T, Nakamura S. Mitochondrial gene mutation, Sonninen P, Savontaus ML. Leber’s “plus”: neurological abnormalities in but not large-scale deletion, is a feature of colorectal carcinomas with mito- patients with Leber’s hereditary optic neuropathy. J Neurol Neurosurg Psy- chondrial microsatellite instability. Int J Cancer 1999;83:625-9. chiatry 1995;59:160-4. 8 Polyak K, Li Y, Zhu H, Lengauer C, Willson JK, Markowitz SD, Trush MA, 22 De Vries DD, Went LN, Bruyn GW, Scholte HR, Hofstra RM, Bolhuis PA, Kinzler KW, Vogelstein B. Somatic mutations of the mitochondrial genome van Oost BA. Genetic and biochemical impairment of mitochondrial com- Nat Genet in human colorectal tumours. 1998;20:291-3. plex I activity in a family with Leber hereditary optic neuropathy and 9 Schapira AH, Warner T, Gash MT, Cleeter MW, Marinho CF, Cooper JM. hereditary spastic dystonia. Am J Hum Genet 1996;58:703-11. Ann Neurol 41 Complex I function in familial and sporadic dystonia. 1997; : 23 Macaya A, Munell F, Burke RE, De Vivo DC. Disorders of movement in 556-9. Leigh syndrome. Neuropediatrics 1993;24:60-7. 10 Benecke R, Strumper P, Weiss H. Electron transfer complex I defect in idio- Ann Neurol 32 24 Lera G, Bhatia K, Marsden CD. Dystonia as the major manifestation of pathic dystonia. 1992; :683-6. Mov Disord 11 Ciulla TA, North K, McCabe O, Anthony DC, Korson MS, Petersen RA. Leigh’s syndrome. 1994;9:642-9. Bilateral infantile cataractogenesis in a patient with deﬁciency of complex I, 25 Sudarsky L, Plotkin GM, Logigian EL, Johns DR. Dystonia as a presenting a mitochondrial electron transport chain enzyme. J Pediatr Ophthalmol feature of the 3243 mitochondrial DNA mutation. Mov Disord 1999;14: Strabismus 1995;32:378-82. 488-91. 12 Pitkanen S, Merante F, McLeod DR, Applegarth D, Tong T, Robinson BH. 26 Fearnley IM, Walker JE. Conservation of sequences of subunits of Familial cardiomyopathy with cataracts and lactic acidosis: a defect in com- mitochondrial complex I and their relationships with other proteins. plex I (NADH-dehydrogenase) of the mitochondria respiratory chain. Biochim Biophys Acta 1992;1140:105-34. Pediatr Res 1996;39:513-21. 27 Vilarinho L, Chorao R, Cardoso ML, Rocha H, Nogueira C, Santorelli FM. 13 Souied EH, Sales MJ, Soubrane G, Coscas G, Bigorie B, Kaplan J, Munnich The ND1 T3308C mutation may be a mtDNA polymorphism. Report of A, Rotig A. Macular dystrophy, diabetes, and deafness associated with a two Portuguese patients. J Inherit Metab Dis 1999;22:90-1. large mitochondrial DNA deletion. Am J Ophthalmol 1998;125:100-3. 28 Sue CM, Tanji K, Hadjigeorgiou G, Andreu AL, Nishino I, Krishna S, 14 Simon DK, Pulst SM, Sutton JP, Browne SE, Beal MF, Johns DR. Familial Bruno C, Hirano M, Shanske S, Bonilla E, Fischel-Ghodsian N, DiMauro multisystem degeneration with parkinsonism associated with the 11778 S, Friedman R. Maternally inherited hearing loss in a large kindred with a mitochondrial DNA mutation. Neurology 1999;53:1787-93. novel T7511C mutation in the mitochondrial DNA tRNA(Ser(UCN)) 15 Pettus EH, Betarbet R, Cotrell B, Wallace DC, Greenamyre JT. Immunocy- gene. Neurology 1999;52:1905-8. tochemical characterization of the mitochondrially-encoded ND1 subunit 29 Clark KM, Taylor RW, Johnson MA, Chinnery PF, Chrzanowska- of complex I (NADH-ubiquinone oxidoreductase) in rat brain. J Lightowlers ZM, Andrews RM, Nelson IP, Wood NW, Lamont PJ, Hanna Neurochem 2000;75:383–92. MG, Lightowlers RN, Turnbull DM. An mtDNA mutation in the initiation 16 Campos Y, Martin MA, Rubio JC, Gutierrez del Olmo MC, Cabello A, Are- codon of the cytochrome C oxidase subunit II gene results in lower levels nas J. Bilateral striatal necrosis and MELAS associated with a new T3308C of the protein and a mitochondrial encephalomyopathy. Am J Hum Genet mutation in the mitochondrial ND1 gene. Biochem Biophys Res Commun 1999;64:1330-9. 1997;238:323-5. 30 Marsden CD, Quinn NP. The dystonias. BMJ 1990;300:139-44.

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A common ancestor for COCH related DFNA9 is the only form of hereditary non-syndromal http://jmg.bmj.com/ hearing impairment where strongly marked vestibular cochleovestibular (DFNA9) patients in involvement has been described. This locus has been Belgium and The Netherlands bearing mapped to chromosome 14q12-q13.4 Progressive sensorineural hearing impairment is present, usually starting the P51S mutation between the ages of 35 and 50 in the high frequencies.5–7 This is paralleled by a gradually deteriorating sense of balance, leading to instability (most notably in the dark).

Some, but not all patients periodically suVer from vertigo on September 26, 2021 by guest. Protected copyright. EDITOR—Hearing impairment is extremely heterogeneous, both phenotypically and genetically. It is the most frequent attacks associated with nausea, tinnitus, or aural fullness, which is reminiscent of the symptoms of Menière’s form of sensory impairment in the western world, aVecting 5 approximately 1/1000 newborns and approximately half of disease. Eventually, after a disease course of approximately the people above the age of 80.12In all these cases heredi- 20 years, patients become severely to profoundly deaf tary factors are a prominent cause. So far, more than 60 across all frequencies and lose their vestibular function. loci for monogenic non-syndromal hearing impairment The identification of the gene responsible for DFNA9 have been described and 14 responsible genes have been was greatly helped by the availability of a cochlear specific 8 identified (Van Camp and Smith, Hereditary Hearing Loss cDNA library. One of the transcripts in this cDNA library Homepage http://dnalab-www.uia.ac.be/dnalab/hhh). was a novel cochlear gene designated COCH-5B2, which Balance problems are also are relatively frequent, but was later renamed COCH. COCH was mapped to chromo- considerably less is known about the causes. Purely genetic some 14q12-13, making it a strong candidate gene for 9 forms of vestibular impairment are extremely rare and no DFNA9. Subsequent COCH mutation analysis in three genes have been identified yet. However, it is commonly DFNA9 families showed a missense mutation in each of known that many hearing impaired people also suVer from them.10 The predicted COCH protein has a length of 550 balance problems. Moreover, it is now recognised that amino acids and consists of a signal peptide, a cysteine rich many syndromes with genetic hearing impairment also domain with homology to the factor C domain of the show a dysfunction of the vestibular system.3 The horseshoe crab Limulus (FCH domain), and two domains prevalence of vestibular dysfunction may be severely with homology to the von Willebrand factor A domain underestimated, as it often remains unnoticed until (vWFA1 domain and vWFA2 domain). All three muta- specialised vestibular tests are performed. Owing to the tions are located in the FCH domain. intimate relationship between the auditory and the Following the identification of the COCH gene, COCH vestibular systems, there are probably many genes with a mutation analysis was performed in several other DFNA9 function in both systems. families. In six Dutch and one Belgian family, a C→T point

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Table 1 COCH families haplotyped in this study J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

No of patients References Haplotype* Family sharing Origin Anamnestic Conﬁrmed† Genetic Clinical

1 7/7 Belgium 48 60 5 (fam 1) 5 2 4/7 The Netherlands 4 4 5 (fam 2) 12 3 1/7 The Netherlands 7 8 5 (fam 3) 14 4 7/7 The Netherlands 8 6 11 (W98-066) 5 4/7 The Netherlands 40 34 11 (W98-011) 6 6 4/7 The Netherlands 17 14 11 (W98-065) 7 7/7 The Netherlands 10 4 11 (W98-094) 18 8 7/7 Belgium 13 24 This study 9 7/7 Belgium 17 5 This study 10 — Belgium 1 1 This study 11 — Belgium 2 2 This study 12 7/7 Belgium 26 14 This study 13 — Belgium 1 1 This study 14 — Belgium 1 1 This study 15 — Belgium 1 1 This study

*The amount of haplotype sharing between a given family and family 1. †Includes presymptomatic carriers. mutation at base pair 151 was found.511 This mutation 15. In family 8, a genome search was performed by the leads to the substitution of a conserved proline for a serine Mammalian Genotyping Center (Marshfield, USA), which at position 51 of the COCH protein (P51S). Just like the mapped the disease locus to chromosome 14q12-13 (data three previously described mutations, this is a missense not shown). mutation residing in the FCH domain, and the substituted As the P51S mutation creates an extra site for the amino acid is conserved between the human, mouse, and restriction enzyme DdeI, the mutation was analysed by chicken COCH homologues. In this study, eight additional PCR amplification followed by restriction enzyme diges- families (or at least one of their members) were shown to tion and gel electrophoresis, as described previously.5 In carry the P51S mutation in the COCH gene. Fine mapping normal controls, the 295 bp PCR product is digested into of the markers from the COCH region and haplotype two bands of 247 and 48 bp, respectively. In heterozygous analysis of markers flanking the COCH gene strongly sug- patients carrying the P51S mutation, two additional bands gested a single common founder for at least nine of these of 143 and 104 bp are visible on agarose gel electrophore- families. sis. Details of the families are shown in table 1. The clinical The Whitehead database (http://www-genome.wi. characteristics of family 1,5 family 2,71213 family 3,14 and mit.edu/) was screened with markers D14S262 and families 4-7611have been described previously. Symptoms D14S1071 flanking the COCH region at 14q12-13, which similar to those described were observed in families 8, 9, yielded YAC contig WC14. YACs 746-F10, 732-A9, 855- 11, and 12, as well as in the isolated patients 10, 13, 14, and C7, 814-E9, 857-D12, 905-B1, 925-C2, 952-D9, and http://jmg.bmj.com/

D14S262 D14S975 COCH D14S1021 D14S257 D14S1071 D14S1040 D14S1034

855 C7

857 D12 on September 26, 2021 by guest. Protected copyright.

905 B1

925 C2

949 A9

732 A9

746 F10

814 E9

952 D9

Figure 1 YAC contig spanning the COCH region. YACs were screened for the diVerent markers by PCR. A positive signal is denoted by a black dot, and a stretch of positive signals is connected by a dashed line. The length of the YACs used for the mapping of the markers gave no clear answer to the physical distance between the markers (not shown). Genetic distance between D14S262 and D14S975 is 3.3 cM on the Généthon map,15 whereas the distance between the other markers is unclear. In the Généthon map, markers D14S975, D14S1021, D14S257, D14S1071, D14S1040, and D14S1034 are located within an interval of 0.9 cM. Therefore, the genetic distance between D14S975 and the most distal marker D14S1034 is probably not larger than 1 cM.

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Table 2 Haplotype sharing around the COCH locus in P51S families J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

Family D14S 262 D14S975 COCH D14S1021 D14S257 D14S1071 D14S1040 D14S1034

1 11P51S22161 21 1 P51S 2 2 445 33 1 P51S 32461 411P51S22161 511P51S22461 611P51S22455 711P51S22161 811P51S22161 911P51S22161 10 2/1 1/2 P51S 2/1 3/2 4/1 6/1 1 11 1 1/2 P51S 7/2 2 4/1 6 1 12 11P51S22161 13 3 1/3 P51S 2/3 2 4/1 6 1 14 1/2 1/3 P51S 4 2/5 2/1 5/6 1 15 11P51S4/2 2 5/1 6/3 1

The area in italics marks the shared segment. In some families, the disease haplotype could not unequivocally be determined. For allele lengths, see table 3.

949-A9 were obtained from the UK Human Genome that on the Généthon map the order of these flanking Mapping Project (HGMP, Harwell, UK). The relative markers was not completely resolved (not shown). In addi- marker order of seven polymorphic Généthon markers, as tion, the exact position of the COCH gene with respect to well as the position of the COCH gene, were determined by these markers was unknown. Therefore, we constructed a STS content mapping through PCR analysis. YAC contig spanning the COCH region and determined Microsatellite markers were analysed using a standard the marker order through PCR analysis on this contig. As a radioactive PCR assay. Primer sequences and allele result of this analysis, the marker order and the position of frequencies for all markers used in this study were retrieved COCH could be unambiguously determined (fig 1). This from the Généthon database.15 marker order is diVerent from the one on the Généthon The P51S mutation in the COCH gene has been found map, which locates D14S1034 proximal to D14S1021, previously in seven families characterised by a combination D14S1040, and D14S1071, and D14S975 distal to of progressive sensorineural hearing impairment and D14S257.15 511 vestibular dysfunction. We analysed the presence of this The results of the haplotype analysis of the markers mutation in a total of 29 additional families or isolated flanking the COCH gene are shown in table 2. Families 1, patients with similar symptoms. All families originate from 4, 7, 8, 9, and 12 show exactly the same haplotype for all Belgium or the southern part of The Netherlands. In eight seven markers examined: 1-1-2-2-1-6-1 (for allele lengths of them, the P51S mutation was present, bringing the total and frequencies, see table 3). number of P51S bearing families to 15. In the remaining This common disease haplotype may also be present in 21 families without the P51S mutation, no further family 11 (two brothers) and in patients 10, 13, and 15, but mutation analysis has been performed, so it is possible that only partly in patient 14. In these cases, the disease haplo-

COCH http://jmg.bmj.com/ they have a diVerent, as yet unidentified mutation. types could not be unequivocally determined. Families 2, General data about the families included in this study are 5, and 6 have the same haplotype as families 1, 4, 7, 8, 9, summarised in table 1. Although all P51S bearing families and 12 for the four markers that most closely flank the originate from the same region, we found no genealogical COCH gene on both sides. Marker D14S1071 has a diVer- evidence for a common ancestor. ent haplotype and therefore the three most distal markers V To investigate a possible founder e ect, we recon- are not shared with the other families. In family 3, only structed the disease haplotypes for polymorphic markers marker D14S975 has the same haplotype as all the other flanking the COCH locus. This was complicated by the fact

families. on September 26, 2021 by guest. Protected copyright. Table 3 Allele lengths of 14q12-13 markers* The P51S mutation in the COCH gene, which is responsible for progressive cochleovestibular impairment Marker Allele No Allele length Allele frequency (DFNA9), had previously been found in seven families. In D14S257 2 182 0.426 this study we have found the same mutation in eight novel 3 184 0.037 families and isolated patients. This could indicate that 5 180 0.037 either all 15 P51S bearing families share a common ances- D14S1040 1 211 0.089 3 227 0.071 tor or that the P51S mutation represents a mutation 4 229 0.196 hotspot that gave rise to several independent mutational 5 231 0.446 6 233 0.107 events. Analysis of the disease associated haplotype of the D14S1034 1 172 0.304 polymorphic markers around COCH showed signiﬁcant 5 182 0.036 haplotype sharing in nine families and six of them (1, 4, 7, D14S1071 1 277 0.222 2 279 0.056 8, 9, 12) shared haplotypes for all seven markers examined. 4 283 0.463 The probability that six independent families would share 5 285 0.037 a seven allele haplotype merely by chance is negligible D14S262 1 198 0.574 2 200 0.037 (table 3). Therefore, families 1, 4, 7, 8, 9, and 12 must be 3 202 0.278 related to and originate from a common ancestor. D14S1021 1 260 0.018 Most probably, families 2, 5, and 6 also originate from 2 266 0.441 3 272 0.429 this common aVected ancestor. These latter families have 4 276 0.018 four alleles in common with families 1, 4, 7, 8, 9, and 12, 7 268 0.071 COCH D14S975 1 174 0.661 which are the ones that most closely ﬂank the gene 2 170 0.214 (table 2). One ancestral recombination between D14S257 3 168 0.125 and D14S1071 can explain the diVerent haplotypes of the *Data are based upon the Généthon database.19 distal markers D14S1071, D14S1040, and D14S1034. Only the alleles mentioned in table 3 are given. Taken together, a total of nine families have the identical

www.jmedgenet.com 64 Letters haplotype (1-1-2-2) for markers D14S262, D14S975, FLORIS L WUYTS† WIM I M VERHAGEN** J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from D14S1021, and D14S257 and it can be concluded that PATRICK L M HUYGEN‡ they all share a common ancestor. WYMAN T MCGUIRT†† Only family 3 has a haplotype that is distinct from the RICHARD J H SMITH†† other families for the markers ﬂanking COCH, with the LIONEL VAN MALDERGEM‡‡ FRANK DECLAU† exception of marker D14S975. However, as allele 1 is a COR W R J CREMERS‡ very common allele for D14S975 (66%), sharing of it PAUL H VAN DE HEYNING† could be a coincidence. It cannot be excluded that the FRANS P M CREMERS¶ GUY VAN CAMP* mutation in family 3 arose independently from the other *Department of Medical Genetics, University of Antwerp (UIA), families, but, on the other hand, this family may be more Universiteitsplein 1, B-2610 Antwerp, Belgium distantly related to the others and the shared interval †Department of Otorhinolaryngology, Antwerp University Hospital around the COCH gene may be very narrow. (UZA), Antwerp, Belgium Families 10, 13, 14, and 15 represent isolated patients ‡Department of Otorhinolaryngology, University Hospital Nijmegen, with the P51S mutation, whereas family 11 consists of two Nijmegen, The Netherlands aVected brothers. Here, the disease haplotype could not be §ENT Department, University Hospital of Leuven (St-Raphael), Leuven, Belgium unambiguously determined. However, with the exception ¶Department of Human Genetics, University Hospital Nijmegen, of patient 15, who is homozygous for allele 4 of marker Nijmegen, The Netherlands D14S1021, the haplotypes observed in these isolated **Department of Neurology, Canisius-Wilhelmina Hospital, Nijmegen, patients indicate that it cannot be excluded that they, too, The Netherlands are a carrier of the 1-1-2-2-1-6-1 haplotype. This question ††Molecular Otolaryngology Research Labs, Department of can only be resolved if more family members become Otolaryngology, University of Iowa, Iowa City, Iowa, USA available. ‡‡Department of Medical Genetics, Institut de Pathologie et de Génétique, The 10 families in which the exact disease associated Loverval, Belgium haplotype could be determined may allow an estimate of Correspondence to: Professor Van Camp, [email protected] the number of generations passed since the common ancestor. Formulae to calculate this age rely on the 1 Gorlin RJ, Toriello HV, Cohen MM. Hereditary hearing loss and its syndromes. New York: Oxford University Press, 1995. probability of having recombinations in an interval with a 2 Morton NE. Genetic epidemiology of hearing impairment. AnnNYAcad known size.16 17 The accuracy of these calculations, Sci 1991;630:16-31. 3 Huygen PLM, Verhagen WIM. Peripheral vestibular and vestibulo-cochlear however, are heavily dependent upon è and thus upon the dysfunction in hereditary disorders. J Vestibular Res 1994;4:81-104. accuracy of the genetic maps. However, the data obtained 4 Manolis EN, Yandavi N, Nadol JB, Eavey RD, McKenna M, Rosenbaum S, Khetarpal U, Halpin C, Merchant SN, Duyk GM, MacRae C, Seidman here indicate that the Généthon genetic map around the CE, Seidman JG. A gene for non-syndromic autosomal dominant progres- COCH locus is not reliable. Therefore, the intermarker sive postlingual sensorineural hearing loss maps to chromosome 14q12-13. Hum Mol Genet 1996;5:1047-50. distances around the COCH gene are unknown and even a 5 Fransen E, Verstreken M, Verhagen WIM, Wuyts FL, Huygen PLM, rough estimate of the age of the mutation is not feasible. D’Haese P, Robertson NG, Morton CC, McGuirt WT, Smith RJ, Declau F, Van de Heyning PH, Van Camp G. High prevalence of symptoms of This question may be resolved in the future by an Menière’s disease in two families with a mutation in the COCH gene. Hum improvement of the map or by the sequencing of the Mol Genet 1999;8:1425-9. 6 Bom SJH, Kemperman MH, de Kok Y, Huygen PLM, Verhagen WIM, COCH region as part of the Human Genome Project. Cremers FP, Cremers CWRJ. Progressive cochleovestibular impairment caused by a point mutation in the COCH gene at DFNA9. Laryngoscope

The geographical spreading of the P51S mutation may http://jmg.bmj.com/ 1999;109:1525-30. be another clue to the age of the mutation. The 15 families 7 Verhagen WIM, Bom SJH, Huygen PLM, Fransen E, Van Camp G, Crem- in which the P51S mutation was found contain more than ers CWRJ. Familial progressive vestibulocochlear dysfunction caused by a COCH mutation (DFNA9). Arch Neurol (in press). 200 patients in Belgium and The Netherlands and there 8 Robertson NG, Khetarpal U, Gutierrez-Espeleta GA, Bieber FR, Morton must be many more unidentified mutation carriers (table CC. Isolation of novel and known genes from a human fetal cochlear cDNA library using substractive hybridization and diVerential screening. 1). This makes the P51S mutation a frequent cause of late Genomics 1994;23:42-50. onset hearing impairment combined with vestibular 9 Robertson NG, Skvorak AB, Yin Y, Weremowicz S, Johnson KR, Kovatch KA, Battey JF, Bieber FR, Morton CC. Mapping and characterization of a dysfunction, at least in the Dutch and the Belgian popula- novel cochlear gene in human and in mouse: a positional candidate gene for Genomics 46 tions. A systematic mutation analysis in other populations a deafness disorder, DFNA9. 1997; :345-54. on September 26, 2021 by guest. Protected copyright. 10 Robertson NG, Lu L, Heller S, Merchant SN , Eavey RD, McKenna M, would indicate whether this mutation is also present Nadol JB, Miyamoto RT, Linthicum FH, Lubianca Neto JF, Hudspeth AJ, elsewhere. A more general occurrence of the P51S Seidman CE, Morton CC, Seidman JG. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic sensorineural deafness with mutation would indicate an ancient origin of the mutation vestibular dysfunction. Nat Genet 1998;20:299-303. or a mutational hotspot. This would imply that COCH is a 11 de Kok Y, Bom SJH, Brunt TM, Kemperman MH, van Beusekom E, van der Velde-Visser SD, Robertson NG, Morton CC, Huygen PLM, Verhagen major gene for cochleovestibular impairment in many dif- WIM, Brunner H, Cremers CWRJ, Cremers FP. A Pro51Ser mutation in ferent populations harbouring a recurrent mutation that the COCH gene is associated with late-onset autosomal dominant progressive sensorineural hearing loss with vestibular defects. Hum Mol Genet can easily be screened for in a diagnostic setup. 1999;8:361-6. 12 Verhagen WIM, Huygen PLM, Joosten EMG. Familial progressive vestibulocochlear dysfunction. Arch Neurol 1988;45:766-9. The authors wish to thank all the families who contributed to this study. Gerard 13 Verhagen WIM, Huygen PLM. Familial progressive vestibulocochlear J te Meerman is acknowledged for his critical remarks on the manuscript. This dysfunction. Arch Neurol 1991;48:262-2. project was funded by a research grant from the Flemish Fund for Scientific 14 Verhagen WIM, Huygen PLM, Theunissen EJ, Joosten EMG. Hereditary Research (FWO-Vlaanderen) to GVC and PVdH, a grant from the University of vestibulo-cochlear dysfunction and vascular disorders. J Neurol Sci Antwerp to GVC and PVdH, and by a grant from The Netherlands 1989;92:55-63. Organisation for Scientific Research to FPMC. The clinical studies in the six 15 Dib C, Faure S, Fizames C, Samson D, Drouot N, Vignal A, Millaseau P, Dutch (Nijmegen) DFNA9 families were funded by grants from the Heinsius Marc S, Hazan J, Seboun E, Lathrop GM, Gyapay G, Morissette J, Weis- Houbolt foundation and the Nijmegen ORL-Research Foundation. The senbach J. A comprehensive genetic map of the human genome based on genome search in family 8 was performed at the Mammalian Genotyping Serv- 5,264 microsatellites. Nature 1996;380:152-4. ice (Marshfield, USA). EF and GVC hold research positions at the 16 Cox TK, Kerem B, Rommens J, Iannuzzi MC, Drumm M, Collins FS, FWO-Vlaanderen. Dean M, Tsui LC, Chakravarti A. Mapping of the cystic fibrosis gene using ERIK FRANSEN* putative ancestral recombinants. Am J Hum Genet 1989;45:A136. MARGRIET VERSTREKEN*† 17 te Meerman GJ, van der Meulen MA, Sandkuijl L. Perspectives of identity by descent (IBD) mapping in founder populations. Clin.Exp. 1995;25:97- STEVEN J H BOM‡ 102. FRANÇOIS LEMAIRE§ 18 Verhagen WIM, Huygen PLM, Bles W. A new autosomal dominant MARTIJN H KEMPERMAN‡ syndrome of idiopathic progressive vestibulo-cochlear dysfunction with YVETTE J M DE KOK¶ middle-age onset. Acta Otolaryngol (Stockh) 1992;112:899-906.

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J Med Genet 2001;38:65–67 J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from The spastic paraplegia SPG10 locus: eral sustained (>5 beats) ankle or knee clonus. Subjects were classified as being possibly aVected if lower limb narrowing of critical region and hyperreflexia was present without other abnormal signs exclusion of sodium channel gene and as being normal if they had an entirely normal neurological examination. Thirteen members of family 4 are SCN8A as a candidate aVected by ADPHSP and the family has a relatively young mean age at onset of 10.8 (SD 9.6) years (range 8-40). We have now genotyped additional markers D12S803, D12S390, D12S270, D12S1618, and D12S355 for EDITOR—The hereditary spastic paraplegias (HSPs) are subjects from family 4, using previously described clinically characterised by progressive lower limb spasticity. methods.10 Primer sequences for these markers are The spasticity may occur in isolation (“pure”) or may be 12 complicated by other major clinical features. Autosomal available from the Généthon microsatellite linkage map dominant, autosomal recessive, and X linked recessive or from the Marshfield Medical Research Foundation inheritance patterns have been described for pure and website. Haplotypes were constructed for these and for complicated forms of HSP.12 previously genotyped markers (fig 1). Obligate recombina- There are loci for ADPHSP on chromosomes 2p (SPG4, tion events in two aVected subjects (III.5 and III.9) MIM 182601),348q (SPG8, MIM 603563),5614q (SPG3, redefine the centromeric boundary of the SPG10 region at MIM 182600),7 15q (SPG6, MIM 600363),8 and 19q D12S270, and an obligate recombinant event in aVected (SPG12).9 In addition, we recently mapped an ADPHSP subject IV.1 redefines the telomeric boundary at D12S355. locus on chromosome 12q13 (SPG10, MIM 604187) in a These recombination events narrow the SPG10 candidate large UK family, family 4.10 This locus was narrowed to a region to 6.95 cM (Marshfield Medical Research Founda- 9.2 cM region between markers D12S368 and D12S83. tion website). Clinical features and diagnostic criteria for family 4 have A database search (Online Mendelian Inheritance in previously been described.10 11 Briefly, subjects were classi- Man) was carried out to identify candidate genes within fied as being aVected if they had lower limb hyperreflexia in the SPG10 critical region. The neuronal sodium channel addition to at least one of the following: progressive spastic gene SCN8A is located at chromosome 12q13, close to gait abnormality, bilateral extensor plantar reflex, or bilat- marker D12S368.13 It is expressed in neurones throughout

I 12

+ ++ +++ +

II http://jmg.bmj.com/ 1 2 34 5678 9 D12S345 2 77 1 1 7 2 7 7 7 2 7 D12S85 1 12 1 1 1 1 1 1 1 1 1 D12S368 3 56 2 9 5 3 5 6 1 3 5 D12S803 6 65 5 5 6 6 6 7 6 6 6 D12S390 1 16 6 5 1 1 1 6 1 1 1 D12S270 5 88 6 1 8 5 8 1 5 5 8 D12S1618 13 91 1 3 9 13 9 12 13 13 9 D121586 12 75 5 5 7 12 7 7 5 12 7 D12S1691 15 711 6 11 7 15 7 13 19 15 7 D12S355 11 13 15 1510 13 11 13 14 15 11 13 D12S83 7 7 6 8 6 7 7 7 3 8 7 7 D12S326 1 1 1 5 1 1 2 1 4 3 2 1 on September 26, 2021 by guest. Protected copyright. + +++++++++ III 1 2 3 4 5 6 7 8 9 10 11 12 D12S345 2 3 2 7 2 1 7 7 2 1 2 3 7 3 2 7 D12S85 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 D12S368 3 5 3 6 3 2 5 6 3 2 3 2 5 3 3 1 D12S803 6 5 6 5 6 5 6 5 6 5 6 7 6 5 6 6 D12S390 1 5 1 6 1 6 1 6 1 6 1 5 1 5 1 1 D12S270 5 6 5 8 5 6 8 8 5 6 5 6 8 1 5 5 D12S1618 13 14 13 1 13 1 13 1 13 1 13 6 13 4 13 13 D121586 12 7 12 5 12 5 12 5 12 5 12 7 12 10 12 5 D12S1691 15 5 15 1115 6 15 11 15 6 15 17 15 6 15 19 D12S355 11 10 11 1511 15 11 15 11 15 11 15 11 4 11 15 D12S83 7 13 7 6 7 8 7 6 7 8 7 7 7 7 7 8 D12S326 1 1 1 1 1 5 1 1 1 5 2 8 2 1 2 3 ++ IV 1 2 3 D12S345 2 12 2 5 D12S85 1 1 1 4 D12S368 3 5 3 1 D12S803 6 5 6 7 D12S390 1 2 1 3 D12S270 5 1 5 1 D12S1618 13 1 13 16 Figure 1 Family tree of ADPHSP family 4, showing haplotypes for markers around the SPG10 region. The D121586 12 7 12 1 marker order is shown alongside each generation. For clarity non-contributory haplotypes are represented by a D12S1691 15 6 15 6 D12S355 10 12 11 16 uniform hatched shading pattern. Recombination events in aVected subjects narrow the SPG10 critical region to a D12S83 13 3 7 5 6.95 cM region between D12S270 and D12S335. Subjects’ sexes have been anonymised for confidentiality reasons. D12S326 1 1 1 6 Filled symbol=aVected, half filled=possibly aVected, quarter filled=clinically normal, +=DNA available.

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AB J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from G ATC III III IV G Glu A G T (Cys)Arg G (T) C NH COOH G 2 Gln A R1026C C

Figure 2 Genetic variation in exon 14. (A) A 643 bp fragment containing exon 14 was amplified from genomic DNA with primers 14F (CAA GAG CCT CTT C TGA GTC TGT CAC G) and 14R (ACA AGC ACC CTG TTT GCT CTC III.5 III.6 III.8 III.9 AC). Patient III.2 is heterozygous foraCtoTtransition within the exon (arrow), resulting in amino acid substitution R1026C and loss of an Fnu4HI restriction site. (B) R1026C is located in cytoplasmic loop 2 of the sodium channel SCN8A. 250 bp Horizontal bars indicate intron/exon boundaries.13 (C) Wild type exon 14 contains Fnu4HI fragments of 116 bp and 134 bp which are replaced by a 250 bp fragment 134 bp in the variant exon. AVected subjects III.6 and III.8 are heterozygous for the 116 bp Fnu4HI site and generate all three fragments, but aVected subjects III.5 and III.9 are homozygous for the wild type allele and do not generate the 250 bp fragment. the central and peripheral nervous systems.14 15 Mice 0/27 Japanese subjects. To our knowledge, this is the first homozygous for null mutations in SCN8A (med and medtg) relatively common coding variant described within an ion develop progressive hind limb paralysis and muscle channel gene. Further studies will be required to determine atrophy, with death in the juvenile period.14 16 The progres- whether this polymorphism has any functional eVect on the sive paralysis in mouse SCN8A null mutants is caused by SCN8A protein, or whether it confers any predisposition to functional denervation of skeletal muscle, with normal polygenic diseases which may arise from ion channel motor neurone number and diameter.17 Although disorders. ADPHSP is pathologically characterised by “dying back” To date, four diVerent classes of genes have been impli- of the terminal ends of corticospinal tract axons,18 we con- cated in HSP. X linked spastic paraplegia is caused by sidered there to be suYcient overlap between the ADPHSP mutations in the cell adhesion molecule L1-CAM19 and phenotype and the SCN8A mouse null mutant phenotypes mutations in a myelin gene, the proteolipid protein gene.20 to investigate this gene as a candidate for the disease. One form of autosomal recessive HSP is caused by muta- The 26 exons (with flanking intronic sequence) of tions in a nuclear encoded mitochondrial gene (paraplegin) SCN8A were amplified by polymerase chain reaction, as which has metalloprotease and chaperone function.21 http://jmg.bmj.com/ previously described,13 using DNA from aVected subject Finally, a gene for the most common form of ADPHSP, III.2. Each exon, with its flanking intronic sequence, was spastin, encodes a protein which may be involved in the sequenced manually, as previously described,13 or with dye structure of the nuclear proteosome.22 The diversity of terminators (Perkin Elmer, Foster City) on an ABI377 HSP genes suggests that the neurodegeneration seen in automated sequencer (Perkin Elmer). Two previously HSPs may be a final common pathway for a number of identified single nucleotide polymorphisms were detected, processes, some of which may involve novel proteins. This a C to T in intron 19, position –27 from exon 20, and a C makes selection of candidate genes problematical. Identifi- 13 to T in exon 22, at position +90. Neither of these variants cation of additional SPG10 linked families will be crucial on September 26, 2021 by guest. Protected copyright. change the amino acid sequence. We also detectedaTfor to narrow the candidate region and facilitate positional C substitution in exon 14 at position +175 (fig 2A). This cloning of the SPG10 gene. new variant results in an arginine to cysteine substitution in the SCN8A protein at amino acid 1026, within a conserved Electronic databases: Centre d’Etude du Polymorphisme Humain (CEPH) website: http://www.cephb.fr (for details of CEPH families). Marshfield Medical region of the second cytoplasmic loop (fig 2B). The T for Research Foundation: http://www.marshmed.org/genetics (for marker genetic C substitution can be detected readily, since it abolishes an locations). Online Mendelian Inheritance in Man (OMIM): http:// Fnu www.ncbi.nlm.nih.gov/Omim (for HSP MIM entries and candidate gene 4HI restriction site. We examined the segregation of search). this new variant in the family by carrying out PCR ampli- The first two authors contributed equally to this work. We thank the members of family 4 for taking part in this study. Marker genotyping and automated fication of exon 14, digestion of PCR product with sequence analysis of SCN8A was carried out in the UK Medical Research Fnu4HI, followed by agarose gel electrophoresis. The T for Council HGMP Linkage Hotel. Patient assessment and sample collection was supported by the UK Medical Research Council. DCR is a Glaxo/Wellcome C variant segregated completely with the disease, except in Research Fellow and ER is a Wellcome Research Training Fellow. ER is aVected family members III.5 and III.9, who were recom- supported by a Sackler Fellowship. binants for this new SNP (fig 2C). These results exclude E REID* SCN8A from the SPG10 candidate region, with a likely A ESCAYG† A M DEARLOVE‡ placing centromeric to D12S1618. This location is D D LEE† consistent with the physical mapping of SCN8A toaYAC M H MEISLER† containing D12S368.13 D C RUBINSZTEIN* The frequency of the exon 14 SNP was determined by *Department of Medical Genetics, Level 4, Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for genotyping 80 parents from the CEPH pedigrees (CEPH Medical Research, Addenbrooke’s Hospital, Cambridge CB2 2XY,UK website) and a panel of unrelated, white United Kingdom †Department of Human Genetics, University of Michigan Medical subjects, black African subjects, and Japanese subjects. The School, Ann Arbor, Michigan, USA T for C substitution was present in 3/80 CEPH parents ‡Medical Research Council Human Genome Mapping Project Resource (3/160 chromosomes, 1.9%), 2/39 of the UK subjects Centre, Cambridge, UK (2/78 chromosomes, 2.6%), in 0/44 black African, and Correspondence to: Dr Rubinsztein, [email protected]

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1 Fink JK, Heiman-Patterson T. Hereditary spastic paraplegia: advances in 12 Dib C, Fauré S, Fizames C, Samson D, Drouot N, Vignal A, Millasseau J, genetic research. Neurology 1996;46:1507-14. Marc S, Hazan J, Seboun E, Lathrop M, Gyapay G, Morissette J, Weissen- J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from 2 Reid E. The hereditary spastic paraplegias. J Neurol 1999;246:995-1003. bach J. A comprehensive genetic map of the human genome based on 5,264 3 Hazan J, Fontaine B, Bruyn RPM, Lamy C, van Deutekom JC, Rime CS, microsatellites. Nature 1996;380:152-4. Dürr A, Melki J, Lyon-Caen O, Agid Y, Munnich A, Padberg GW, de 13 Plummer NW, Galt J, Jones JM, Burgess DL, Sprunger LK, Kohrman DC, Recondo J, Frants RR, Brice A, Weissenbach J. Linkage of a new locus for Meisler MH. Exon organisation, coding sequence, physical mapping, and autosomal dominant familial spastic paraplegia to chromosome 2p. Hum polymorphic intragenic markers for the human neuronal sodium channel Genomics 54 Mol Genet 3 gene SCN8A. 1998; :287-96. 1994; :1569-73. 14 Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, Spear B, 4 Hentati A, Pericak-Vance MA, Lennon F, Wasserman B, Hentati F, Juneja Meisler MH. Mutation of a new sodium channel gene, Scn8a, in the mouse T, Angrist MH, Hung WY, Boustany RM, Bohlega S, Iqbal Z, Huether mutant “motor endplate disease”. Nat Genet 1995;10:461-5. CH, Ben Hamida M, Siddique T. Linkage of a locus for autosomal domi- 15 Schaller KL, Krzemien DM, Yarowsky PJ, Krueger BK, Caldwell JK. A nant familial spastic paraplegia to chromosome 2p markers. Hum Mol Genet novel, abundant sodium channel expressed in neurons and glia. J Neurosci 1994;3:1867-71. 1995;15:3231-42. 5 Hedera P, Rainier S, Alvarado D, Zhao X, Williamson J, Otterud B, Leppert 16 Kohrman DC, Harris JB, Meisler MH. Mutation detection in the med and M, Fink JK. Novel locus for autosomal dominant hereditary spastic medj alleles of the sodium channel Scn8a: unusual splicing due to a minor paraplegia, on chromosome 8q. Am J Hum Genet 1999;64:563-9. class AT-AC intron. J Biol Chem 1996;271:17576-81. 6 Reid E, Dearlove AM, Whiteford ML, Rhodes M, Rubinsztein DC. 17 Porter JD, Goldstein LA, Kasarkis EJ, Bruecker JK, Spear BT. The neuronal Autosomal dominant spastic paraplegia: reﬁned SPG8 locus and further voltage-gated sodium channel, Scn8a, is essential for postnatal maturation genetic heterogeneity. Neurology 1999;53:1844-9. of spinal, but not oculomotor, motor units. Exp Neurol 1996;139:328-34. 7 Hazan J, Lamy C, Melki J, Munnich A, de Rocondo J, Weissenbach J. Auto- 18 Behan WMH, Maia M. Strümpell’s familial spastic paraplegia: genetics and somal dominant familial spastic paraplegia is genetically heterogeneous and neuropathology. J Neurol Neurosurg Psychiatry 1974;37:8-20. one locus maps to chromosome 14p. Nat Genet 1993;5:163-7. 19 Jouet M, Rosenthal A, Armstrong G, MacFarlane J, Stevenson R, Paterson J, Metzenberg A. X-linked spastic paraplegia (SPG1), MASA syndrome 8 Fink JK, Wu CB, Jones SM, Sharp GB, Lange BM, Lesicki A, Reinglass T, Nat Varvil T, Otterud B, Leppert M. Autosomal dominant familial spastic and X-linked hydrocephalus result from mutations in the L1 gene. Genet 7 paraplegia: tight linkage to chromosome 15q. Am J Hum Genet 1994; :402-7. 56 20 Saugier-Veber P, Munnich A, Bonneau D, Rozet JM, Le Merrer M, Gil R, 1995; :188-92. Boespﬂug-Tanguy O. X-linked spastic paraplegia and Pelizaeus- 9 Reid E, Dearlove AM, Osborn O, Rogers MT, Rubinsztein DC. A locus for Merzbacher disease are allelic disorders at the proteolipid protein locus. autosomal dominant “pure” hereditary spastic paraplegia maps to chromo- Nat Genet 1994;6:257-62. some 19q13. Am J Hum Genet (in press). 21 Casari G, De Fusco M, Ciarmatori S, Zeviani M, Mora M, Fernandez P, De 10 Reid E, Dearlove AM, Rhodes M, Rubinsztein DC. A new locus for Michele G, Filla A, Cocozza S, Marconi R, Durr A, Fontaine B, Ballabio A. autosomal dominant “pure” hereditary spastic paraplegia mapping to Spastic paraplegia and OXPHOS impairment caused by mutations in paraple- chromosome 12q13, and evidence for further genetic heterogeneity. Am J gin, a nuclear-encoded mitochondrial metalloprotease. Cell 1998;93:973-83. Hum Genet 1999;65:757-63. 22 Hazan J, Fonknechten N, Mavel D, Paternotte C, Samson D, Artiguenave F, 11 Reid E, Grayson C, Rogers MT, Rubinsztein DC. Locus-phenotype corre- Davione CS, Cruaud C, Durr A, Wincker P, Brottier P, Cattolico L, Barbe lations in autosomal dominant pure hereditary spastic paraplegia: a clinical V, Burgunder JM, Prud’homme JF, Brice A, Fontaine B, Heilig B, Weissen- and molecular genetic study of 28 United Kingdom families. Brain bach J. Spastin, a new AAA protein, is altered in the most frequent form of 1999;122:1741-55. autosomal dominant spastic paraplegia. Nat Genet 1999;23:296-303.

J Med Genet 2001;38:67–73 Cytogenetic and molecular study of a in these disorders, Dandy-Walker malformation is always one feature of a larger spectrum of anomalies. Various jumping translocation in a baby with cytogenetic abnormalities have been reported, but to date Dandy-Walker malformation no consistent chromosomal aberration has been recognised.78The present study combines molecular and cytogenetic investigations, including FISH and PRINS label-

ling procedures, to characterise a constitutional jumping http://jmg.bmj.com/ EDITOR—Jumping translocations are rare chromosomal translocation. events in which a donor chromosome segment is The patient is a baby boy, the second child of healthy, translocated to various recipient chromosome sites. Jump- consanguineous parents (the parents are double third ing translocations were initially described in constitutional cousins). The family history is otherwise unremarkable. chromosome syndromes,12 but the majority of published Consent was obtained from the parents for genetic studies. cases have been observed in haematological malignancies, At the time of birth, the mother and father were 26 and 34 where their presence has been related to poor prognosis.34 years old respectively. There was no history of maternal

Most jumping translocations involve acrocentric chro- exposure to environmental hazards during pregnancy. The on September 26, 2021 by guest. Protected copyright. mosomes and a characteristic feature is that breakpoints pregnancy was uneventful until sonographic examination usually concern areas of repetitive DNA (telomeric, at 39 weeks’ gestation suggested partial agenesis of the ver- centromeric, or heterochromatic regions). Recipient chro- mis. Fetal sampling for chromosome analysis was not mosome involvement seems to be randomly distributed but undertaken. The baby was delivered at 40.5 weeks’ with a preferential involvement of telomeric segments. This gestation; birth weight, length, and head circumference has led to the hypothesis that repetitive telomeric were 3260 g, 50 cm, and 34 cm, respectively. Apgar scores sequences could be implicated in the occurrence of jump- were 8 at one minute and 9 at ﬁve minutes. The baby was ing translocations, as suggested by FISH studies which referred to our genetics service at the age of 1 month. At have reported the presence of interstitial telomeric this time, weight, height, and head circumference were sequences at the junction sites of jumping translocations.56 5250 g (+3 SD), 57 cm (+2 SD), and 40.5 cm (+2 to +3 However, the molecular basis underlying these complex SD), respectively. He showed no facial dysmorphism; chromosomal rearrangements is not well understood. physical examination showed a small median mass of the Constitutional jumping translocations are extremely rare occipital soft tissues and bilateral deep plantar furrows, but and are usually associated with various phenotypic abnor- was otherwise normal. Skull x ray showed an occipital bone malities. We report the ﬁnding of a jumping translocation defect. MRI showed (1) hypoplasia of the cerebellar vermis in a baby with Dandy-Walker malformation. Dandy- (only a part of the posterior and anterior segments are Walker malformation consists of the triad (1) hypoplasia or Table 1 Distribution of diVerent cell lines in patient tissues absence of the vermis, (2) upward displacement of the falx, lateral sinuses, and torcular, and (3) a large, thin walled Total cells retrocerebellar cyst formed by the roof of the fourth ventri- Tissues examined t(1;2) t(2;5) t(2;6) t(2;12) cle. Most Dandy-Walker malformations are sporadic. The Blood Possum database reports six autosomal dominant syn- First sample 126 108 2 2 14 Second sample (3 weeks later) 72 65 1 2 4 dromes and 37 autosomal recessive syndromes which can Skin 49 47 1 0 1 be associated with Dandy-Walker malformation. However,

www.jmedgenet.com 68 Letters J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from Idiogram indicating Partial R banded karyotype the specific breakpoints

36.2 35 25.2 34.2 33 24 32.2 31.3 22 31.1 16 22.2 14 21 12 13.2 12 12 12 14.1 21.2 14.3 22 21.2 22 24 24.1 24.3 31 32.1 32.2 32.3 41 42.2 34 43 36 37.2

2 der(2)1der(1) 2 der(2) 1 der(1) t(1;2)(p36;p12)

25.2 24 22 16 14 15.2 14 12 13.2 12 12 12 14.1 14.3 13.2 21.2 14 22 21 24.1 24.3 23.1 23.3 32.1 31.2 32.3 32 33.2 34 34 36 35.2 37.2

2 der(2)5 der(5) 2 der(2) 5 der(5) t(2;5)(p12;q35)

25.2 24 22 24 16 22.3 14 22.1 12 21.2 12 12 12 14.1 14 14.3 16.1 21.2 16.3 22 22.1 24.1 22.3 24.3 23.2 24 32.1 25.2 http://jmg.bmj.com/ 32.3 26 34 36 37.2

2 der(2)6 der(6) 2 der(2) 6 der(6) t(2;6)(p12;q27) on September 26, 2021 by guest. Protected copyright.

25.2 24 22 16 14 13.2 12.3 12 12.1 12 12 14.1 13.2 14.3 14 21.2 21.1 22 21.3 24.1 23 24.3 24.2 32.1 24.32 32.3 34 36 37.2

2 der(2)12 der(12) 2 der(2) 12 der(12) t(2;12)(p12;q24)

Figure 1 Partial idiograms and R banded karyotypes of the chromosome pairs involved in the jumping translocation. visible), (2) upward displacement of the cerebellar falx, and guage skills were acquired normally, and school perform- (3) a retrocerebellar cyst communicating with the fourth ance is normal for age. ventricle. These features led to a diagnosis of Dandy- Initial cytogenetic investigations in the patient were per- Walker malformation. Neurological developmental mile- formed on peripheral blood lymphocytes and then stones were normal at the age of 1 month. At the age of 5 subsequently repeated on a second blood sample and skin years, neurological examination and psychomotor develop- biopsy. Chromosomes were studied according to standard ment were normal. The patient walked at 16 months, lan- procedures for RHG banding.9

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Figure 2 Examples of dual colour painting of donor chromosome 2 (in red) and the four recipient chromosomes (in green): (A) translocation t(1;2)(p36;p12), (B) translocation t(2;5)(p12;q35), (C) translocation t(2;6)(p12;q27), (D) translocation t(2;12)(p12;q24). Dual colour FISH labelling was performed with (60°C) and 20 minutes at 72°C in order to allow the in situ commercially available whole chromosome paints for nucleotide chain elongation. At the end of the reaction, chromosomes 1, 2, 5, 6, and 12, according to the suppli- slides were washed in 2 × SSC-0.5% Tween 20 and then http://jmg.bmj.com/ ers’ instructions (Oncor, Gaithersburg, MD). Specific directly counterstained with propidium iodide in Vecta- painting probes were combined as follows: biotinylated shield antifade solution (Vector Laboratories, Burlingame, chromosome 2 specific paint with chromosome 1, 5, 6, or CA). 12 specific paint labelled with digoxigenin. Biotinylated Combined FISH and PRINS experiments were also painting was visualised using fluorescein-avidin whereas performed according to the previously reported protocol.11 detection of digoxigenin was done with antidigoxigenin- We used a centromeric repeat probe specific for chromo- rhodamine. some 2 (Oncor) and the consensus telomeric primer

Specific labelling of subtelomeric regions 1p, 2q, 5q, 6q, (CCTAA)7. on September 26, 2021 by guest. Protected copyright. and 12q was performed using subtelomeric specific probes. Fluorescent signals generated by FISH or PRINS were Probes for 2p, 5q, 6q, and 12q were direct fluorophore visualised by using a Leitz DMRD microscope equipped labelled probes provided by Vysis (Vysis Inc, Downers with appropriate filters. Combined FISH and PRINS Grove, IL). The YAC probe 762B5, specific for the subte- labelling experiments were analysed by confocal micros- lomeric region 1p, was a gift from Dr Rocci (Barri, Italy). copy. Optical sections were sampled with a confocal Nikon This probe was labelled by nick translation with microscope. Images were transferred to a Power Macintosh digoxigenin-11-dUTP (Boehringer Mannheim, Meylan, computer and standard software tools (Adobe Photoshop France) and used under the same conditions as the 5.0) were used for image analysis. purchased subtelomeric probes. The subtelomeric probes The parental origin of chromosome 2 was investigated correspond to loci estimated to be within 300 kb of the end by PCR analysis of two microsatellite markers, D2S337 of the chromosomes. and D2S2232, located on both sides of the breakpoint Telomere repetitive sequences were labelled by PRINS 2p12 (respectively in 2p14 and 2p11). DNA was extracted using the telomeric consensus primer (CCTAA)7. PRINS from peripheral blood of the proband and his parents. experiments were performed as previously described.10 Oligonucleotide primer sets were obtained from the Fifty µl of a reaction mix composed of the telomeric Genethon. PCRs were performed in a total volume of 50 consensus primer, a nucleotide mixture including µl containing 200 ng genomic DNA, 5 µl of 1 × PCR buVer fluorescein-dUTP, Ta q DNA polymerase buVer, and 2 (10 mmol/l Tris-HCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l units of Ta q DNA polymerase (Boehringer Mannheim) was MgCl2, 0.01% (w/v) gelatin), 200 µmol/l each of dNTPs, used. PRINS reactions were performed on a programma- 0.15 µl dUTP-R110, 0.2 µmol/l primers, and1UofTa q ble thermal cycler equipped with a flat plate block. The DNA polymerase). After an initial denaturation (96°C for denatured slides were put on the plate block and the reac- 21⁄2 minutes), the PCR was carried out for 30 cycles con- tion mixes placed on the slides and overlaid with coverslips. sisting of 20 seconds at 94°C, 20 seconds at 55°C, 40 sec- The reaction cycle consisted of two programmed steps: 10 onds at 72°C, followed by a final extension step of 72°C minutes at the specific annealing temperature of the primer for 10 minutes. One µl of each reaction product was mixed

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Figure 3 Results of combined in situ hybridisation of the subtelomeric speciﬁc probes for region 2p, labelled in green, and the four regions 1p (A), 5q (B), 6q (C), and 12q (D), labelled in red. In each case, lack of labelling of the der(2) can be seen. http://jmg.bmj.com/ with 2 µl of formamide, 0.5 µl of molecular weight marker, mary of the cytogenetic data is given in table 1 and partial and 0.5 µl of loading buVer. The mix was denatured for ideograms and RHG banded karyotypes are presented in three minutes at 90°C and 1 µl was loaded onto a ﬁg 1. This constitutional mosaicism was consistent with a denaturing 4% polyacrylamide gel (36 cm × 20 cm × 0.2 jumping translocation involving almost the entire short mm thick). The electrophoresis conditions were one hour arm of chromosome 2 (donor chromosome) alternately 40 seconds at 3000 V and 51°C. The analysis was carried transferred to the telomeric regions of four recipient chro-

out with the Genescan 2.1 software (Applied Biosystems) mosomes (chromosomes 1, 5, 6, and 12). The predomi- on September 26, 2021 by guest. Protected copyright. on an ABI 377. nant cell line in both initial and control samples was Chromosome studies on the initial blood sample and t(1;2)(p36;p12) (table 1). The parents’ chromosomes were control blood and ﬁbroblast cultures showed four diVerent normal with RHG banding. Derivative chromosomes cell lines: 46,XY,t(1;2)(p36;p12)/46,XY,t(2;5)(p12;q35)/ resulting from the jumping translocation were conﬁrmed 46,XY,t(2;6)(p12;q27)/46,XY,t(2;12)(p12;q24). A sum- in metaphase spreads by the use of whole chromosome

Figure 4 Detection of telomeric sequences by the PRINS technique showing the presence of interstitial telomeric sequences (arrow) at the junction of chromosome arm 2p12-pter and the recipient chromosomes 1 (A) and 6 (B).

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Figure 5 Confocal analysis of combined FISH and PRINS labelling on the derivative chromosome 2. (A) The centromere of the derivative chromosome 2 was identified with a rhodamine labelled centromeric specific probe. (B) Telomeric repetitive sequences were detected on both ends of the broken chromosome 2 (arrows) by PRINS with a telomeric consensus primer and fluorescein-dUTP.(C) The combined picture shows the overlapping of the two signals close to the 2p12 breakpoint. painting probes (fig 2). No normal or unbalanced chromo- The results of our classical cytogenetic investigations some complements were found. However, dual colour and whole chromosome painting suggested a simple trans- whole chromosome painting did not show evidence of position with fusion between the 2p12 segment and the translocation of chromosomal material from recipient telomeres of recipient chromosomes rather than a recipro- chromosomes 1, 5, 6, and 12 onto another chromosome. cal exchange, since recipient chromosomes appeared Specific subtelomeric FISH labelling (fig 3) showed that cytogenetically intact. The observed subtelomeric labelling these sequences were not translocated between chromo- was in accord with this interpretation since no transloca- some 2 and the four recipient chromosomes, whereas tion of subtelomeric sequences was observed between consensus PRINS labelling of telomeric repetitive se- chromosome 2 and the four recipient chromosomes. quences clearly indicated the presence of interstitial telo- Nevertheless, this does not rule out the possiblity of trans- meric sequences on the recipient chromosomes (at the locations occcurring distal to subtelomeric sequences since junction sites of the translocation) (fig 4), and at both ends the subtelomeric probes are usually located between 40 of the derived chromosome 2 (fig 5). and 300 kb from the telomeres. The results of microsatellite marker analysis of chromo- PRINS labelling showed interstitial telomeric sequences some 2 are given in fig 6. Both paternal and maternal alle- at the telomeric breakpoint of the recipient chromosomes les were present in the proband for each locus. This and at the broken end of chromosome 2. These data may heterozygosity was not in favour of uniparental disomy for suggest that the telomeric repeat sequences of the recipient chromosome 2. chromosomes were partially deleted and translocated onto The proband is the product of a consanguineous union. the proximal end of the broken chromosome 2, which

He shows signs of Dandy-Walker malformation and would be in favour of a translocation event. However, for http://jmg.bmj.com/ normal neurological evaluation at the age of 1 month. the jumping translocation we report, this event would have Repeat chromosome studies in blood and in ﬁbroblasts, as to occur four times which seems unlikely. well as chromosome painting experiments, show de novo An alternative explanation for the presence of telomeric mosaicism for a jumping translocation in which chromo- sequences at the proximal end of der(2) is the de novo for- some 2p is alternately transferred to various recipient mation of telomeric sequence at the end of the der(2). This chromosomes (1, 5, 6, and 12). There is no evident has been shown to occur through healing of broken chro- relationship between the phenotype and the chromosome mosomal ends by seeding of telomeric sequences and 16 abnormality in our patient. Subtelomeric rearrangement requires the expression of telomerase in the cell. The sta- on September 26, 2021 by guest. Protected copyright. does not seem to contribute to the phenotype; however, bilisation of broken chromosomes could also result from mosaicism cannot be totally excluded and it is also possible the recognition by telomerase of internal telomere-like that the translocation breakpoint on chromosome 2 has sequences proximal to the breakpoint.17 In many eukaryo- disrupted the sequences of one (or more) genes implicated tes, the ability to heal broken chromosome ends by telo- in Dandy-Walker syndrome. As shown in a recent system- meric addition is a well documented phenomenon and atic study, cryptic deletions may be an important cause of appears to be controlled by developmental factors.18 This disease in patients with apparently balanced chromosome phenomenon occurs early in embryonic development, rearrangements.12 The possibility of uniparental disomy of shortly after zygote formation when telomerase activity is chromosome 2, which might be related to the malforma- still eYcient.19 tion of the proband, has been minimised by the microsat- This information contributes to an understanding of ellite analysis. when our proband’s rearrangement occurred. Mosaicism At present, little is known about the origin of jumping was found in blood and skin cultures of the patient. For translocations. In three other reports of constitutional both tissues, there is one largely predominant cell line, jumping translocations, interstitial telomeric sequences involving chromosomes 1 and 2. No chromosomally have been observed.51314 Interstitial telomeric sequences normal cells were found. It seems likely that chromosome thus seem to be a common feature of constitutional jump- 2 is prone to breakage. Breakage could have occurred in ing translocations occurring at telomeric recipient sites. four (or more) diVerent cell lineage precursors and the The presence of interstitial telomeric sequences was not broken chromosome 2 fragment translocated to the end of systematically observed in cases of jumping translocations diVerent chromosomes. associated with haematological disorders.15 Nevertheless, The presence of interstitial telomeric sequences is failure to observe interstitial telomeric sequences may be considered to represent a form of chromosome instability because of an inherent limitation of in situ labelling proce- and, consequently, these sequences could play a role in dures for the detection of submicroscopic telomeric generating the observed mosaicism.20 Although the telo- sequences. meric repeat nucleotide sequences are similar in telomeres

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Figure 6 Results of PCR analysis of chromosome 2 microsatellite markers D2S337 and D2S2232 on DNA extracted from peripheral blood of the proband and his parents. For each locus, the two parental alleles were found in the proband’s DNA (A and B). and intrachromosomal areas, their diVerent locations telomere-like sequences in chromosome breakage, rear- (terminal or interstitial) could contribute to diVerential rangement, and instability could be the result of quantita- organisation of the chromatin in the two domains, as shown tive or qualitative diVerences in the constitution of proteins by the variable eVect of exonuclease on terminal and inter- associated with telomeric repeat sequences according to stitial telomeric sequences.21 In jumping translocations, the their location. It would be interesting to determine whether presence of interstitial telomeric sequences on a receptor specific telomeric proteins such as TRF1 and TRF2 are chromosome makes this chromosome unstable and makes present in interstitial telomere sequence regions.22 the transferred fragment susceptible to “jumping” from one The elucidation of the underlying mechanism and the telomere to another. An alternative suggestion is that pref- role of telomeres in the jumping translocation process will erential involvement of telomeric regions in jumping trans- require studies combining cytogenetic and molecular tech- locations could result from the stabilisation of chromosome nologies. In the future, the understanding of jumping breakage by a telomeric capture process. Our report translocation processes will greatly benefit by the molecu- provides further evidence that the jumping process and the lar cloning of breakpoints as well as the search for specific presence of interstitial telomeric sequences could be genes or sequences involved in the occurrence of these rare related. The preferential involvement of interstitial chromosomal events.

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We wish to thank Dr Mariano Rocci for providing the YAC probe 762B5. This 11 Pellestor F, Quenesson I, Coignet L, Girardet A, Andréo B, Lefort G,Char- work was supported by the Association Française Contre les Myopathies (AFM). lieu JP. FISH and PRINS, a strategy for rapid chromosome screening: J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from Cytogenet Cell GENEVIÈVE LEFORT* application to the assessment of aneuploidy in human sperm. Genet 1996;72:34-6. PATRICIA BLANCHET† 12 Wirth J, Nothwang HG, van der Maarel S, Menzel C, Borck G, ANNE MARIE CHAZE* Lopez-Pajares I, Brondum-Nielsen K, Tommerup N, Bugge M, Ropers ANNE GIRARDET‡ HH, Haaf T. Systematic characterisation of disease associated balanced PIERRE SARDA† chromsome rearrangements by FISH: cytogenetically and genetically JACQUES DEMAILLE*‡ anchored YACs identify microdeletions and candidate regions for mental FRANCK PELLESTOR‡ retardation genes. J Med Genet 1999;36:271-8. *Cytogenetics Laboratory, CHU Montpellier, France 13 Rossi E, Floridia G, Casali M, Danesino C, Chiumello G, Bernardi F, Mag- †Clinical Genetics Department, CHU Montpellier, France nani I. Types, stability and phenotypic consequences of chromosome rear- ‡CNRS-UPR 1142, IGH, 141 rue de la Cardonille, F-34396 Montpellier rangements leading to interstitial telomeric sequences. J Med Genet Cedex 5, France 1993;30:926-31. 14 Devriendt K, Petit P, Matthijs G, Vermeesch JR, Holvoet M, de Muelenaere Correspondence to: Dr Pellestor, [email protected] A, Marynen P, Cassiman JJ, Fryns JP. Trisomy 15 rescue with jumping translocation of distal 15q in Prader-Willi syndrome. J Med Genet 1997;34: 395-9. 1 Gimelli G, Porro E, Santi F, Scappaticci S, ZuVardi O. “Jumping” satellites 15 Gray BA, Bent-Williams A, Wadsworth J, Maiese RL, Bhatia A, Zori RT. in three generations: a warning for paternity test and prenatal diagnosis. Fluorescence in situ hybridization assessment of the telomeric regions of Hum Genet 1976;34:315-18. 2 Lejeune J, Maunoury C, Prieur M, Van den Akker J. Translocation sauteuse jumping translocations in a case of aggressive B-cell non-Hodgkin (5p;15q), (8q;15q), (12q;15q). Ann Genet 1979;22:210-13. lymphoma. Cancer Genet Cytogenet 1997;98:20-7. 3 Reis MD, Dube ID, Pinkerton PH, Chen-Lai J, Robinson JB, Klock RJ, 16 Flint J, Wilkie AO, Buckle VJ, Winter RM, Holland AJ, McDermid HE. The Senn JS. “Jumping” translocations involving band 3q13.3 in a case of acute detection of subtelomeric chromosomal rearrangements in idiopathic men- monocytic leukemia. Cancer Genet Cytogenet 1991;51:189-94. tal retardation. Nat Genet 1995;9:132-40. 4 Ben-Neriah S, Abramov A, Lerer I, Polliack A, Leizerowitz R, Rabinowitz R, 17 Meltzer PS, Guan XY, Trent JM. Telomere capture stabilizes chromosome Abeliovich D. “Jumping translocation” in a 17-month-old child with breakage. Nat Genet 1993;4:252-5. mixed-lineage leukemia. Cancer Genet Cytogenet 1991;56:223-9. 18 Blackburn EH. Developmentally programmed healing of chromosomes. In: 5 Park VM, Gustashaw KM, Wathen TM. The presence of interstitial telom- Blackburn EH, Greider CW, eds. Telomeres. New York:Cold Spring Harbor eric sequences in constitutional chromosome abnormalities. Am J Hum Laboratory Press, 1995:203-18. Genet 1992;50:914-23. 19 Cooke H. Non-programmed and engineered chromosome breakage. In: 6 Vermeesch JR, Petit P, Speleman F, Devriendt K, Fryns JP, Marynen P. Blackburn EH, Greider CW, eds. Telomeres. New York:Cold Spring Harbor Interstitial telomeric sequences at the junction site of a jumping transloca- Laboratory Press, 1995:219-45. Hum Genet tion. 1997;99:735-7. 20 BouZer SD, Morgan WF, Pandita TK, Slijepcevic P. The involvement of 7 Humbertclaude VT, Coubes PA, Leboucq N, Echenne BB. Familial Dandy- telomeric sequences in chromosomal aberrations. Mutat Res 1996;366:129- Walker malformation and leukodystrophy. Pediatr Neurol 1997;16:326-8. 8 Ulm B, Ulm MR, Deutinger J, Bernaschek G. Dandy-Walker malformation 35. diagnosed before 21 weeks of gestation: associated malformations and 21 Fernandez JL, Goyanes VJ, Ramiro-Diaz J, Gosalvez J. Evidence of a diVer- chromosomal abnormalities.Ultrasound Obstet Gynecol 1997;10:167-70. ential organization of chromatin containing terminal or interstitial (TTAG- 9 Dutrillaux B, Lejeune J. Sur une nouvelle technique d’analyse du caryotype GG)n repeats by in situ digestion with nucleases. Cytogenet Cell Genet 1998; humain. C R Acad Sci (D) (Paris) 1971;272:2638-40. 82:195-8. 10 Pellestor F, Girardet A, Lefort G, Andréo B, Charlieu JP. The use of PRINS 22 Broccoli D, Smogorzewska A, Chong L, de Lange T. Human telomeres con- technique for a rapid in situ detection of chromosomes 13, 16, 18, 21, X tain two distinct Myb-related proteins, TRF1 and TRF2. Nat Genet 1997; and Y. Hum Genet 1995;95:12-17. 17:231-5.

J Med Genet 2001;38:73–76 Clinical and cytogenetic have suggested that most of the phenotypic features of DS are the result of the triple dose of the genes contained in the characterisation of a patient with region around marker D21S55. This region is called the http://jmg.bmj.com/ Down syndrome resulting from a Down syndrome critical region (DSCR).4–6 However, recent evidence suggests that genes outside this region may 21q22.1→qter duplication also contribute to the DS phenotype.7 To evaluate DS patients clinically, a checklist of 25 clinical traits was ﬁrst reported by Jackson et al8 and later revised by Epstein et al.9 The accurate and exhaustive clinical evaluation of the EDITOR—Trisomy of human chromosome 21 is one of the

patients, along with the characterisation of the extent of on September 26, 2021 by guest. Protected copyright. most frequent aneuploidies in humans and results in Down each trisomy, should help to establish genotype/phenotype syndrome (DS), aVecting approximately 1 in 700 live 1 correlations. Comparison of the clinical findings in DS births. DS is a major cause of mental retardation and con- patients with similar partial trisomies is now possible genital heart disease in humans, but is also associated with through the database created by J Delabar (http:// other major features, such as characteristic facies, skeletal www.infobiogen.fr/services/aneu21), which includes the abnormalities, and an increased risk of leukaemia and Alzheimer’s disease. In most cases (95%), the trisomy of chromosome 21 involves the whole chromosome through maternal (most frequent) or paternal non-disjunction, whereas in some cases (4%) Down syndrome is the result of an unbalanced translocation. Among the latter, a very small proportion of cases are the result of partial trisomy of chromosome 21 arising either from non-Robertsonian translocations or from intrachromosomal duplications.2 The DS phenotype is also found in cases (1%) of mosaicism of trisomy 21.3 During the last decade, considerable progress has been made towards discovering the gene content of chromosome 21, but the functions of most of these genes and their specific contributions to the final DS phenotype still remain unknown. The identification and characterisation Figure 1 Patient with a duplication 21q22-qter at the age of cases of partial trisomy of chromosome 21 has allowed a of 2 years 11 months. Note the flat face, brachycephaly, phenotypic map of this chromosome and DS to be palpebral fissures, epicanthic folds, and malformed and low constructed. Clinical and molecular studies of these cases set ears.

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Loci YAC J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

D21S226 D21S213 D21S300 814C1 D21S301 D21S302 D21S303 D21S305 D21S310 280B1 D21S306 D21S304 D21S263 D21S307 731H10 D21S309 D21S93 13 SOD1 D21S389 12 D21S390 p D21S311 11.2 D21S261 690F12 D21S312 D21S313 11.1 D21S314 D21S404 D21S262 667B10 11.1 D21S315 D21S316 D21S391 11.2 D21S317 246C2 D21S392 D21S319 D21S216 D21S320 876D4 GART D21S323 IFNAR D21S58 VNO2 D21S324 21 KCNE1 D21S325 D21S332 DSCR1 72H9 73D10 D21S65 AML1 D21S393 D21S211 A222A12 D21S17 q CBR D21S33 230E8 D21S334 D21S396 D21S395 D21S167 549A9 D21S267 SODI D21S335 D21S336 152F7 D21S394 22.1 D21S270 DYRK1A http://jmg.bmj.com/ D21S337 GART 238B1 D21S55 AMLI D21S233 D21S341 374B8 ERG D21S259 ERG 22.2 D21S338 221B9 D21S339 D21S3 ETS2 D21S3 767B3 D21S343 D21S220 D21S344 D21S345 on September 26, 2021 by guest. Protected copyright. 22.3 D21S346 D21S168 COL6A1/ D21S15 D21S23 COL6A2 D21S349 D21S53 BACE2 552A3 D21S64 D21S355 D21S359 MX2 MX1 D21S19 D21S113 CBS Figure 2 Schematic representation of human chromosome 21, indicating the region involved in the partial trisomy of the patient with dup(21q22.1→qter). A panel of 18 YACs covering the proximal region involved in the duplication is shown. Circled YACs were used in FISH experiments. The markers near the duplication breakpoint (D21S304, D21S306, D21S310, and D21S305) are indicated in bold. Other markers outside the duplication breakpoint are shown as reference loci. molecular characterisation and clinical findings of patients but also lacks some of the characteristic features of the dis- with aneuploidy of chromosome 21. FISH analysis has order. The extent of the duplication was characterised by proven very useful for characterising the extent of partial FISH. It extends from marker D21S304 to the telomere trisomies of chromosome 21, since a contig of YACs and the centromeric breakpoint is localised in the chromo- covering the whole long arm of the chromosome is somal region encompassed by YAC 280b1, centromeric to available10 11 and the sequenced clones are constantly the SOD1 gene. released to the public databases. The patient is a boy from Sâo Paulo, Brazil. The family We report here the clinical findings of a patient with a history is unknown and the patient is currently in a Shelter partial trisomy of chromosome 21 resulting from a House of the Brazilian Government waiting for adoption. dup(21q22.1-qter). The patient has some clear signs of DS The patient was first seen at the age of 1 year 10 months.

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Table 1 Clinical features of Down syndrome in patients with partial trisomy of chromosome 21q22-qter J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from

Cases

Present Phenotypic features DL* SOL† t(15;21)‡ case

Short stature ? +/− ++ + Mental retardation ? + + + Microcephaly ? + + − Brachycephaly ? + + + Oblique eye fissures + + + + Epicanthic folds + + + + Brushfield spots + − − − Flat nasal bridge + − + + Flat face ? + + + Open mouth ? + + - High arched palate ? + + + Furrowed tongue ? − − − Malpositioned ears ? + − + Small, dysmorphic ears + + + + Short neck + + + − Short and broad hands + + + + Clinodactyly 5th finger + + + + Transverse palmar crease − − + − Abnormal dermatoglyphics + ? + − Hypotonia ? − + +/− Congenital heart defect + − − − Other visceral anomalies − − − −

+, presence of the feature. −, absence of the feature. ?, unknown. *Delabar et al,6 †Korenberg et al,7 ‡Nadal et al.14

The first examination showed some clinical features suggestive of DS, such as oblique eye fissures, low set ears, high and narrow palate, mild hypotonia, and protruding tongue, as well as delayed psychomotor milestones. A karyotype was performed then and showed 46,XY,21q+. This cytogenetic result prompted a further clinical examination of the patient according to the protocols described by Epstein et al.9 At this second examination, the patient was 2 years 11 months old (fig 1). Chromosome analyses were performed on standard phytohaemaglutinin (PHA) stimulated lymphocyte cul- http://jmg.bmj.com/ tures from peripheral blood. Some chromosome spreads were G banded and 5-bromo-2'-deoxyuridine (BrdU) was Figure 3 FISH analysis on metaphase spreads of a patient with Down added to two cultures to perform R banding. We generated syndrome phenotype and a dup(21q22.1→qter). (A) Metaphase of the patient showing a duplication in one of the chromosome 21 homologues painting probes from chromosome 21 in order to elucidate when hybridised with YAC280b1. In the left corner, R banding image of the origin of the extra material. We used the hybrid dup(21) and its homologue. (B) Metaphase of the same patient hybridised (mouse-human) cell line WAV-17 that has chromosome 21 with YAC 814c1 showing only one signal on both chromosomes 21, as its only human component.12 We extracted the DNA and indicating that the partial trisomy does not include the markers contained 13 in this YAC. performed inter Alu-PCR. To define the partial trisomy on September 26, 2021 by guest. Protected copyright. further, YACs from the long arm of chromosome 21 were of chromosome 21, showed that the patient had a duplica- selected10 and also amplified by inter Alu-PCR. A total of tion 21q22.1→qter. The contig of YACs used showed that 14 YACs were hybridised (fig 2). Probes were labelled and the breakpoint was located just proximal to SOD1, within hybridised as described elsewhere.14 Twenty metaphases YAC 280b1, which contains markers DS21S304 to and 50 interphase nuclei were studied for each probe. D21S306 (fig 2). All distances between the double signals The clinical evaluation of the patient at the age of 2 years on the dup(21) were identical for all YACs studied. All the 11 months showed short stature, brachycephaly, flat facies, metaphases analysed by FISH showed the duplication, dis- oblique palpebral fissures, epicanthic folds, flat nasal counting the possibility of mosaicism observed using bridge, high palate, malformed and low set ears, short and banding techniques (fig 3). broad hands with clinodactyly of the fifth finger, and mod- We have identified a case of partial trisomy of erate mental retardation. The patient also had delayed psy- chromosome 21 and a DS phenotype caused by duplica- chomotor development; he sat at the age of 8 months and tion of 21q22.1→qter. Using FISH with YAC probes from walked at 16 months (table 1, fig 1). Dermatoglyphics were the long arm of chromosome 21, we have identified the normal and an electrocardiogram showed that the patient extent of this duplication as well as its orientation. The tri- had an incomplete block of the right branch (with no somic region spans marker D21S213 to the telomere, charge), but no major congenital heart defect. Abdominal which covers about 13 Mb of the chromosome, as judged scan and audiometry were normal. The cytogenetic study from the physical map.15 Comparison of the clinical performed with both G and R banding showed that out of features resulting from partial trisomy of chromosome 21 45 metaphases analysed, 37 had extra material on chromo- has provided the basis for construction of the DS some 21q. A more detailed study of the case by FISH using phenotypic map. The coverage of chromosome 21 by a a painting probe that covers the whole long arm of panel of YAC clones and the use of these YACs in FISH chromosome 21 showed that all the extra material attached analyses allows us to combine the phenotypic information to one chromosome 21 was derived from the same from our DS patient with a fairly accurate molecular defi- chromosome. FISH performed with YACs along the q arm nition of the duplicated region. This approach is much

www.jmedgenet.com 76 Letters faster and more precise than the dosage studies or DECIO BRUNONI§ J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from polymorphic marker methods previously used.67The phe- MELANIE PRITCHARD*¶ 7 XAVIER ESTIVILL* notypic map of DS constructed by Korenberg et al *Centre de Genètica Mèdica i Molecular-IRO, Avenida Castelldefels Km assigned 25 features to regions spanning 2 to 20 Mb and 2.7, L’Hospitalet de Llobregat, 08907 Barcelona, Spain they concluded that DS is a contiguous gene syndrome †Laboratorio de Genética Humana, Universidad Nacional Pedro Ruiz with duplications distinct from distal 21q22 contributing Gallo, Lambayeque, Perú to the main features of DS. Given that the partial trisomy ‡Laboratorio de Citogenética, UNIFESP-EPM, Brasil §Centro de Genética Médica, UNIFESP-EPM, Brasil of chromosome 21 in the patient does not involve any other chromosome, it further supports the hypothesis that the Correspondence to: Dr Estivill, [email protected] genes contained in the region from 21q22 to the telomere ¶Present address: Centre for Functional Genomics and Human Disease, are responsible for the majority of the features of DS, as Monash University Medical School, Clayton, Victoria, Australia previously reported by Korenberg et al.7 Three other cases have been reported with a similar duplicated region.6714As 1 Hassold T, Jacobs P. Trisomy in man. Annu Rev Genet 1984;18:69-97. 2 Epstein C. Down syndrome. In: Scriver CR, Beaudet AL, Sly WS, Valle D, shown in table 1, the case reported here further confirms eds. The metabolic and molecular bases of inherited disease. 7th ed. Chap 18. that the majority of the phenotypic features of DS are con- New York: McGraw-Hill, 1995:749-94. 3 Hook EB. Unbalanced Robertsonian translocations associated with Down’s tained in the region triplicated in the four cases. However, syndrome or Patau’s syndrome: chromosome subtype proportion inherited, the penetrance of the majority of the clinical features of DS mutation rates, and sex ratio. Hum Genet 1981;59:235-9. 4 Niebuhr E. Down’s syndrome: the possibility of a pathogenic segment on is not complete, so to establish the correlation only the chromosome No 21. Hum Genet 1974;21:99-101. presence (not the absence) of a given trait should be taken 5 McCormick MK, Schindel A, Petersen MB, Stetten G, Driscoll DJ, Cantu ES, Tranebjaerg L, Mikkelsen M, Watkins PC, Antonarakis SE. Molecular into account. When the three published cases of DS with a genetic approach to the characterization of the ‘Down syndrome region’ of similar partial trisomy and the present case are compared, chromosome 21. Genomics 1989;5:325-31. 6 Delabar JM, Theophile D, Rahmani Z, Chettouh Z, Blouin JL, Prieur M, we can see that almost all the physical traits typical of DS Noel B, Sinet PM. Molecular mapping of twenty-four features of Down are present, perhaps with the exception of the furrowed syndrome on chromosome 21. Eur J Hum Genet 1993;1:114-24. 7 Korenberg JR, Chen XN, Schipper R, Sun Z, Gonsky R, Gerwehr S, tongue which has an overall frequency of 55% in the DS Carpenter N, Daumer C, Dignan P, Disteche C, Graham JM, Hugdins L, population.16 One of the diYculties in the construction of a McGillivray B, Miyazaki K, Ogasawara N, Park JP, Pagon R, Pueschel S, Sack G, Say B, SchuVenhauer S, Soukup S, Yamanaka T. Down syndrome phenotypic map of DS, based on cases of partial trisomy, is phenotypes: the consequences of chromosomal imbalance. Proc Natl Acad that a large proportion of these cases have in addition other Sci USA 1994;91:49970-5001. 8 Jackson JF, North ER, Thomas JG. Clinical diagnosis of Down’s syndrome. chromosomal abnormalities which may contribute to the Clin Genet 1976;5:483-7. clinical findings. Our patient presents no other chromo- 9 Epstein CJ, Korenberg JR, Anneren G, Antonarakis SE, Ayme S, Courchesne E, Epstein LB, Fowler A, Groner Y, Huret JL, Kempter TL, somal abnormality, so all his clinical traits can be assumed Lott IT, Lubin BH, Magenis E, Opitz JM, Patterson D, Priest JH, Pueschel to be associated with the duplication of chromosome 21. SM, Rapoport SI, Sinet PM, Tanzi RE, de la Cruz F. Protocols to establish genotype-phenotype correlations in Down syndrome. Am J Hum Genet However, we have established comparisons with other 1991;1:207-35 cases with a very small partial monosomy of another chro- 10 Chumakov I, Rigault P, Guillou S, Ougen P, Billaut A, Guasconi G, Gervy P, LeGall I, Soularue P, Grinas L, Bougueleret L, Bellanné-Chantelot C, mosome, which may have a very small contribution to the Lacroix B, Barillot E, Gesnouin P, Pook S, Vaysseix G, Frelat G, Schmitz A, final phenotype of the patients. Sambucy JL, Bosch A, Estivill X, Weissenbach J, Vignal A, Riethman H, Cox D, Patterson D, Gardiner K, Hattori M, Sakaki Y, Ichikawa H, Ohki With the continued development of molecular cyto- M, Le Paslier D, Heilig R, Antonarakis S, Cohen D. Continuum of overlap- genetic tools and the availability of methods and probes for ping clones spanning the entire human chromosome 21q. Nature 1992;359:380-6.

the accurate determination of the chromosomal rearrange- 11 Nizetic D, Gellen L, Hamvas R, Mott R, Grigoriev A, Vatcheva R, Zehetner http://jmg.bmj.com/ ments in each case, phenotype/genotype correlations can G, Yaspo ML, Dutriaux A, Lopes C, Delabar JM, Van Broeckhoven C, Potier MC, Lehrach H. An integrated YAC-overlap and ‘cosmid-pocket’ be obtained with a high degree of accuracy and diagnosis map of the human chromosome 21. Hum Mol Genet 1994;3:759-70. can be performed at the molecular level with a high degree 12 Raziuddin A, Sarkar FH, Dutkowski R, Shulman L, Ruddle FH, Gupta SL. Receptors for human alpha and beta interferon but not gamma interferon of certainty. are specified on human chromosome 21. Proc Natl Acad Sci USA 1984,81: 5504-8. 13 Tagle DA, Collins FS. An optimized Alu-PCR primer pair for human The first two authors contributed equally to this work. We thank D Patterson for specific amplification of YACs and somatic cell hybrids. Hum Mol Genet the gift of the human chromosome 21 somatic hybrid cell lines and D Cohen, I 1992;1:121-2. Chumakov, and D Patterson for YAC clones. This work was supported in part 14 Nadal M, Moreno S, Pritchard M, Preciado MA, Estivill X, Ramos-Arroyo by the Fundació Síndrome de Down/Marató TV3, SAF99-0092-C02-01 the MA. Down syndrome: characterisation of a case with partial trisomy of on September 26, 2021 by guest. Protected copyright. Comisión Interministerial de Investigación Científica y Técnica and by the chromosome 21 owing to a paternal balanced translocation (15;21)(q26; Servei Català de la Salut and the Generalitat de Catalunya CIRIT/1997/SGR/ q22.1) by FISH. J Med Genet 1997;34:50-4. 00085 grant. 15 Ichikawa H, Hosoda F, Arai Y, Shimizu K, Ohira M, Ohki M. A NotI Nat Genet MARGA NADAL* restriction map of the entire long arm of human chromosome 21. 1993;4:361-5. CÉSAR GUZMÁN VIGO† 16 Pueschel SM, Sassaman EA, Scola PS, Thuline HC, Stark AM, Horrobin MARIA ISABEL MELARAGNO‡ M. Biomedical aspects in Down syndrome. In: Pueschel SM, Rynders JE, JOYCE A D ANDRADE‡ eds. Down syndrome. Advances in biomedicine and behavioral sciences. LUIS GARCIA ALONSO§ Cambridge, MA: Ware, 1982:169.

J Med Genet 2001;38:76–79 Stable non-Robertsonian dicentric tion of anaphase bridge during cell division. Therefore, breakage of the dicentric can occur with subsequent cell chromosomes: four new cases and a death.1 Stability can be achieved when the centromeres are review very close together and form only one heterochromatin block, or when one of them is inactivated. One report by Vianna-Morgante and Rosenberg2 shows a rarer mechanism of stabilisation, the deletion of one centromere. Sev- EDITOR—Dicentric autosomes are rarely encountered as eral mechanisms can lead to dicentric chromosomes: mei- stable constitutional chromosomes in humans, with the otic recombination within a paracentric inversion loop, exception of Robertsonian translocations. The presence of isochromatid break with U shaped rejoining, mitotic cross- two alpha satellite sequences on the same chromosome ing over, Robertsonian translocation, and non-homologous leads to a high risk of attachment of the same chromatid to non-Robertsonian translocation. Non-homologous dicen- the mitotic spindle from opposite poles and to the forma- tric autosomes are expected to be formed by the latter. We

www.jmedgenet.com Letters 77

compatible with intrauterine death at 15 weeks. External J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from and internal pathological examination showed no malformation. Therefore, post-amniocentesis intrauterine fetal death was not excluded. Case 2 was ascertained through fetal tissues received for IUFD at 32 weeks of gestation. The mother was a 29 year old gravida 3, para 1, aborta 1. The second trimester screening ultrasound at 18 weeks’ gestation showed an isolated choroid plexus cyst. At 32 weeks, IUFD was diagnosed. Necropsy showed a growth retarded male fetus with a weight of 650 g and with crown-heel length of 32 cm. The phenotype was consistent with trisomy 18, including mild hirsutism, small mouth, overlapping fingers, rocker bottom feet, short hallux, nail hypoplasia, interventricular septal defect, and Meckel’s diverticulum. The tissue cultures showed two abnormal and discordant G banded cell lines: 46,XY,+18,dic(14;18) (p11.2;p11.3)[12]/45,XX,dic(14;18)(p11.2;p11.3)[12]. One cell line was an unbalanced male karyotype composed of a dicentric chromosome formed by an apparently balanced translocation between the short arms of chromosomes 14 and 18, as well as two normal chromosomes 18, resulting in trisomy 18. The other cell line was female, carrying the same dicentric with a chromosome count of Figure 1 Dicentric chromosomes of our cases. Panels A-D: chromosomes 45 as a result of the formation of the dicentric. This find- G banded at the 450-500 band level of resolution from cases 1-4, ing could be explained by the presence of a female resorbed respectively. In all four panels, the dicentric chromosome is placed in twin or by maternal contamination. The latter was more between its normal monocentric homologues. Panels E and F: subtelomeric sequence specific to the long arm of 16q and 14/22 alpha satellite likely since the phenotypically normal mother did indeed hybridised, respectively, to chromosomes of case 1 (hybridisation signals carry the dicentric (fig 1B). The grandmother’s karyotype indicated by arrows). Panels G and H: 13/21 alpha satellite sequence and was normal. The maternal grandfather could not be 18 alpha satellite sequence hybridised, respectively, to chromosomes of case 3 (hybridisation signals indicated by arrows). C banded dicentric of case 2 reached. The dicentric showed only one primary constric- (centromeric heterochromatin indicated by arrows). tion at the site of chromosome 18 centromere in all cells analysed from the fetus and the mother. C banding have found 22 reported cases of cytogenetically recognis- confirmed the presence of two blocks of centromeric able, non-homologous, non-Robertsonian dicentric heterochromatin (fig 1I). autosomes.1–19 We present four new cases of non- Case 3 was born at 40 weeks of gestation after an homologous, non-Robertsonian dicentric autosomes with uneventful pregnancy. The birth weight was 3960 g (75th centile), birth length was 54 cm (90th centile), and OFC centromeres distinguishable by standard cytogenetic tech- http://jmg.bmj.com/ niques; two were inherited from asymptomatic carriers and was 36.5 cm (75th centile). Apgar scores were 8, 9, and 10 two occurred de novo in children with deletion 18p at one, five, and 10 minutes respectively. Karyotyping was syndrome phenotype. requested at the age of 41⁄2 years because of psychomotor Metaphase chromosomes were obtained from peripheral retardation. He was globally delayed with severe diYculties blood or amniocytes harvested according to standard pro- in language. At the age of 41⁄2 years, he could walk, hop, tocols. GTG banding was performed on all cases. C band- and ride a tricycle but had diYculty with his balance, could ing using Ba(OH)2 was used. Fluorescence in situ hybridi- only speak 20 words with no sentences, and would not play sation (FISH) of alpha satellite sequences was performed with other children. Physical examination showed mild on September 26, 2021 by guest. Protected copyright. on metaphase chromosomes according to the protocols dysmorphic features, including a long face, bilateral provided by ONCOR. FISH with a 16q subtelomeric epicanthic folds, bulbous nose, large and slightly an- sequence was performed according to the protocol teverted ears, high arched and narrow palate, microretrog- provided by AL Technology. nathia, pectus excavatum, and bilateral fifth finger Case 1 was ascertained through amniocentesis at 14 clinodactyly. Thyroid evaluation was normal. The karyo- weeks’ gestation for advanced maternal age. The mother type, 45,XY,dic(13;18)(p12;p11.2), showed an unbal- was a 41 year old gravida 3, para 2, aborta 0. The couple’s anced translocation between the short arms of chromo- family history was unremarkable. The GTG banded somes 13 and 18 creating a dicentric chromosome with a karyotype at a resolution of approximately 350 bands deletion of the distal band of chromosome 18p, 18p11.3 showed the presence of an apparently balanced transloca- (fig 1C). There was only one primary constriction tion between the telomeric regions of 16q and 22p, creat- corresponding to chromosome 18 centromere in all cells ing a dicentric 45,XY,dic(16;22)(q24;p11.2). The pheno- analysed. FISH with probes against the alpha satellites typically normal father was proven to carry the same 13/21 (fig 1G) and 18 (fig 1H) confirmed the presence of translocation which, in his case, had arisen de novo (fig two centromeres. 1A). The dicentric showed only one primary constriction Case 4 was first evaluated at 20 months of age because of on G banding in all cells analysed in the fetus and his developmental delay and dysmorphic features. She was father, corresponding to chromosome 16 centromere. It born at 37 weeks of gestation after an uneventful hybridised with probes for the alpha satellite sequences of pregnancy. Her birth weight was 2565 g (5th-10th centile), chromosomes 14 and 22 (fig 1F) and for the subtelomeric birth length was 47 cm (10th centile), and OFC was 30.8 sequence of the long arm of chromosome 16 (fig 1E). After cm (<5th centile). At birth she was diagnosed with genetic counselling, the couple decided to continue the transposition of the great arteries and operated on as a pregnancy. However, in the 20th week of gestation, intra- neonate. At 3 months of age she suVered from a urinary uterine fetal death (IUFD) was diagnosed. The fetus tract infection and renal ultrasound showed mild bilateral showed severe autolytic changes and had measurements hydronephrosis. Her psychomotor development was de-

www.jmedgenet.com 78 Letters layed.She started to crawl at 14 months and was not walk- number of hypotheses can be advanced to explain the non- J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from ing at 20 months; she was not speaking any words at the random participation of acrocentrics in the formation of time of the first evaluation. At that age, she weighed 9.0 kg non-Robertsonian heterodicentric chromosomes. There (<5th centile), her length was 79 cm (10th-25th centile), may be an increased rate of dicentric formation with acro- and her OFC was 45.5 cm (2nd centile). Physical centrics based on non-random nuclear positioning of examination showed dysmorphic features, including bilat- chromosomes. However, the latter explanation does not eral ptosis of the eyelids (right>left), mild bilateral epican- account for the randomness of the non-acrocentric thic folds, bulbous nose, large and anteverted simple ears, chromosomes involved in the formation of the dicentrics. central dimple on the chin, slightly short neck, mild pectus The predominance of acrocentics is probably more a excavatum, bilateral 5th finger clinodactyly, partial 2-3 reflection of the higher likelihood of stability of the dicen- syndactyly of the toes, and a blind sacral dimple. Ophthal- tric formed. Deletion of the short arm of an acrocentric is mological evaluation showed myopia. Growth hormone often present, so the distance between the centromere of and IgA levels were normal. The karyotype, the acrocentric and the translocation breakpoint is always 45,XX,dic(13;18)(p11.2;p11.2), showed an unbalanced relatively small, which is in favour of dicentric stability. translocation between the short arms of chromosomes 13 Moreover, absence of a phenotype related to a deletion of and 18 creating a dicentric chromosome with a deletion of the p arm of an acrocentric is in favour of embryonic the distal band of chromosome 18p, 18p11.3 (fig 1D). All viability. It has been suggested by Roberts et al12 that there cells analysed showed one primary constriction at the level may be a tendency for the centromeres of acrocentrics to of chromosome 18 centromere. The derivative chromo- become inactivated, making those dicentrics most likely to some hybridised with alpha satellite sequences of chromo- be stable and therefore visible in patients. somes 13/21 and 18. In the majority of cytogenetically recognisable heterodi- The four cases reported here bring to 26 the total centric autosomes, only one primary constriction is seen number of non-Robertsonian heterodicentric autosomes (14/18) in all cells of a given subject. The primary reported since the seventies (table 1). Many of these are constriction corresponds to the activity of the centro- associated with a deletion of the chromosomes involved mere.20 Even if this is a crude estimate, this suggests that (13/26) and, consequently, with phenotypic abnormality. only one centromere is active. Immunostaining against This significant number of unbalanced cases could be the active centromere kinetochore specific protein (CENPs) is result of a bias of ascertainment. In those cases parental more reliable in assessing centromeric activity. Unfortu- karyotypes were normal, as expected. Phenotypically nately, it was not possible to perform these studies in our normal people have also been observed to carry a balanced cases. Earnshaw et al21 have shown the presence of only one heterodicentric autosome (12 cases). However, among immunostaining signal against CENP-C on a dicentric those, infertility was present in three cases21419 caused chromosome consistent with the presence of one active either by gonadal dysgenesis or severe oligospermia associ- centromere. The mechanism by which centromere inacti- ated with abnormal sperm morphology. One other case was vation occurs is still unknown. The primary constriction is ascertained through recurrent spontaneous abortions. found at the site of the non-acrocentric centromere in most Four cases were found in skin cultures established from instances (12/14). Only two cases showed one primary phenotypically normal induced abortuses.15 Including two constriction at the site of the acrocentric centromere.818

cases presented here, only four cases of non-Robertsonian Among non-Robertsonian heterodicentric autosomes, http://jmg.bmj.com/ heterodicentric autosomes were transmitted over two or there were four cases of mosaicism for centromeric more generations. Their heritability indicates their unique activity.124 Intercentromeric distance seems to be one stability. In these cases there is a possibility of transmitting important factor in determining if a dicentric will be func- unbalanced gametes by non-disjunction as seen in case 2. tionally dicentric or monocentric.22 To be stable and lead to In the vast majority of cases (22/26), the short arm of an a viable embryo, dicentrics with distanced centromeres acrocentric chromosome is involved in the translocation. A have to inactivate one centromere very soon after Table 1 Summary of cases reported to carry non-homologous non-Robertsonian heterodicentric autosomes on September 26, 2021 by guest. Protected copyright.

Reference Dicentrics Acrocentric Primary constriction Phenotype Inheritance

10 (5;13)(p12;p12) + Chrom 5 &13 (34%), chrom 5 Abnormal De novo (54%), chrom 13 (10%) 19 (12;14)(p13;p13) + Chrom 12 Primary amenorrhoea De novo 13 (19;20)(p?;p?) − NA Abnormal NA 9 (7;15)(p21;p11) + Chrom 7 Abnormal De novo 16 (13;18)(p1;p11) + NA NA De novo 12 (8;22)(p23;p13) + Chrom 8 Abnormal De novo 14 (15;18)(p12;p11) + NA Oligospermia De novo 3 (13;18)(p12;p11.2) + Chrom 18 Normal Familial 3 (9;22)(p22;p11) + Chrom 9 Abnormal NA 1 (5;15)(p31;p11) + Chrom5&15(85%),chrom 15 Abnormal De novo (8%), chrom 5 (7%) 8 (14;18)(p1?;q22) + Chrom 14 Abnormal De novo 15 (2;22)(p25;p12) + NA Normal NA (1;19)(p36;q13) − NA Normal NA (12;17)(p13;q23) − NA Normal NA (13;18)(q36;q23) + NA Normal NA 6 (6;19)(pter;qter) − Chrom 19 Abnormal De novo 4 (9;13)(p22;p13) + Chrom9&13 Abnormal De novo 7 (13;18)(p13;p11.32) + Chrom 8 Normal Familial 17 (13;18)(p11;p11)+r13 + NA Abnormal NA 2 (13;20)(p12;q13) + Chrom 20 80% Primary amenorrhoea De novo 11 (15;20)(ter;ter) + Chrom 20 in lymphocytes Abnormal De novo 18 (4;21)(p16;q22) + Chrom 21 Normal (miscarriage) De novo Case 1 (16;22)(q24;p11.2) + Chrom 6 Normal Familial Case 2 (14;18)(p11.2;p11.3) + Chrom 18 Normal Familial Case 3 (13;18)(p12;p11.2) + Chrom18 Abnormal De novo Case 4 (13;18)(p11.2;p11.2) + Chrom 18 Abnormal De novo

NA: not available.

www.jmedgenet.com Letters 79 formation. However, when the centromeres are close to 2 Vianna-Morgante AM, Rosenberg C. Deletion of the centromere as a mechanism for achieving stability of a dicentric chromosome. Cytogenet Cell J Med Genet: first published as 10.1136/jmg.38.1.43 on 1 January 2001. Downloaded from each other, the inactivation process can occur later, leading Genet 1986;42:119-22. to mosaicism for centromere inactivation. Vig and 3 Daniel A. Single Cd band in dicentric translocations with one suppressed Zinkowski23 observed centromere separation in dicentric centromere. Hum Genet 1979;48:85-92. 4 Daniel A, Ekblom L, Phillips S, FitzGerald JM, Opitz JM. NOR activity and chromosomes at the metaphase-anaphase point. In pro- centromere suppression related in a de novo fusion tdic(9;13)(p22;p13) metaphase, most dicentrics showed two primary constric- chromosome in a child with del(9p) syndrome. Am J Med Genet 1985;22: tions. However, 18% already showed premature centro- 577-84. 5 Daniel A, Perel ID, Clarke AJ, Saville T. Familial dicentric translocation mere separation of one centromere, suggesting the activity t(13;18)(p13;p11.2) ascertained by recurrent miscarriages. J Med Genet of only one centromere. There was consistency from cell to 1979;16:73-5. 6 Drets ME, Therman E. Human telomeric 6;19 translocation chromosome cell with respect to which centromere separated early. By with a tendency to break at the fusion point. Chromosoma 1983;88:139-44. metaphase, 95% of the dicentrics showed premature sepa- 7 Howard PJ, Berry AC. Familial transmission of a non-Robertsonian translo- ration of one centromere. cation dicentric. Clin Genet 1986;29:246-50. 8 Lambert JC, Ferrari M, Bergondi C, Galliana A, Ayraud N. 18q- syndrome After the acrocentrics, chromosome 18 is most fre- resulting from a tdic(14p;18q). Hum Genet 1979;48:61-6. quently involved in non-Roberstonian heterodicentrics (10 9 Nakagome Y, Teramura F, Kataoka K, Hosono F. Mental retardation, mal- cases). The high frequency of involvement of this chromo- formation syndrome and partial 7p monosomy (45,XX,tdic(7;15)(p21; p11)). Clin Genet 1976;9:621-4. some may reside in the fact that both 18p− and 18q− are 10 Niebuhr E. A 45,XX,5-,13-, dic+ karyotype in a case of cri-du-chat viable syndromes. Other chromosomes are involved more syndrome. Cytogenetics 1972;11:165-77. 11 Rivera H, ZuVardi O, Maraschio P, Caiulo A, Anichini C, Scarinci R, or less randomly (table 1). Vivarelli R. Alternate centromere inactivation in a pseudodicentric In conclusion, our cases indicate further the predomi- (15;20)(pter;pter) associated with a progressive neurological disorder. J nance of acrocentric chromosomes in stable dicentric Med Genet 1989;26:626-30. 12 Roberts SH, Howell RT, Laurence KM, Heathcote ME. Stable dicentric autosomes. Most of them will reach stability by inactivating autosome, tdic(8;22)(p23;p13), in a mentally retarded girl. J Med Genet one centromere and will be functionally monocentric. If an 1977;14:66-8. acrocentric is involved, its centromere is most often the 13 Sekhon GS, Hillman LS, Kaufman RL. Identiﬁcation of a 19/20 translocation by G-, Q- and C-banding. Birth Defects 1975;11:237-40. inactivated one. 14 Singh-Kahlon DP, Serra A, Bova R. A complex mosaic with D/E translocation tdic(15;18)(p12;p11) in an oligospermic male with apparently total infertility. Clin Genet 1977;11:342-8. V The excellent technical assistance of the sta of the Cytogenetics Laboratory of 15 Sit KH, Wong HB. Translocation dicentric chromosomes in prostaglandin the Montreal Children’s Hospital is gratefully acknowledged. This work has been partially funded by a grant from the Fonds de la Recherche en Santé du E2 induced abortuses and possible aneusomy through asynchronous Québec. centromeric divisions. Cytogenet Cell Genet 1981;29:60-4. 16 Skovby F, Niebuhr E. Centromere inactivation in dicentric human chromosomes. EMMANUELLE LEMYRE*† Amsterdam: Excerpta Medica International Congress Series, 1976:153-4. VAZKEN M DER KALOUSTIAN†‡ 17 Uehara M, Kida M. A complex mosaic with tdic(13;18)(p11;p11),+13p- ALESSANDRA M V DUNCAN*† ,+18p-,r(13) etc in a male infant. I. Centromere inactivation and dissocia- *Division of Cytogenetics, Department of Pathology, Montreal Children’s tion of dicentric chromosome. Jpn J Hum Genet 1986;31:27-35. Hospital and McGill University, Montreal, Canada 18 Wang F, Li Y. A new stable human dicentric chromosome, tdic(4;21)(p16; †Department of Human Genetics, McGill University, Montreal, Quebec, q22), in a woman with ﬁrst trimester abortion. J Med Genet 1993;30:696. Canada. 19 Warburton D, Henderson AS, Shapiro LR, Hsu LYF. A stable human dicentric chromosome, tdic(12;14)(p13;p13), including an intercalary sat- ‡F Clarke Fraser Clinical Genetics Unit, Department of Pediatrics, ellite region between centromeres. Am J Hum Genet 1973;25:439-45. Montreal Children’s Hospital and McGill University, Montreal, Canada 20 Therman E, Susman M. Human chromosomes. Structure, behavior and eVects. New York: Springer-Verlag, 1993:100. Correspondence to: Dr Duncan, Cytogenetics Laboratory, Montreal Children’s 21 Earnshaw WC, Ratrie H III, Stetten G. Visualization of centromere proteins Hospital, 2300 Tupper Street, Montreal, Quebec, Canada H3H 1P3, CENP-B and CENP-C on a stable dicentric chromosome in cytological [email protected] spreads. Chromosoma 1989;98:1-12. http://jmg.bmj.com/ 22 Sullivan BA, Willard HF. Stable dicentric X chromosomes with two 1 Dewald GW, Boros SJ, Conroy MM, Dahl RJ, Spurbeck JL, Vitek HA. A functional centromeres. Nat Genet 1998;20:227-8. tdic(5;15)(p31;p11) chromosome showing variation for constriction in the 23 Vig BK, Zinkowski RP. Sequences of centromere separation: a mechanism centromeric regions in a patient with cri du chat syndrome. Cytogenet Cell for orderly separation of dicentrics. Cancer Genet Cytogenet 1986;22:347- Genet 1979;24:15-26. 59. on September 26, 2021 by guest. Protected copyright.

Correction

In the February 2000 issue of the journal, on page 88, in the paper “Haim-Munk syndrome and Papillon-Lefèvre syndrome are allelic mutations in cathepsin C”, we regret that Dr Zlotogorski’s name was misspelt.