American Journal of Medical Genetics 443288-292 (1993)

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Molecular and Cytogenetic Characterization of 9p - Abnormalities Ahmad S. Teebi, Lisa Gibson, James McGrath, M.S. Meyn, W. Roy Breg, and Teresa L. Yang-Feng Department of Genetics, Yale University School of Medicine, New Haven, Connecticut

We report on 2 girls with terminal deletion of the short arm of chromosome 9 with concur- rent duplication unrecognizable by routine chromosome studies. The phenotype of the patients was not specifically suggestive of the 9p- syndrome in the absence of trigono- cephaly and long philtrum as cardinal mani- festations. In addition to psychomotor retar- dation, their manifestations were mild and include upward slant of palpebral fissures and dolichomesophalangy which are charac- teristic of del(9p). Chromosome abnormalities were de novo in both cases.

The two rearranged chromosomes 9 exhibit similar G-banding patterns and suggested the possible duplication of distal 7p. Fluores- cence in situ hybridization (FISH) with a chro- mosome-7 specific library probe indeed iden- tified that one derivative chromosome 9 was the result of a translocation between chromo- somes 7 and 9 [der(9)t(7;9)(~15.3;~24] but failed to detect a signal on the other derivative 9. In the second case, the concurrent abnor- mality was an inverted duplication of proxi- mal 9p and deletion of distal 9p [inv dup(9)(p13+p22::p22-,qter)l confirmed by FISH using a chromosome 9 specific library probe. FISH clearly identified the origin of these 2 abnormal chromosomes 9 and pro- vided crucial information for clinical evalua- tion. We emphasize the importance of utilizing updated cytogenetic and molecular tech- niques in the precise delineation of subtle or complex abnormalities where there are no useful phenotypic clues. o 1993 Wiley-Liss, Inc.

KEY WORDS: deletion, duplication, chromo- somes 9p and 7p, high-resolu- tion cytogenetics, FISH

~ Received for publication August 7, 1992; revision received November 25, 1992.

Address reprint requests to Teresa L. Yang-Feng, Ph.D., Cyto- genetic Laboratory, Yale University School of Medicine, P.O. Box 3333, 333 Cedar Street, New Haven, CT 06510.

0 1993 Wiley-Liss, Inc.

INTRODUCTION Routine cytogenetic techniques sometimes fail to pre-

cisely characterize subtle chromosome rearrangements. The introduction of molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) may permit a more precise characterization of such rear- rangements. Here we report on our application of high resolution G-banding and FISH analysis in 2 patients with de novo chromosome abnormalities involving a partial deletion of 9p and a concurrent duplication. The initial GTG banding had failed to demonstrate these abnormalities.

CLINICAL REPORTS Patient 1, K.H., was born at term by repeat C-section

to a 28-year-old mother. The pregnancy was unremark- able except for mild third trimester vaginal spotting. At birth she weighed 2,900 g and underwent phototherapy for 5 days for neonatal hyperbilirubinemia. Since early life she was noted to be mildly hypotonic and at age 5 months she developed a seizure disorder. She was subse- quently found to have intermittent strabismus, signifi- cant psychornotor developmental delay, and a partial hearing loss. An initial G-banded chromosomal analysis showed 46,XX, - 9, + der 9 t(?;9)(?;p22). The parents' chromosomes were apparently normal. At age 26 months when evaluated by us she was able to say a few single words and to walk without assistance. Her weight was 11 kg (15th centile), length was 85 cm (25th centile), and head circumference ( O F 0 was 48 cm (25th centile). Craniofacial manifestations (Fig. 1) included dolicho- cephaly with long narrow face, bilateral epicanthic folds, and telecanthus. There was an alternating eso- tropia and clear corneae. The nasal bridge was de- pressed and the palate was highly arched and narrow. Her ears were of normal size with a prominent antihelix and small ear canals. Her internipple distance was 12.5 cm (90th centile) and chest circumference was 46.5 (25th centile). There was bilateral fifth finger clinodactyly. Her palm length was 5.2 cm (3rd centile) and middle finger length was 5 cm (75th centile). Apart from mild hypotonia, neurological status was normal. Examina- tion of other organs showed no significant abnormality.

Patient 2, K.M., was born at 36 weeks gestation by normal vaginal delivery to a 31-year-old mother. During pregnancy, intrauterine growth retardation was noted

9p - Abnormalities 289

Fig. 1. Patient 1, K.H. Fig. 2. Patient 2, K.M.

ultrasonographically. A chromosome analysis of am- niocytes was reportedly normal. The birth weight was 1,900 g. She was evaluated for developmental delay at age 9% months. At this age she was noted to be small with weight, length, and OFC below the 3rd centile. She was able to hold objects, roll over, transfer things from hand to hand, verbalize dada and mama, but could not sit or stand. Her craniofacial manifestations (Fig. 2) included narrow forehead with metopic ridging, a small anterior fontanelle, small nose, midface hypoplasia, mild upward slant of palpebral fissures, mild epicanthic folds, thin and long eyebrows with mild synophrys, thin lips, and a short neck. There was a relative excess of subcutaneous fat in the face and trunk. The hands were small with relatively long tapering fingers and mild fifth finger clinodactyly. Neuromuscular examination showed hypotonia with slightly reduced deep tendon reflexes. Examination of other organs documented no significant abnormalities.

Initial G-banded chromosomal analysis showed 46, XX, - 9 + der 9 t(?7:9)(p21;p22). The parents’ chromo- somes were normal. Follow-up evaluation at age 20 months showed no significant change in craniofacial manifestations. Her weight, height, and OFC remained below the 3rd centile for age. The OFC and length were at the 50th centile for a 7-month-old girl while the weight was at the 50th centile for a 10-month-old girl. There was a small umbilical hernia. At this age she was able to crawl, stand, cruise, eat crackers by herself, but could not walk by herself, and could not speak more than a few single words.

CYTOGENETICS AND MOLECULAR CYTOGENETICS

Analysis of Giemsa-banded chromosomes from per- ipheral blood showed a derivative chromosome 9 in each

patient (Fig. 3). The aberrant chromosomal segments could not be identified with certainty using standard cytogenetic methods. Despite different breakpoints, they appeared similar in these 2 patients, and consistent with the banding pattern of the distal portion of 7p.

Epstein-Barr virus-transformed lymphoblastoid cell lines were established and FISH analysis was performed as described previously [Lichter et al., 19901. Chromo- some-specific DNA libraries (painting probes) (provided by Joe Gray at University of California, San Francisco) were used to determine the chromosomal origin of the abnormal segments. Chromosome specific painting and centromere probes were labeled with biotin-1 1-dUTP by nick translation and hybridized to chromosome prepara- tions at concentrations of 50 and 5 nglkl, respectively. Hybridization sites were detected by fluorescein isothio- cyanate-conjugated avidin (FITC) and chromosomes were counterstained with propidium iodide (PI). Chro- mosomes were visualized and photographed with a pho- tomicroscope equipped with FITC and PI epifluores- cence optics and Kodak TMax 400 film.

Chromosomes of patient 1, K.H., showed a signal a t the end of one chromosome 9 (identified by an alphoid DNA centromere probe, provided by Antonio Baldini) when hybridized to the chromosome 7-specific painting probe (Fig. 4). This finding confirms the result sus- pected from high resolution G-band analysis, 46,XX, - 9, + der(9)t(7;9)(~15.3;~24).

Chromosomes of patient 2, K.M., did not show hybrid- ization signal on her derivative chromosome 9. Further FISH analysis documented a signal along the entire chromosome 9 when hybridized to the chromosome 9-specific painting probe (Fig. 5). Reevaluating her G-banded chromosomes, it was determined that the de- rivative chromosome is the result of an inverted duplica- tion of the proximal portion of the short arm (9p13-p22) and a deletion of the distal portion (9~22-pter).

290 Teebietal.

p24 * r I R

KH

K M

7 9

p22B-

a p13 8- II

c k! 9

1 der(9)

- 8 a E 8

inv dup(9)

Fig. 3. G-banded chromosomes 7 and 9 from two metaphases of patients K.H. and K.M. Triangles indicate the derivative chromosome 9s. Arrows pointing to the ISCN ideogram of chromosomes 7 and 9 indicate the breakpoints of chromosome rearrangements.

DISCUSSION

Craniofacial manifestations in 9p - syndrome include trigonocephaly and upward slant of palpebral fissures and the clinical diagnosis is often suspected because of their presence [de Grouchy and Turleau, 19841. Huret et al. [19881 reviewed 39 patients with 9p - as a sole anom- aly and 41 patients with 9p - anomalies associated with another unbalanced chromosome segment. The authors found that individual cases with only 9p- exhibited trigonocephaly, an upward slant of the palpebral fis-

sures (more pronounced than in cases of trisomy 211, a long philtrum, a short nose, a short, broad and webbed neck, dolichomesophalangy, and abnormal der- matoglyphics with extra whorls on the fingers. Among the cases with an associated unbalanced segment the defect in most of them was the result of malsegregation of a parental balanced translocation. In these individ- uals, trigonocephaly, a long philtrum, dolichomesopha- langy, and inguinal as well as umbilical hernias were often encountered.

In our patients, the clinical manifestations (Table I)

Fig. 4. Metaphase spread of patient 1, K.H., after FISH with the chromosome 7 painting probe and centromere probe homologous to both chromosomes 4 and 9. Arrow indicates the hybridization signal at the end of one chromosome 9.

9p - Abnormalities 291

TABLE I. Manifestations of 9p - in W o Patients

Manifestations Patient 1 Patient 2

Fig. 5. Metaphase spread of patient 2, K.M., after FISH with the chromosome 9 painting probe. Arrows indicate the normal and abnor- mal chromosomes 9.

were not specifically diagnostic of 9p - anomalies since both cases lack 2 of the cardinal manifestations, namely the trigonocephaly and a long philtrum. However, the presence of upslanting palpebral fissures and dolicho- mesophalangy in both individuals is in agreement with the diagnosis of 9p- syndrome.

In patient 1 there was duplication of 7p in addition to 9p - . The presence of dolichocephaly and a high-arched palate in this patient may be the result of 7p duplication as these traits are among the main phenotypic mani- festations of dup(7p) syndrome along with psychomotor retardation [Milunsky et al., 19891. Such manifesta- tions were extrapolated from reviewing 20 possible pa- tients with different breakpoints. Other manifestations of 7p duplication include microbrachycephaly, large fon- tanelles and wide sagittal or metopic sutures, large, apparently low-set, angulated ears, micrognathia and high arched palate, joint dislocations, either contrac- tures or hyperextensibility of joints, and cardiac septa1 defects. Specifically in 4 patients with similar duplica- tion of 7p21-pter but with different concurrent chro- mosomal abnormalities [Larson et al., 1977; Rockman- Greenberg et al., 1982; Gabarron et al., 19881 the phe- notype showed only a little similarity to our patient in particular of some individual nonspecific traits such as high arched palate and epicanthic folds in 3 patients, hypotonia in 2 patients, and telecanthus in only one patient.

In patient 2, who had an inverted duplication of 9p, there was growth retardation of prenatal onset and psy- chomotor retardation. These manifestations are present in the dup(9p) syndrome. This cytogenetic abnormality has been reported in at least 100 patients and the spec- trum of physical and developmental manifestations is remarkably consistent despite the varying sizes of the

Trigonocephaly

Brachycephaly Upward slant of

Hypertelorism Epicanthic folds Very arched

eyebrows Flattened nasal

bridge Short nosel

anteverted nares High arched palate Long philtrum Apparently low set

Short, broad neck Neck webbing/low

posterior hair line Abnormal

dermatoglyphics Long fingers and

toes Widely spaced

nipples Umbilicalhnguinal

hernia Hypotonia Developmental

delay/MR Karyotype

palpebral fissures

ears

- (dolicocephaly)

- +

- (Telecanthus) + -

+ -

(small ear canal) -

+

t

+

+ +

46.XX. - 9. + der(9)

- (metopic ridge)

+ (mild) -

- + (mild) +

( + synophrys) -

t

+ -

+

+ + +

46.XX.inv duD

duplicated segments [Nichols, 19901. In most cases, the trisomic segment comprises the entire short arm of chro- mosome 9 (9pter+9p12). However, proximal duplica- tions similar to the one observed in patient 2 have been frequently described with a clinical presentation which is similar but less severe [F'ryns et al., 1979; Cuoco et al., 1982; Montegi et al., 19851. In our patient the craniofa- cia1 changes were mild and the diagnosis of 9p duplica- tioddeletion was not made on clinical information.

In both of the present patients described in this report a chromosome anomaly was suspected, but the precise characterization was not possible until after the com- bined utilization of high-resolution banding and FISH. Both cases were de novo and therefore no information could be obtained from studies of the parents. We would like to emphasize that the combined application of re- cent molecular and cytogenetic techniques may permit the characterization of subtle unbalanced and balanced rearrangements in persons with unrecognized multiple congenital anomalies/developmental delay.

ACKNOWLEDGMENTS We thank Jackie Benoit for typing the manuscript.

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