Prader Willi syndrome (PWS) occurs with an incidence of 1:10,000 to 30,000 births(1) and is associated with developmental as well as behavioral problems. Most common genetic defect is paternal deletion of 15q11-ql3 region (70% of case)(2); second mechanism is maternal uniparental disomy (UPD; 25% of cases)(3). About 1-5% of patients have biparental inheritance of chromosome 15, but show abnormal methylation pattern and gene expression(4). These patients have mutation in the gene involved in imprinting process(4). The test of methylation pattern of SNRPN exon 1 detects 95% of PWS patients(5) irrespective of mechanisms. Microsatellite analysis of loci within and outside PWS/AS region dis-tinguishes between the genetic mechanisms (deletion, UPD, or imprinting mutation). Fluorescent in situ hybridization (FISH) is a useful tool to detect deletion cases not recognized through conventional or high resolution banding cytogenetics. FISH, being the only way to analyze each cell or metaphase individually, has the ability to diagnose mosaicism very easily and reliably. Deletional mosaicism can also be diagnosed by high resolution banding technique in some cases as well as by methylation-specific PCR followed by denaturing high performance liquid chromatography(6). The later has the ability to reveal a low cell mosaicism (as low as 8% deleted cell lines) like FISH. However, those are laborious and require experts as well as high technology. In this report we describe a female child with clinical features of atypical Prader Willi syndrome and FISH analysis showed mosaicism for a deletion in the PWS critical region, probably the first on mosaic PWS from India.

A 9-year-female child was referred to us for marked weight gain and temper tantrum. She was born to a 35-year-old gravida 2 para 1 at full term by cesarian section for face presentation. Her elder brother was normal. Her weight, length and head circumference were normal at birth. There was no history of perinatal complications and suckling in infancy was normal. Her milestones were normal and she was active and thin until 3+ years of age when she developed increased urge for food and irritable behavior with food denial. Her weight gain was gradual initially but later (at the age of 8 years) aggressively (16 kg in a span of 1 month). She was put on an appetite suppressant therapy. At present her weight is 78.5 kg (>3 SD) and height is 148.1 cm (>3 SD).

She was diagnosed to be suffering from a bipolar affective disorder and rapid mood changes at the age of 8 years for which she is undergoing conservative management. She also had an adenoidectomy (tonsillar pillar) for sleep apnea at the age of 8+ years. Hearing impairment is also present since 3+ years of age for which gourmet insertion is done every six months.

Physical examination revealed morbidly obese child of normal intelligence. She had no dysmorphism except skin picking, straight ulnar border and hypoplastic labia minora. She did not have any other characteristics of Prader Willi syndrome including that of face. Extensive endocrinological work up including that of growth hormone was normal and con-ventional cytogenetics using high resolution banding was inconclusive from two different laboratories.

Since the patient had some findings suggestive of Prader-Willi syndrome, FISH study to detect a possible deletion in the critical PWS/AS region was done. FISH was done using the manufacturers protocols (Vysis Inc) using probes for D15S10 and SNRPN and two control probes D15Z1 (localized to 15p11.2) and PML (localized to15q22). A total of 40 metaphases were scored (20 in each culture aparted by 5months). Metaphase FISH result showed 15% (i.e., 6 out of 40) metaphases have deletion with both probes (SNRPN & D15S10) i.e., mosaic PWS. Interpretation of interphase cells was inconclusive due to probes designing (too many signals/split signals).

Mosaicism in Prader Willi Syndrome is rare and only a few cases have been described in literature (1,7-9). Cassidy in 1984(1) was the first to describe mosaicism in Prader- Willi syndrome. They described two patients who were mosaic for a chromosome 15 deletion using a high resolution banding technique. The clinical manifestations of these two patients did not differ from those of the rest of the population they had studied. Mowery-Rushton, et al.(7) reported four mosaic cases detected by FISH. Malzac,k et al.(8) reported another case of typical Prader Willi syndrome and 20% deletion detected by FISH in peripheral blood lymphocytes. Recently, Golden et al.(9) also reported one more case with atypical Prader Willi findings and deletion of SNRPN probe in 78% of peripheral lymphocytes. Our case is probably the first case of FISH detected mosaicism in PWS using both SNRPN and D15S10 probes without any facial dysmorphism from India. Although our case did not have typical facial dysmorphism, we suspected PWS because of presence of following major and minor characteristics laid by Holm, et al.(10).

Major criteria presented in our case were marked weight gain, temper tantrum, hyperphagia and hypoplastic labia minora. Minor criteria included bipolar affective disorder, rapid mood changes, sleep apnea and skin picking.

Laboratory approach to diagnose typical PWS should be methylation specific PCR as first step (along with conventional cytogenetics) and later FISH for confirmation of micro-deletion, which is about 70%. In case of negative FISH one should go for micro satellite analysis for confirmation of uni-parental disomy, which are about 25%.

However, ascertainment of low level mosaic deletion by either microsattelite repeats or methylation would be difficult due to the presence of a normal cell line, which would mask the appearance of deleted chromosome 15. Since FISH analysis can assess individual cells, this technique can pick up low level of mosaicism. Mosaicism can affect the phenotype of the disorder and may be an important factor in clinical variability; mild or atypical cases may not be studied if they do not meet the diagnostic criteria. Our case emphasizes the importance of FISH in the diagnosis of such cases i.e., mosaicism. It is well accepted that DNA methylation test is useful as an initial screening test for a typical PWS/AS(ASHG/ACMG 1996) cases. However, in atypical case a negative methylation test cannot rule out possibility for a mosaicism. In such atypical cases, FISH analysis should be performed to rule out deletional mosaicism. To establish mosai-cism, a repeat blood sample should always be analyzed and along with a double blind FISH study, preferably in two centers. Skin fibroblasts should always be analyzed wherever possibe(11) however, we were unable to do so due to non co-operation from the patient.

Contributors: AH coordinated work-up, carried out FISH and drafted the paper; he will act as the guarantor of the paper. VC and SA carried out clinical work up and collected the literature. VC, SA prepared the initial drafting of the paper. SP was in-charge of the patient and carried out conventional cytogenetics study.

Funding: We are grateful to the institute i.e., SGPGIMS for financing various FISH probes including that of PWS and FISH imaging system as part of standardization of molecular cytogenetics facility.

Competing interests: None stated.

11. Nicholas RD. Mosaicism in Prader Willi syndrome. Am J Med Genet 2000; 90: 175-176.