Helen Stuart, Cardiff

1. Interesting case of a symptomatic male with 5 duplicated regions of Xq28 including a partial MECP2 duplication detected by array CGH and MLPA Helen Stuart, Cardiff

2. Case summary • Male patient (DOB 09/11/1991; refs 6M4446 C06-3708 GC19825) • Referred from Consultant Clinical Geneticist age 5 years (1996) • Symptoms: delayed motor development, behavioural problems, hyperphagia and micropenis • EDTA and lithium heparin samples received • Fragile X testing and karyotyping initially requested – both normal • Further testing: PWS, FISH for 22q11del (DiGeorge), FISH for subtelomeric rearrangements – all normal • Referred for microarray research study in 2006 - 5 duplicated regions on Xq including duplication at Xq28 (encompassing MECP2 gene) • MECP2 MLPA performed to confirm MECP2 duplication – MECP2 exon 4 duplication identified (and dup IRAK1, LICAM, IDH3G + SLC6A8)

3. Molecular report: Fragile X • Report date 25/07/1996 SUMMARY: PCR ANALYSIS FOR THE TRIPLET REPEAT EXPANSION KNOWN TO CAUSE FRAGILE X (A) SYNDROME:- NORMAL MALE Molecular testing for the Fragile X (A) syndrome in this male has been completed. PCR amplification of the CCG repeat within the FMR1 gene has revealed the presence of a triplet repeat of normal size. This result excludes the Fragile X (A) syndrome in 99% of cases. Due to the wide variety of syndromes that are known to give rise to mental impairment, some of which are detectable at a chromosomal level, it is recommended that cytogenetic screening is carried out in all cases which are negative for the Fragile X (A) test.

4. Cytogenetics report: karyotype • Report date 14/09/1996 46XY = normal male No abnormality was detected in 6 cells examined from a lymphocyte culture established from the blood of this patient. No screening for fragile X lesions was undertaken since the diagnosis of fragile X syndrome is now carried out by molecular techniques in the DNA laboratory. The results of this analysis has been sent to you.

5. Molecular report: PWS • Report date: 18/11/1997 SUMMARY:-METHYLATION PATTERN AT THE PRADER WILLI LOCUS IS NORMAL Molecular testing for Prader Willi Syndrome, using the SNRPN methylation probe has been completed on this patient and a normal result has been obtained. The test is able to detect virtually all cases of Prader Willi syndrome. Approximately 60% of cases of Prader Willi syndrome are due to small deletions of the paternally derived chromosome 15 (15q11-q13), and 30% have uniparental disomy with 2 maternal chromosomes present, rather than the normal situation of a chromosome having been inherited from each parent. Among the remaining 10%, a substantial amount have chromosomal abnormalities involving chromosome 15. The molecular test performed in this instance is able to detect loss of paternal imprinting at 15q11-q13. The result revealed both maternal and paternal imprinting, indicating that the chromosomes 15 in the patient were of both maternal and paternal origin. Thus there is no evidence of a microdeletion or maternal uniparental disomy involving this region of chromosome 15. In view of the residual risk of chromosomal abnormality it is recommended that this patients karyotype be reviewed.

6. Cytogenetics report: FISH for 22q11del (DiGeorge syndrome) • Report date 07/10/2003 • Note: Fewer than 5% of individuals with clinical symptoms of the 22q11.2 deletion syndrome have normal routine cytogenetic studies and negative FISH testing. They may have variant deletions of DiGeorge syndrome that may be detectable on a research basis only or with other more advanced clinical testing method. Further to the request of Dr. S Davies, fluorescence in situ hybridization (FISH) studies to test for 22q11 Deletion syndrome were carried out. Analysis with the probe TUPLE1 showed that there was no molecular deletion of the sequences recognised by this probe. There was therefore no evidence of loss of the region associated with 22q11 Deletion syndrome. Due to the small amount of stored material, only 7 cells rather than our usual 10 could be analysed. A normal male karyotype has previously been reported on this, 17/03/98, and other samples, 96.2707, 96.4095, 98.0622 and 99.0787. Normal FISH tests for Prader Willi syndrome and Smith Magenis syndrome (17p11.2 deletion) were reported on sample nos. 98.0667 and 99.0787 respectively.

7. Cytogenetics report: FISH for subtelomeric rearrangements • Report date 05/11/2004 • Note: Submicroscopic subtelomeric chromosome defects have been found in 7.4% of children with moderate to severe mental retardation and in 0.5% of children with mild retardation (ref: de Vries et al 2001). Fluorescence in situ hybridisation (FISH) studies were carried out to test for subtelomeric rearrangements. Analysis with the Vysis ToTelVysion kit showed that there was no evidence of any subtelomeric rearrangements. A normal male karyotype and normal FISH analysis for Prader Willi, Smith Magenis and 22q11 Deletion syndromes have been reported on previous samples from this patient (report nos. 990781, 980667 etc.).

8. Cyto results: array CGH (BAC array v1.1) • Referred for microarray research study in 2006 • Array studies carried out using the BlueGnome CytoChip BAC array (v1.1) identified the presence of three duplicated regions on the long arm of the X chromosome: • 1) A single clone duplication detected by the BAC RP1-29A6 at Xq27.3; • 2) A 0.39Mb interstitial duplication of the sequences detected by the BACs RP3-433G13, RP6-224C24, RP11-37P24 and RP11-161L9 at Xq27.3-q28; • 3) A 0.40Mb interstitial duplication of the sequences detected by the BACs RP11-54I20 and RP11-617G06 at Xq28.

9. Cyto results: array CGH (v2.0) • Array CGH analysis of DNA, Molecular Laboratory number 6M4446, from the above patient using the BlueGnome CytoChip Oligo ISCA 8x60k (v2.0) array showed five duplicated regions detected on the long arm of the X chromosome. These duplications were detected by oligonucleotides for the sequences : • 1) 133,977,853bp-135,036,406bp. A 1Mb duplication located at Xq26.3; • 2) 142,727,023bp-143,516,409bp. A 790kb duplication located at Xq27.3; • 3) 146,041,236bp-148,008,630bp. A 2Mb duplication located at Xq27.3->q28; • 4) 151,966,264bp-152,548,983bp. A 583kb duplication located at Xq28; • 5) 152,848,020bp-152,947,909bp. A 100kb duplication also located at Xq28. • Within the duplicated regions there were 30 HGNC and 9 OMIM genes including SLC9A6, FMR1, TREX2, BGN, ATP2B3, RENBP, HCFC1, IRAK1 and MECP2. • An array CGH chart for the X chromosome is enclosed. Please also refer to the enclosed Ensembl overview chart for these regions. BASIS OF TEST: • Array comparative genomic hybridisation was carried out using a BlueGnome CytoChip Oligo ISCA 8x60K (v2.0) array. • Base pair positions are based on NCBI build 36. • Test DNA was referenced against same sex control DNA and data analysed using BlueFuse Multi (v2.2) software, with data filtered on 3 consecutive probes and software algorithm smoothing to 350kb and 100kb for backbone and disease regions, respectively. • Oligonucleotide probes were called as copy number changes if sample and control Cy dye ratios were outside thresholds of 3x SD of the autosomal median log 2 ratio (the Derivative Log Ratio Spread, DLRS, is <0.25). • Limitations: This test is still under development. It will not detect balanced rearrangements and may not detect mosaic imbalances. • Note: All three duplicated regions found using the v1.1 array were confirmed using the CytoChip Oligo ISCA 8x60k array.

10. Molecular report: MECP2 MLPA • Report dated 30/07/2007 • MECP2 exon 4 duplication identified (and dup IRAK1, LICAM, IDH3G + SLC6A8)

11. Molecular report for mother of 6M4446: MECP2 MLPA • Asymptomatic mother of 6M4446 • Report date 16/06/2008

12. Cyto report: array CGH (v2.0) • Report dated 21/10/2010 The most distal duplicated region at Xq28 found in # (from 152,848,020bp-152,947,909bp) includes the MECP2 gene. Multiplex Ligation-dependent Probe Amplification (MLPA) studies carried out by the Molecular Genetics Laboratory using the MRC Holland Salsa MLPA kit P015C, which contains probes that map to MECP2, detected a duplication of exon 4 of the MECP2 gene. Please refer to the Molecular Genetics Laboratory report no. 6M4446, a copy of which is enclosed. Further MLPA studies found that #'s mother is also a carrier of the same MECP2 duplication. Please refer to Molecular Genetics report no. 7M2796. # therefore, appears to have inherited this duplication from his mother. Duplications of MECP2 are associated with clinical problems including speech delay, psychomotor delay and developmental delay. There are, however, apparently no reports in the literature of males with partial MECP2 gene duplications, as seen in this case. The significance of this finding is therefore uncertain. The exact clinical significance of the other four duplicated regions on the X chromosome in this patient is also uncertain, although partial duplication of the X chromosome in a male would be expected to lead to functional disomy and result in clinical problems, including intellectual impairment. A recent paper published by Rio et al., 2010, in the European Journal of Human Genetics 18:285-290, reports a large family with X-linked syndromic mental retardation in which affected males had a 5.1Mb interstitial duplication at Xq27.3q28 encompassing the FMR1 gene but not MECP2. A copy of this publication is enclosed. The clinical features of the affected males included neonatal hypotonia, poor motor skills and mild learning difficulties. #'s duplication at Xq27.3 to q28 overlaps the region reported in this publication. This patient has been entered into the DECIPHER database, patient no. CAR004571, a copy of which is enclosed. No other patients entered into DECIPHER appear to have similar duplications. We would, in due course, be pleased to carry out further investigations in #'s motherto determine if all duplicated regions have been inherited. This would enable us to assess the clinical significance of our findings and determine any future risks.

13. Further reading: functional disomy • In males, duplication of any part of the X chromosome leads to structural and functional disomy of the corresponding genes. It may result from an intrachromosomal duplication, an unbalanced translocation or 'aneusomie de recombinaison' (= complex but balanced chromosome rearrangement that can be transmitted as such in a family but can also give rise through recombination during meiosis to a nonbalanced rearrangement with duplication/deficiency, e.g. following an uneven number of crossing overs within a pericentric inversion). • Functional disomy of part of the X chromosome is a rare event. It is usually lethal except in a mosaic state resulting from X-inactivation in girls or when the segment involved is very small. • Functional disomy for the Xq28 chromosome region results in excess gene dosage and yields a recognizable phenotype including distinctive facial features, major axial hypotonia, severe feeding difficulties, abnormal genitalia and proneness to infections. Severe developmental delay is almost constant. Ref: Sanlaville et al 2005 Eur J of Hum Genet 13, 579-585 • Partial duplications of Xq in males are usually inherited from heterozygous mothers in whom preferential inactivation of the dup(X) chromosome leads to a skewed X-inactivation pattern and results in a normal or mildly abnormal phenotype (ref: Rio et al 2010 Eur J of Hum Gen 18 285-290).

14. Further reading: Xq dup cases • “Duplications involving the X chromosome, in which the duplicated region is not subject to inactivation, are rare. We describe four distal Xq duplications, in three males and one female, in which the duplicated X chromosomal material is active in all cells. The infantile phenotype bears some resemblance to that of the Prader–Willi syndrome, presenting with initial feeding difficulties, hypotonia and, sometimes, with cryptorchidism. However, the severity of the phenotype is not simply related to the size of the duplication and so variations in gene expression (i.e. functional disomy causing increased dosage), gene disruption (i.e. genes at breakpoints) or position effects from breakpoints should be considered as explanations.” Ref: Lachan et al 2004 Human Genetics 15 (5) 399-408

15. Detection of partial Xq duplications • Until recently, most cases of functional disomy have been detected using standard cytogenetic methods because of intrachromosomal duplications, an unbalanced X/Y, X autosome translocation or X pericentric inversion. • The availability of array CGH has increased the ability to detect cryptic interstitial chromosome X imbalances.

16. References • de Vries et al 2001 J Med Genet 38: 145-150 • Lachan et al 2004 Human Genetics 15 (5) 399-408 • Rio et al 2010 Eur J of Hum Gen 18 285-290 • Sanlaville et al 2005 Eur J of Hum Genet 13, 579-585