Velo-cardio-facial syndrome associated with chromosome 22 deletions encompassing the DiGeorge locus

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REFERENCES 1. Fernando

R.

Preventable

acute

organophosphate

poisoning

deaths.Ceylon Med J 1989; 34: 139-42. Senanayake N, Karalliedde L. Pattern of acute poisoning in a

N N Y Y H H H H H

medical unit in central Sri Lanka. Forensic Sci Int 1988; 36: 101-04. 3. Senanayake N, Karalliedde L. Neurotoxic effects of organophosphorus insecticides: an intermediate syndrome. N Engl J Med 1987; 316: 761-63. 4. Milby TH. Prevention and management of organophosphate poisoning. JAMA 1971; 216: 2131-33. 5. Namba T, Nolte CT, Jackrel J, Grob D. Poisoning due to 2.

546

543

2

organophosphate insecticides: acute and chronic manifestations. Am J Med 1971; 50: 475-92. 6. Hayes WJ. Toxicology of pesticides. Baltimore: Williams & Wilkins, 1975: 410-517. 7. Lotti M, Becker CE. Treatment of acute OP poisoning: evidence of a direct effect on central nervous system by PAM-2. J Toxicol Clin Toxicol 1982; 19: 121-27. 8. Durham WF, Hayes WJ. Organic phosphorus poisoning and its therapy. Arch Environ Health 1962; 5: 21-53. 9. Kusic R, Jovanovic D, Randjelovic S, et al. HI-6 in man: efficacy of the oxime in poisoning by organophosphorus insecticides. Hum Exp Toxicol 1991; 10: 113-18. 10. Sanderson DM. Treatment of poisoning by anticholinesterase insecticides in the rat. J Pharm Pharmacol 1961; 13: 435-39. ADDRESSES: Department of Medicine, Faculty of Medicine, University of Peradeniya, Kandy, Sri Lanka (H. J. de Silva, DPhil, R. Wijewickrema, MB, Prof N. Senanayake, FRCP). Correspondence to Dr H. J. de Silva.

Velo-cardio-facial syndrome associated with chromosome 22 deletions encompassing the DiGeorge locus

Fig 1-Detection with probe 22.71 of 22q11 deletion in VCF patients. Lanes NY4and NY5= patients; lanes H2- H6 =controls. Control probe signal strength for NY4 is greater than control signal in H3 but it can be seen that 22.71 signal is reduced in NY4 compared with this control (both XV2c bands are used as controls). Similarly, NY5 control probe signal is stronger than that in H2, but 22.71 signal is clearly less in NY5 than H2 Densitometry was used to calculate a mean signal strength ratio (patient: control values) of0.46 for NY4 and 0 41 for NY5 (2271 signals repeated at different exposure to show signal in NY5.)

the

large clinical overlap between DiGeorge syndrome and velo-cardio-facial syndrome suggests an aetiological connection. DiGeorge syndrome is The

associated with microdeletions of chromosome 22q1 1 and is therefore likely to be caused by reduced dosage of genes within this region. We present preliminary data that velocardiofacial syndrome patients have similar chromosome deletions, a finding consistent with the hypothesis that these disorders represent part of a spectrum of abnormalities seen with monosomy for 22q11.

hypothesis that the clinical spectrum of this syndrome

encompasses DGS and has a related causation. DNA probes which flank the DGS locus have been isolated. HP500 is a subclone from the Lawrence Livermore library LL22NS01; 22.71 is a clone from a microdissection and microcloning of 22q 11 ;3,5,7 sc4.1 is a cosmid clone containing the HP500 sequence. Together, these markers detect hemizygosity in 95% of DGS patients.3-5 Five patients with VCF were ascertained with typical features of VCF, but none had evidence of thymic or parathyroid involvement. One patient NW35 had a normal high-resolution (at least 850 bands) karyotype; no high-resolution karyotypes were available on the other patients. Genomic DNA from the subjects and a series of controls was digested with PMJI and Southern blotted. In each experiment the same membrane was hybridised with a control probe from chromosome 7 (XV2c) and the test probe. Several control lanes with different quantities of DNA were run to ensure that an appropriate control was available for each test lane comparison; multiple exposures were used to ensure that comparisons are done within the linear range of the film. Experiments were done in triplicate. Membranes were either simultaneously hybridised with test probe and control probe, or

(when more than one test probe was to be examined) hybridised sequentially without ’stripping’ the membrane between probings.

syndrome (VCF; McKusick index 19243)1 multiple anomaly disorder commonly with cleft palate, heart defects, cognitive presenting and a characteristic facial appearance; over thirty disorder, clinical features of the syndrome have been reported.2 The spectrum of abnormality invites comparison with DiGeorge syndrome (DGS; McKusick index 18840)1 in which the classic presentation includes cardiac outflow tract defect, hypoparathyroidism, and cell-mediated immune deficiency. Velo-cardio-facial is

Most

cases

a

of DGS

are

associated with monosomy for

a

region of chromosome 22q11, and DNA markers within the deleted region provide the most sensitive means of detecting this hemizygosity.3-5 Two patients examined in our studies of DGS have been retrospectively diagnosed as VCF by others.6 We therefore examined patients with VCF to test

An example of one quantitative hydridisation experiment is shown in fig 1. One patient (NW35) was examined by fluoresence in situ hybridisation (FISH).8 Fig 2 shows a representative result demonstrating that sc4.1 is hemizygous in this patient; HP500 also detected a deletion in this patient using quantitative Southern analysis. All five patients were deleted for at least one of the DNA probes. HP500 detected hemizygosity in patients NW35, NY11 and NY4, but was dizygous in patients NY2 and NY5. 22.71 detected hemizygosity in patients NY2, NY4 and NY5, but dizygous in NY1(not tested for patient NW35). This suggests that the VCF locus is flanked by HP500 and 22.71 and therefore maps to the same region of 22q 11 as the DGS locus.3,4 .

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Carey AH, Roach S, Williamson R, et al. Localisation of 27 DNA markers to the region of human chromosome 22q11-pter deleted in patients with the DiGeorge syndrome and duplicated in the der22 syndrome. Genomics 1990; 7: 299-306. 8. Lichter P, Tang C-J, Call K, et al. High resolution mapping of human chromosome 11 by in situ hybridisation with cosmid clones. Science

7.

1990; 64-68.

Sharkey AM, McLaren L, Carroll M, et al. Isolation of anonymous DNA markers for human chromosome 22q1 1 from a flow sorted library, and mapping using hybrids from patients with DiGeorge syndrome. Hum Genet (in press). 10. Wilson DI, Cross IE, Goodship JA, et al. DiGeorge sequence, isolated aortic coarctation and isolated ventricular septal defect in three sibs with a 22q11 deletion of maternal origin. Br Heart J 1991; 66: 308-12. 9.

Fig 2-FISH studies: patient NW35.

cosmid sc4.1

Bars indicate chromosomes 22, signal both chromatids of single chromosome.

detecting deletion in (yellow spot) detected

on

One patient studied previously was retrospectively diagnosed as VCF (cell line GM07939, available from NIGMS)and a somatic cell hybrid retaining the deleted chromosome 22 has been established from the patients lymphoblasts.9 Probe HP500 is deleted from this hybrid (not shown) and high resolution cytogenetics reveals an interstitial deletion (NIGMS catalogue) which corroborates the conclusion that VCFS is associated with deletions of 22qll. We therefore propose that VCF and DGS are aetiologically related, and are two different manifestations of haploinsufficiency for the same gene or group of genes. We have drawn attention to the wide range of phenotypes associated with hemizygosity for 22ql 1, a pattern which can be found within a family as well as between individuals.10 The abnormalities found in patients with monosomy 22Il 1, including VCF, are consistent with a defect within the mesencephalic and cardiac neural crest. The anomalies may occur alone rather than as part of a syndrome and we anticipate that a proportion of patients with isolated features of DGS and VCF have a deletion or gene mutation at 22ql 1. We would welcome referral of patients who have isolated congenital defects which form part of these syndromes so that DNA investigations may be conducted.

Supported by Action Research, British Heart Foundation, Medical Research Council, and Dunhill Medical Research Trust. FISH work was done in the laboratory of Dr Antonio Baldini and with his generous assistance (ICRF Laboratories, London). We also thank the patients and families for their cooperation, and D. Ward’s laboratory for computer software. REFERENCES 1. McKusick VA. Mendelian inheritance in Man. 1990; 9th ed. Johns Hopkins Univ Press, Baltimore: entries 19243 and 18840. 2. Shprintzen RJ, Velo-cardio-facial syndrome. In: Buyse MA, ed. Birth defects encyclopedia. Dover, Mass: Center for Birth Defects Information Services, 1990: 1744-45. 3. Carey AH. Molecular genetic analysis of the DiGeorge sequence. PhD thesis, University London, UK, 1991. 4. Scambler PJ, Carey AH, Wyse RK, Roach S, Dumanski JP, Nordenskjold M, Williamson R. Microdeletions within 22q11 associated with sporadic and familial DiGeorge syndrome. Genomics 1991; 10: 201-06. 5. Carey AH, Claussen U, Ludecke H-J, et al. Interstitial deletions in DiGeorge syndrome detected with microclones from 22q11. Mammal Genome (in press). 6. Stevens CA, Carey JC, Shigeoka AO. DiGeorge anomaly and velocardiofacial syndrome. Pediatrics 1990; 85: 526-30

ADDRESSES: Department of Biochemistry and Molecular Genetics, St Mary’s Hospital Medical School, London W21PG, UK (P. J. Scambler, MD, D. Kelly, BSc, E. Lindsay, PhD, Prof R. Williamson, PhD); Center for Craniofacial Disorders, Montefiore Medical Centre, Bronx, NY, USA (R Goldberg, MS, R. Shprintzen, MD); and Division of Human Genetics and Cytogenetics, University of Newcastle upon Tyne, Newcastle-upon-Tyne, UK (D. I. Wilson, MRCP, J. A. Goodship, MD, I. E. Cross, BSc, Prof J. Burn, FRCP). Correspondence to Dr P J. Scambler.

Viral-associated

haemophagocytosis with parvovirus-B19-related pancytopenia Viral-associated haemophagocyte syndrome in response to infection with human parvovirus B19 was seen in 2 patients with hereditary spherocytosis.

Depressed reticulocyte response during acute parvovirus infection is a known cause of hypoproliferative crises in patients with reduced erythrocyte lifespan; the observation of parvovirusassociated haemophagocytosis could account for the pancytopenia that may accompany human parvovirus B19 infection. Infection with human parvovirus B 19 can cause a hypoproliferative crisis in patients with sickle-cell disease,! and can also cause similar crises in other conditions with reduced red-blood-cell lifespan.2 Haemopoietic cell infection with human parvovirus is specific and confined to the erythroid series,3 with failure of the reticulocyte response during acute viraemia.4 Infection of myeloid and megakaryocyte elements has not been seen, but most parvovirus-infected patients show transient falls in neutrophil and platelet counts;4°5 spontaneous recovery of neutrophils may be delayed for 14 days,4 which might further increase the risk of infection in sickle-cell disease. Virus-associated haemophagocytic syndrome, a nonmalignant histiocytosis characterised by pancytopenia, marrow hypoplasia, disseminated intravascular coagulation, and lymphadenopathy,6,7 has been seen in association with human parvovirus B 19 infection in a child with no previous haematological disorder.8 We report 2 patients with occult hereditary spherocytosis in whom viral-associated haemophagocytic syndrome was associated with human parvovirus B19 infection. A 19-year-old man, previously well, presented with a 4-day history of malaise, anorexia, and night sweats. On examination he had cervical and axillary lymphadenopathy and splenomegaly (8 cm