American Journal of Medical Genet ics 4R114-117 (1993)

VACTERL With Hydrocephalus: Spontaneous Chromosome Breakage and Rearrangement in a Family Showing Apparent Sex-Linked Recessive Inheritance

Hungshu Wang, Alasdair G.W. Hunter, Brian Clifford, Margaret McLaughlin, and Diane Thompson Division of Genetics (H.W., A.G.W.H., B.C., M.M., D.T.), Children’s Hospital of Eastern Ontario, and Department of Pediatrics (H. W., A.G. W.H.), University of Ottawa, Ottawa, Canada -

The rate of spontaneous and mitomycin C in- duced chromosome breakage and sister chro- matid exchange (SCE) was studied in three related cases diagnosed with VACTERL-H syndrome. There have been recent reports of sporadic patients with VACTERL-H in whom high rates of chromosome breakage were ob- served. This has led to the suggestion that some of these patients may represent the se- vere expression of Fanconi anemia. The pat- tern of inheritance in our family is highly sug- gestive of X-linked recessive inheritance supporting the hypothesis that VACTERL-H is, at least in some cases, a syndrome and not an association. o 19% Wiley-Liss, Inc.

KEY WORDS: VACTERL-H, hydrocephalus, X-linked inheritance, chromo- some breakage

INTRODUCTION The VACTERL association is a well-established id-

iopathic condition in which central nervous system ab- normalities are relatively uncommon [Aleksic et al., 19841. However, a number of cases with hydrocephalus (VACTERL-H) have been reported and a significant pro- portion appear to be clinically at the severellethal end of the spectrum and to show familial occurrence [Briard et al., 1984; Sujansky and Leonard, 19831. However, Iafolla et al. [1991] suggested that such cases may not all be lethal or have an extremely poor prognosis. In a 1989 review, Evans et al. noted a possible excess of affected male sets of sibs. They added an affected brother (Fig. 1, 111-5) to the family originally reported by Hunter and MacMurray [19861 and suggested causal heterogeneity

Received for publication October 15, 1992; revision received March 15, 1993.

Address reprint requests to Dr. Hungshu Wang, Division of Genetics, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, K1H 8L1.

0 1993 Wiley-Liss, Inc.

of VACTERL-H with some cases showing X-linked re- cessive inheritance. Additional X-linked cases were re- ported by Neri et al. [19931. It seems likely that the three brothers reported as “VACTERL plus hydrocephalus’’ by Kunze et al. [19921 had a different syndrome in that they lacked hydrocephalus and had other complex cere- bral anomalies.

Recently, Sommer et al. [19891 and Porteous et al. “19921 have reported sporadic cases with findings com- patible with a diagnosis of VACTERL-H, but who had cytogenetic findings suggestive of severe Fanconi ane- mia. Toriello et al. [19911 reported a patient with similar cytogenetic findings and malformations compatible with VACTERL. That patient had a prenatal ultrasound diagnosis of porencephalic cyst but this was not further defined by any postnatal study. These reports have led to the conviction that some cases of VACTERL-H may rep- resent the severe end of the Fanconi anemia spectrum, although hemoglobin F levels have not been reported, and pancytopenia had not yet developed in the one child who had survived to age 2% years [Porteous et al., 19921. Evans and Chodirker [personal communication] have studied a case of VACTERL-H with a negative breakage response to mitomycin C, and they again em- phasize the heterogeneity of the VACTERL association. This latter point is further emphasized by the fact that while in most cases the hydrocephalus is due to aque- ductal stenosis, the patients of Evans and Chodirker and of Neri et al. [1993] had hydrocephalus secondary to Arnold-Chiari malformation.

A maternal aunt (11-7) of the propositus (1114) from the family that we reported previously [Hunter and MacMurray, 1986; Evans et al., 19891 was recently found to be carrying a fetus with ultrasound findings of VACTERL-H. Cells studied from fetal skin obtained at the time of termination of the pregnancy showed a high rate of spontaneous chromosome and chromatid break- age and rearrangement. Review of the chromosome worksheets from the previously reported cases (111-4, 111-5) in the family showed similar rates ofbreakage and rearrangement. Given the X-linked recessive pattern of inheritance in our family, it seems clear that not all cases of VACTERL-H with high levels of chromosome breakage represent severe Fanconi anemia.

VACTERL-H 115

specific chromosome involved more frequently than others. During this period, 1985-1986, the laboratory routine recorded all chromosome breaks on the work- sheet and these were entered into a computer database [Speevak et al., 19891. The average number of chromo- some breaks observed over this period was ~ 0 . 1 per cell.

Analysis of the rate of spontaneous and 0.32 Fg/ml mitomycin C (MMC) induced chromosome breakage and t? sister chromatid exchange (SCE) was carried out on the mothers (11-5,11-7) of the affected children, on a repeat sample of 111-8, and on Cont-1 and 2 (Table 11). The controls were sex and age matched with the patients. A subsequent sorting of our stored cell lines uncovered the existence of amniocytes of 111-5. A repeat experiment of

,

%l'?:"#ib% 4 5

HYDROCEPHALUS + VATER 0 Fig. 1. Pedigree of the family.

CLINICAL REPORTS The pedigree is shown in Figure 1. The histories of 11-8

and 111-4 [Hunter and MacMurray, 19861 and of 111-5 [Evans et al., 19891 were reported previously and are summarized in Table I.

SS (11-7) became pregnant in 1992 and was counselled that she was at 50% risk to be a carrier of this apparently X-linked gene. Ultrasound evaluations carried at a high risk centre, at 11.5 weeks and 15.5 weeks, were reported as normal. However, a t 19 weeks 6 days the male fetus was noted to have hydrocephalus, abnormal forearms with flexed wrists, no stomach bubble, and possibly ab- normal kidneys. An adequate cardiac evaluation was unobtainable. The couple elected termination which precluded complete evaluation at autopsy. However, the fetus was noted to have a 3 vessel cord, absent thumbs, and radii and 2-3 syndactyly of the right hand.

CHROMOSOME STUDIES A skin culture was established from 111-8 after the

pregnancy was terminated. Analysis of 15 cells showed a normal 46,XY karyotype with over 50% of the cells showing breakage (1.10 breaks per cell). The rate of breakage observed among the routine cultures in the laboratory at the time was less than 10% of fibroblast cells. This observation led to a review of the worksheets recorded at the time of the studies on 111-4 and 111-5 carried out between 1985 and 1986. Routine G-banded chromosome analysis on blood lymphocytes (111-4) and amniocytes (111-5) were both reported as normal 46,XY. However, multiple breaks were recorded in 50% of the 24 cells (0.87 breaks per cell) analyzed in 111-4, and 50% of the 20 cells (0.55 breaks per cell) analyzed in 111-5. Simple chromosome rearrangements such as deletions, translocations, and fragments were also observed at lower frequencies. The chromosome breaks and rear- rangements appeared randomly distributed with no

TABLE I. Summarv of C1

chromosome breakage and SCE on this sample and two controls Wont-3 and -4) was carried out. The two tech- nologists who performed the analyses were blinded as to the source of the slides and the results are presented in Table 11. For each treatment in each case, 30 cells were scored for breakage including rearrangements, and SCE.

Case 111-5 and 111-8 again showed approximately 50% of cells with spontaneous breakage (1.15 and 0.92 breaks per cell, respectively). The rate was significantly increased in the MMC treated cells in both samples; 100% of cells showed a high rate of isolated breakage (chromosomes not involved in chromatid exchanges) and multiple chromatid exchanges (Fig. 2). The high rate of MMC-induced SCEs coupled with a normal rate of MMC-induced breakage in the two lymphocyte con- trols (Cont-1 and -3) is evidence of normal DNA repair efficiency. In the fibroblast and amniocyte controls (Cont-2 and -41, although isolated breakage was ob- served in a high proportion of the MMC treated cells, no complex chromatid exchanges were observed. The back- ground rate of breaks is considerably higher in am- niocytes and fibroblasts cultured in our Laboratory [Speevak et al., 19891 than in lymphocyte cultures. This may relate to the increased length of time in culture. In both fibroblast and amniocyte controls the cells were growing exponentially and in different stages of the cell cycle. DNA damage would be observed as breaks and aberrations either in the current cell division, or accu- mulated into the next generation of cells. As a result the rate of breakage in fibroblasts tends to be higher than in lymphocytes, which have a shorter culture time and more synchronous growth. In addition, repeated freez- ing and thawing procedures may have rendered the cells more sensitive to chemical DNA-damaging agents which was reflected in the higher rate of MMC induced isolated breaks. However, the characteristic figures of multiple chromatid exchanges, as seen in the patients,

linical Sirms in Our Cases ~~

Hydro- Micro- Cleft Anal Abs. Thumb Renal Outcome cephalus phthalmia palate TEF atr radii anom anom 111-4 Term, SGA, died f a + + + + + + + 11-8 Died + ? ? ? + + + + 111-5 Aborted + ? ? ? + + + ? 111-8 Aborted + ? ? ? +? + + +? a Aqueductal stenosis.

116 Wang et al.

TABLE 11. Rate of Spontaneous and Mitomycin C Induced Breakage and SCE

Percentage of cells Number of SEC with breakage per cell

cases Specimens SeX Spont MMC Spont MMC 11-5 Lymphocytes F 7 10 11 39

(0.07") (0.33")

(0.05) (0.40)

(0.97) (5.7) [4.3b]

(1.15) (6.4) [3.8]

(0.07) (0.06)

(0.09) (1.31)

(0.04) (0.07)

(0.06) (1.07)

11-7 Lymphocytes F 5 10 12 40

111-5 Amniocytes M 57 100 14 NR'

111-8 Fibroblasts M 45 100 13 NR

Cont-1 Lymphocytes F 7 6 9 30

Cont-2 Fibroblasts M 9 65 9 32

Cont-3 Lymphocytes F 4 7 9 30

Cont-4 Amniocytes M 6 57 10 33

Figure in 0 brackets denotes average number of isolated breaks per cell. Figure in [I brackets is the average number of multiple chromatid exchanges per cell. ' NR, no result.

were never observed. The rate of spontaneous or MMC induced breakage was not different between the mothers and Cont-1 and -3. While not striking, the rate of sponta- neous and MMC induced SCE/cell was higher in the two mothers and in the affected children than in the four controls. The rate of MM-C-induced SCE was unattain- able in 111-5 and 111-8 due to cell damage as evidenced by a suppression of mitosis and a high rate of chromosome breakage and rearrangement.

DISCUSSION The rate of spontaneous chromosome breakage ob-

served in our three affected cases and the mitomycin C-induced increment of breakage and complex rear- rangements observed in cases 111-5 and 111-8 is in keep- ing with the results reported by Sommer et al. [19891, Toriello et al. [19911, and Porteous et al. [19921. These previous reports have all been sporadic cases and have included both male and female patients. As previously mentioned the patient of Toriello et al. [1991] may not be an example of VACTERL-H. As many of the malforma- tions overlap with Fanconi anemia, which is known to be associated with such chromosomal changes, the authors have suggested that their patients may represent severe expression of Fanconi anemia. However, pigmentary ab- normalities which are quite common to Fanconi anemia have yet to be reported in the VACTERL-H patients. In addition, tracheoesophageal fistula, esophageal atresia, imperforate anus, and aqueductal stenosis, which are common to these patients, are not common in Fanconi anemia. Thus far only one of the VACTERL-H patients with known chromosome breakage has survived the neonatal period and at 2 V 2 years was reported to have a normal hemoglobin.

suggestive of X-linked recessive inheritance, thus not

Fig. 2. mica1 metaphase showing a) spontaneous breakslgaps (long arrow) and rearrangement (short arrow). b) MM-C-induced iso- lated breaks (short arrow) and complex chromatid exchanges (long arrows).

The pattern Of inheritance in Our is

VACTERL-H 117

compatible with that of Fanconi anemia. Although the termination of pregnancies by dilatation and extraction in two of our cases precluded a complete assessment of the anomalies present, the clinical findings in our cases seem quite constant and not clearly distinguishable from those of the previous patients with VACTERL-H and abnormal chromosome breakage. Thus, the female patient of Sommer et al. [19891 points to further hetero- geneity. The identification of the Fanconi anemia gene (FAU may allow testing of the hypothesis that some cases of VACTERL-H represent severe Fanconi anemia.

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(1984): Neural defects in Say-Gerald (VATER) syndrome. Child Brain 11:255-260.

Briard ML, Le Merrer M, Plauchu H, Dodinval P, Lambotte C, Moraine C, Serville F (1984): Association VACTERL et hydrockphalie: Une nouvelle entit6 familiale. Ann Genet 27:220-223.

Evans JA, Stranc LC, Kaplan P, Hunter AGW (1989): VACTERL with hydrocephalus: Further delineation of the syndrome(s). Am J Med Genet 34: 177- 182.

Evans JA, Chodirker BN (1993): Letter to the Editor: Absence of excess chromosome breakage in a patient with VACTERL-hydrocephalus. Am J Med Genet 47:112-113.

Hunter AGW, MacMurray B (1987): Malformations of the VATER association plus hydrocephalus in a male infant and his maternal uncle. Roc Greenwood Genet Cntr 6146-147.

Iafolla AK, McConkie-Rase11 A, Chen YT (1991): VATER and hydro- cephalus: Distinct syndrome? Am J Med Genet 38:46-51.

Kunze J, Huber-Schumacher S, Vogel M (1992): VACTERL plus hydro- cephalus: A monogenetic lethal condition. Eur J Pediatr 151:467-468.

Neri G, Genuardi M, Chiurazzi P, Capelli A (1993): X-linked VACTERL with hydrocephalus. 2nd International Workshop on Fetal Genetic Pathology. In Opitz JM (ed): “Blastogenesis: Normal, Abnormal.” New York: Wiley-Liss for the National Birth Defects Foundation- March of Dimes. BDOAS, in press.

Porteous MEM, Bross I, Burn J (1992): VACTERL with hydrocephalus: One end of the Fanconia anemia spectrum of anomalies: Am J Med Genet 43:1032-1034.

Sommer A, Harmel R, Zwick D (1989): Multiple congenital anomalies: Fanconi pancytopenia syndrome? Roc Greenwood Genet Cntr 8

Speevak MD, Hunter AGW, Cox DM, Wang HS (1989): The use of computer database for storage and retrieval of detailed cytogenetic data. Karyogram 15:51-53.

Sujansky E, Leonard B (1983): VACTERL association with hydro- cephalus: A new recessive syndrome? Am J Hum Genet 119A.

Toriello HV, Pearson G, Sommer A (1992): Verification of the existence of a severe form of Fanconi pancytopenia. Proc Greenwood Gen Cntr 11:142.

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