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Keywords:

  • Chromosomal abnormalities;
  • Absences;
  • Epilepsy;
  • Genetics

Abstract

  1. Top of page
  2. Abstract
  3. CASE REPORTS
  4. CYTOGENETIC AND MOLECULAR STUDIES
  5. DISCUSSION
  6. REFERENCES

Summary:  Purpose: Several studies attempted to clarify the genotype–phenotype correlations in patients with inverted duplication of chromosome 15 [inv dup(15)], which is usually characterized by severe mental retardation and epilepsy in individuals with large duplications including the Prader–Willi/Angelman region. We report two patients with inv dup(15) who, in spite of a large duplication, had a mild phenotype including adult-onset epilepsy. This report may help to define the milder spectrum of the syndrome.

Methods: A 25-year-old girl with mild mental retardation had a 6-year history of absence seizures, with occasional head drop. Interictal EEG revealed diffuse spike–wave complexes. Epilepsy was well controlled by a combination of lamotrigine (LTG) and valproate (VPA). The other patient, a 27-year-old man with mild mental retardation, had a 5-year history of rare generalized tonic–clonic seizure during sleep, and frequent episodes of unresponsiveness, which appeared to be atypical absence seizures on video-EEG recordings. A combination of VPA and LTG led to a remarkable improvement, although no complete control.

Results: Molecular analysis revealed a large inv dup15 in both patients.

Conclusions: The discrepancy between the mild phenotype and the severe chromosomal abnormality detected in these two patients further supports the notion that the site of breakpoint might be contributory to the inv dup(15) phenotype. Inv dup(15) should be considered in atypical cases of generalized epilepsy of adult onset without clear-cut etiology.

Inverted duplication of chromosome 15 [inv dup(15)] represents the most common subtype of the heterogeneous group of the extrastructurally abnormal chromosomes syndromes, which alone may account for approximately half of these cases (1). Inv dup(15) chromosomes are variable with respect to the size and genetic composition of the chromosome and in their phenotypic effects (2,3).

Patients with small inv dup(15) may have no abnormal phenotype, whereas patients with large inv dup(15) may have multiple abnormalities. The main clinical manifestations usually occurring with large inv dup(15) are severe mental retardation, dysmorphisms, and behavior disorders, usually associated with epilepsy (4). The severity of clinical manifestations, however, is highly variable, and in the last few years, several studies attempted to clarify the possible correlation of phenotype with genotype (2,3,5,7). Many genetic mechanisms have been hypothesized to explain the clinical variability. Some authors claimed that in addition to the extent of the chromosomal duplication, other factors, such as the dosage of the genes located within the duplication (5) and the maternal origin (8), are contributory to the patient's phenotype. Moreover, it seems that epilepsy mostly depends on the involvement of γ-aminobutyric acid (GABA) receptors (9).

Here we report two patients with a mild epileptic disorder and mild mental retardation, who surprisingly carried a very large inv dup(15) extending to band q14 with tetrasomy of 15q11-14.

CASE REPORTS

  1. Top of page
  2. Abstract
  3. CASE REPORTS
  4. CYTOGENETIC AND MOLECULAR STUDIES
  5. DISCUSSION
  6. REFERENCES

Patient 1

The patient, a 25-year-old right-handed woman, was born normally. Pregnancy was complicated by first-trimester bleeding. Her parents were not consanguineous. There was no family history of neurologic disease or epilepsy. She had normal or slightly delayed developmental milestones. Mild mental retardation became apparent at the school age. At age 19 years, she began to have daily absences. Longer absences were occasionally accompanied by head drops or, rarely, by drop attacks.

General physical examination was unremarkable. Neurologic examination revealed convergent strabismus.

Routine hematologic and biochemical investigations were normal. Brain computed tomography (CT) scan and magnetic resonance imaging were unremarkable.

EEG study

When first seen at our center, at age 23 years, video-EEG recording showed normal background activity, associated with generalized, rhythmic spike–wave complexes at 3.5–4 Hz, lasting 4–6 s, which were not associated with clear-cut clinical manifestations (Fig. 1). There was a good response to a bitherapy with phenytoin (PHT) and valproate (VPA), even if rare absences with head drops persisted. Because of seizure recrudescence, PHT was replaced with lamotrigine (LTG) at the dosage of 300 mg/day, with subsequent almost complete control. Nevertheless, she still continues to have rare episodes with head drop.

image

Figure 1. Patient 1. Note the normal background activity. Generalized, rhythmic spike-and-wave discharges at 3.5–4 Hz, lasting 4–6 s, were not associated with clear-cut clinical manifestations.

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Patient 2

This 27-year-old left-handed man was born after an uneventful pregnancy as the fourth son of healthy parents. There was no family history of neurologic disorders. Developmental milestones were mildly delayed. As a small child, he was described as being less bright than his older brothers. He attended mainstream school with special support and learned to read and count. From age 15 years, he attended a center for unskilled workers. At age 22 years, he had his first generalized tonic–clonic seizure during sleep. A few more similar episodes ensued in the following months. Carbamazepine (CBZ) treatment was started with subsequent permanent remission of convulsive seizures. About 4 months later, he started to have episodes of motionless stare with unresponsiveness lasting 5–8 s and appearing several times per month. Gabapentin (GBP), clobazam (CLB), topiramate (TPM), and VPA, used as add-on bitherapy in combination with CBZ, were ineffective. Episodes of unresponsiveness became progressively more frequent, reaching a frequency of up to two to three per day. Some episodes could be accompanied by drop attacks or by a rightward gyratory component and postictal confusion.

General clinical and neurologic examination was unremarkable. No dysmorphic features were noticed. Cognitive testing at age 20 years by using the Wechsler adult intelligence scale and Raven Progressive Matrices was consistent with the low range of mild mental retardation. Brain CT scan was normal.

EEG study

Interictal EEG showed normal background activity associated with generalized bursts of spike and slow wave (Fig. 2). Two episodes of unresponsiveness were captured during EEG recording and were accompanied by generalized rhythmic slow wave and slow spike-and-wave discharges.

image

Figure 2. Patient 2. Interictal recordings with normal background activity and generalized bursts of spike-and-slow wave discharges.

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The patient was then started on a combination of VPA and LTG with remarkable improvement, although no complete seizure control.

CYTOGENETIC AND MOLECULAR STUDIES

  1. Top of page
  2. Abstract
  3. CASE REPORTS
  4. CYTOGENETIC AND MOLECULAR STUDIES
  5. DISCUSSION
  6. REFERENCES

Peripheral blood cultures were set up according to a standard protocol, and staining was performed by QFQ banding. Metaphase examinations provided information about MC morphology and any presence of mosaicism.

We used chromosome 15–specific biotinylate painting probe (Cambio) and single-locus cosmid probes for the detection of the deletion responsible for Prader–Willi/Angelman Syndrome (Vysys) and YAC probes of chromosome 15. YACs were provided by the YAC screening center (DIBIT-HSR and IGBE-CNR, Milan).

Fluorescence in situ hybridization (FISH) with commercial probes was performed according with the manufacturer's instructions. Moreover, Mega YAC DNAs were isolated by using the standard lyticase and detergent lysis procedure, purified by a salt extraction, and concentrated by isopropanol. YAC DNAs were labeled by nick-translation with biotin-16-dUTP (Boehringer-Mannheim) and precipitated with ethanol. Labeled DNA probes were resuspended in 50% formamide/2 ×SSC/10% dextran sulfate at a final concentration of 50 ng/μl.

The procedure for FISH was performed as described (Lichter and Cremer, 1992). The hybridizations were detected with Avidina-Cy3 (Amersham). Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 200 ng/ml) and analyzed with fluorescence microscope Olympus BX70 equipped with a cooled CCD Video Camera image point, Photometrics; the image analyses were carried out with PSI MacProbe software.

FISH analysis using painting probe of chromosome 15 (Cambio) showed an homogeneous hybridization of the little supernumerary chromosome in all metaphases examined (Fig. 3). No other painted segments were observed. These observations ruled out a translocation and suggested a partial chromosome 15 duplication.

imageimage

Figure 3. Fluorescence in situ hybridization with the YAC probe 810f11 in patient 1 (A) and patient 2 (B). Thin arrows, the locus D15S144 on the normal chromosomes 15. Thick arrow, the breakpoint on the little supernumerary chromosome.

To confirm this hypothesis and to determine the extent of the duplication region, FISH analysis was performed with different YAC probes. Results are summarized in Table 1. For both patients, the double spots of probes D15S10 and SNRP indicated tetrasomy for 15q11q13 region, whereas the single spot resulting from the probe 810f11 showed the breakpoint of the little supernumerary chromosome in the 15q14 band. Further characterization of the supernumerary metacentric bisatellite chromosome with FISH by using the GABRB3 probe and the PML chromosome 15q22 control probe (ONCOR, Inc) confirmed that the Prader–Willi/Angelman region was conserved.

Table 1.  Results of FISH analysis on both patients
ProbeLocusCytogenetic bandHybridization spot
  1. FISH, fluorescence in situ hybridization.

D15S10D15S1015q11q13Duplicated
SNRPNSNRPN15q11q13Duplicated
810f11D15S14415q11q14Single spot

DISCUSSION

  1. Top of page
  2. Abstract
  3. CASE REPORTS
  4. CYTOGENETIC AND MOLECULAR STUDIES
  5. DISCUSSION
  6. REFERENCES

The two patients presented here had a number of clinical characteristics usually encountered in inv dup(15) syndrome, such as mental retardation and epilepsy (2,3). The most striking observation, however, was the discrepancy between the mild phenotype and the rather severe genetic abnormality, characterized by a large inv dup(15) extending to q14 or 15 with tetrasomy. Patients with large inv dup(15) usually have severe epilepsy and severe EEG abnormalities, including slow background activity. Our patients had mild cognitive impairment and epilepsy with infrequent generalized seizures. Generalized interictal EEG abnormalities were accompanied by normal background activity. Both rare seizures and mild cognitive impairment may explain why background EEG activity was not abnormal, as is usually seen in patients with large inv dup(15).

Another peculiar aspect of our patients was the lack of facial dysmorphisms, which are usually encountered in patients with inv dup(15). Therefore it is conceivable that, in addition to the extent of the chromosomal duplication (2,3) and dosage effect from genes located within the duplication (5), other factors may be contributory to the patient's phenotype.

As suggested by our patients, we believe that the site of the breakpoint might play a role in determining the severity of inv dup(15) phenotype. In most inv dup(15) patients who display a severe phenotype, the site of the breakpoint is quite homogeneous, being immediately proximal to the Prader–Willi/Angelman syndrome (PWS/AS) region, mapping within bands 15q11q13 (10). Several genes coding for neurotransmitter receptor subunits (i.e., α7-nicotinic receptor subunit and β3, α5, and γ3 subunits of the GABA receptor) map within the PWS/AS region (10). It has been postulated that these genes are involved in determining the severity of the phenotype associated with inv dup(15), including that of epilepsy (9). More distally, an additional gene could potentially be implicated in the pathogenesis of epilepsy, the SLC12A6 (solute carrier, family 12, member 6) gene, coding for a cation chloride cotransporter and expressed in the brain, heart, skeletal muscle, and kidney (11).

It seems reasonable to hypothesize that the more distal site of breakpoint in our patient might have had a limited deleterious influence on the previously mentioned class of genes. In addition, our patients illustrate that a mild epileptic phenotype does not rule out a diagnosis of inv dup(15), which should therefore be considered as a possible cause, although rare, of atypical cases of generalized epilepsy with no clear-cut etiology.

Acknowledgment: This work was partially supported by a grant from Telethon (EO 827).

REFERENCES

  1. Top of page
  2. Abstract
  3. CASE REPORTS
  4. CYTOGENETIC AND MOLECULAR STUDIES
  5. DISCUSSION
  6. REFERENCES
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