1of6 ELECTRONIC LETTER J Med Genet: first published as 10.1136/jmg.39.10.e59 on 1 October 2002. Downloaded from Molecular screening for Smith-Magenis syndrome among patients with mental retardation of unknown cause J L Struthers, N Carson, M McGill, M M Khalifa ............................................................................................................................. J Med Genet 2002;39:e59(http://www.jmedgenet.com/cgi/content/full/39/5/e59) mith-Magenis syndrome is a rare, multiple congenital anomaly/mental retardation syndrome (MCA/MR) asso- ciated with interstitial deletion of chromosome 17p11.2. S 1 Smith et al first described this condition in two patients. To date, more than 150 cases have been described. Patients with SMS display a variable expression of subtle dysmorphic features, MR, short stature, brachydactyly, visual and auditory impairment, behavioural problems, sleep disturbance, and cardiac and renal malformations.23 Almost all cases are de novo, non-mosaic, and of either maternal or paternal origin.45Although the abnormalities associated with SMS are well described, their subtlety and variable expression make clinical diagnosis often difficult, particularly in neonates and young infants.67 The SMS deleted region ranges in size from <1.5 Mb to 9 Mb and the majority of patients have a ∼5 Mb deletion (∼10- 11% of chromosome 17).48In general, there is no obvious cor- relation between the size of the deletion and the severity of the phenotype.8 In the majority of cases, this deletion is visible on careful routine cytogenetic analysis. Despite this, in several cases the deletion has been missed.79Using a FISH probe spe- cific for SMS has enhanced detection of the syndrome, especially in equivocal cases.10–12 Elsea et al13 reported a patient with a typical SMS phenotype and a normal karyotype at 650 http://jmg.bmj.com/ band resolution. This patient was subsequently diagnosed Figure 1 Densitometric analysis of D17S258:DXS70 allelic ratios with SMS by FISH.12 of SMS and normal subjects. (A) Diagram of the predicted banding patterns using the two EcoRI non-polymorphic probes; the X Current estimates of the incidence of SMS, based on ascer- 4 chromosome control probe, DXS70, corresponds to the 5.2 kb band taining cases in genetics centres, are between 1 in 25 000 and and the SMS deletion specific probe, D17S258, corresponds to the 1in5000014 births. Because the diagnosis of SMS can be 4.9 kb band. Grey shading indicates a single copy of the probe, missed both clinically and cytogenetically, it is generally black shading is indicative of a double copy. The D17S258/DXS70 believed that this syndrome might be underdiagnosed. In this density ratio for each lane is shown at the bottom. (B) Southern blot study, we screened a large population of patients with MR/DD of DNA from normal male and female controls and male and female on September 29, 2021 by guest. Protected copyright. SMS patients digested with EcoRI and cohybridised with DXS70 and D17S258 probes. The D17S258:DXS70 allelic ratios in normal males and females were approximately twice that of their SMS Key points counterparts, illustrating the ability of this screening method to detect the SMS deletion. • Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with of unknown cause to determine the frequency of SMS in this deletion 17p11.2. The prevalence of this syndrome in group and to estimate its prevalence in the general population. the general population and its frequency among patients with mental retardation/developmental delay MATERIAL AND METHODS (MR/DD) is unknown. It is generally believed that this Study population syndrome might be underdiagnosed because of its phe- A total of 1618 DNA samples were screened in this study, notypic and cytogenetic variability. which was approved by the Queen’s University Research Eth- 15 • In this study we screened a large number of patients with ics Board. These samples were referred during the period unexplained MR/DD to determine the frequency of SMS between 1996 and 1999 for FRAX testing. among this population. The screening involved Southern Screening strategy for detecting SMS deletion blotting and dosage comparison between non- A several stage screening strategy for detection of SMS was polymorphic markers, SMS deletion specific and control formulated using available patient material. The screening probes. All samples suspected to have the SMS deletion were subjected to confirmatory testing. • Of 1205 patients successfully screened, we detected ............................................................. two SMS patients. The prevalence of SMS in the general Abbreviations: SMS, Smith-Magenis syndrome; MCA/MR, multiple population is estimated. congenital anomaly/mental retardation; MR, mental retardation; DD, developmental delay www.jmedgenet.com 2of6 Electronic letter J Med Genet: first published as 10.1136/jmg.39.10.e59 on 1 October 2002. Downloaded from Table 1 DNA markers used for molecular screening by Southern hybridisation Probe Locus Location Enzyme Allele size (kb) Heterozygosity p1516-R4 D17S258 17p11.2 EcoRI* 4.9 0%† p18-55 DXS70 Xp22.3 EcoRI* 5.2 0%† pYNZ22 D17S5 17p13.3 EcoRI/BssHII* 3.05–4.20 83.5%† Data obtained from GDBTM Human Genome Database (database online). *Additional digestion with BssHII did not alter the allele size or heterozygosity. †Present study. Table 2 DNA markers used for PCR analysis of SMS deletion interval Marker Sequence name Location* Heterozygosity Product size (bp) Label D17S1857 AFM357yg9a/m Distal to common deletion 65.0% 177–187 HEX D17S2206 YL63-64 Within deletion, distal to middle SMS-REP 91.7%* 141–165 6-FAM D17S2207 YL601-602 Within deletion, distal to middle SMS-REP 42.0%* 135–155 HEX D17S805 AFM234ta1a/m Within deletion, proximal to middle SMS-REP 58.8% 216–228 6-FAM D17S841 AFM238vb10a/m Proximal to common deletion 65.4% 253–273 HEX Data obtained from GDBTM Human Genome Database (database online). *Potocki et al.3 involved dosage comparison between SMS deletion specific at 65°C, followed by a stringent wash in 0.1 × SSC, 0.1% SDS (D17S258) and X chromosome control (DXS70) markers. Ini- at 65°C for 20 minutes.17 tially, Southern blots, originally prepared for FRAX testing and Band intensity indicating the number of copies for each containing EcoRI/BssHII digested DNA were lightly stripped probe was initially estimated visually. Densitometry was from the FRAX hybridisation and then cohybridised with performed using scanned autoradiographs into Corel Photo- radiolabelled D17S258 and DXS70 probes. Because these Paint software version 9 as described previously.18 To estimate markers are non-polymorphic for the enzyme(s) used, each the number of copies of the SMS allele, the intensity of the probe produced a single band. These probes were chosen autosomal band was divided by the intensity of the X because the resultant bands were close in size (D17S258 = 4.9 chromosome band. kb band, DXS70 =5.2 kb band), which facilitated dosage To minimise the risk of missing any SMS patients, all sam- comparison. Normal males (46,XY) display two copies of the ples, which showed even minimal deviation from normal band D17S258 band relative to a single copy of DXS70. Normal intensity patterns, were selected for further evaluation. females (46,XX) display two copies of each band. SMS males would display only a single copy of the D17S258 allele and a Fluorescence in situ hybridisation (FISH) single copy of the X chromosome allele, while SMS females Patients who consistently exhibited an unexplainable dosage http://jmg.bmj.com/ would display a single copy of the D17S258 allele and two difference on repeat screening were contacted through their copies of the DXS70 allele. This method was validated before referring physicians to obtain fresh blood samples for FISH. screening using normal male and female controls and SMS Metaphase FISH analysis was performed according to the patients, equal dpm of each labelled probe, and variable expo- manufacturer’s protocols using a Vysis HYBriteTM Hybridiza- sures for each blot to ensure optimal representation of signal tion System and the Oncor D17S258 Smith-Magenis Chromo- intensities (fig 1). For some experiments, another chromo- some Region Probe or the dual-colour Vysis Locus Specific some 17 probe (D17S5) was also included for dosage control. Identifier Smith-Magenis Microdeletion Probe. In the second stage, patients who deviated from the on September 29, 2021 by guest. Protected copyright. expected banding patterns and were suspected of having the PCR analysis of DNA markers within and flanking the SMS deletion were subjected to a repeat of the screening assay, SMS deletion region using their banked DNA and new Southern blots. Finally, PCR heterozygosity analysis was performed for cases where a patients consistently exhibiting suspicious results were inves- fresh blood sample could not be obtained, or repeat screening tigated further using confirmatory testing by FISH and/or PCR was not adequately performed owing to poor DNA quality. microsatellite genotyping. Characteristics of the markers used Fluorescent labelled primers for highly polymorphic se- in this study are listed in tables 1 and 2. quences within and flanking the SMS common deletion were used for DNA amplification (table 2). Normal subjects were Southern blot analysis and densitometry expected to display two alleles at most loci tested, while SMS Southern blotting was performed as described previously.16 A patients were expected to be hemizygous for all loci within the total of 50 ng of probe insert was labelled with 50 µCi α-(32p) deletion interval. Amplified fragments were electrophoresed dCTP (3000 Ci/mmol) by random priming according to the on a 5% denaturing polyacrylamide gel in an ABI 373 DNA manufacturer’s instructions (Random Primers Labeling Sys- Sequencer and recorded by computer assisted laser. The tem, GIBCO BRL). Blots were hybridised in 1 mol/l NaCl, 1% forward primer of each pair, end labelled with either 6-FAM SDS, 10% dextran sulphate, and 0.1 mg/ml salmon sperm (blue) or HEX (green) dye, was synthesised by the University DNA at 65°C overnight.