Different-sized duplications of Xq28 including MECP2 in three males with mental retardation absent or delayed speech and recurrent infections.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:799 –806 (2008) Different-Sized Duplications of Xq28, Including MECP2, in Three Males With Mental Retardation, Absent or Delayed Speech, and Recurrent Infections M. Smyk,1 E. Obersztyn,1 B. Nowakowska,1,2 M. Nawara,1 S.W. Cheung,2 T. Mazurczak,1 P. Stankiewicz,1,2 and E. Bocian1* 1 Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 2 In XY males, duplication of any part of the X chromosome except the pseudoautosomal region leads to functional disomy of the corresponding genes. We describe three unrelated male patients with mental retardation (MR), absent or delayed speech, and recurrent infections. Using highresolution comparative genomic hybridization (HR-CGH), whole genome array comparative genomic hybridization (array CGH), fluorescent in situ hybridization (FISH), and multiplex ligation probe amplification (MLPA), we have identified and characterized two different unbalanced Xq27.3-qter translocations on the Y chromosome (approx. 9 and 12 Mb in size) and one submicroscopic interstitial duplication (approx. 0.3–1.3 Mb) involving the MECP2 gene. Despite the differences in size of the duplicated segments, the patients share a clinical phenotype that overlaps with the features described in patients with MECP2 duplication. Our data confirm previous observations that MECP2 is the most important dosage-sensitive gene responsible for neurologic development in patients with duplications on the distal part of chromosome Xq. ß 2007 Wiley-Liss, Inc. KEY WORDS: array CGH; HR-CGH; gene dosage; psychomotor retardation; absent speech Please cite this article as follows: Smyk M, Obersztyn E, Nowakowska B, Nawara M, Cheung SW, Mazurczak T, Stankiewicz P, Bocian E. 2008. Different-Sized Duplications of Xq28, Including MECP2, in Three Males With Mental Retardation, Absent or Delayed Speech, and Recurrent Infections. Am J Med Genet Part B 147B:799– 806. of the corresponding genes, whereas females with Xq duplications are usually phenotypically normal [Schmidt et al., 1991; Sanlaville et al., 2005]. Duplications involving the distal region of the long arm of X chromosome are rare. To date, at least 19 males and females with terminal Xq duplications have been described [Lachlan et al., 2004; Novelli et al., 2004; Sanlaville et al., 2005]. These patients shared clinical symptoms of prenatal growth retardation, severe mental retardation (MR)/ developmental delay, hypotonia, hypoplastic genitalia, and/or cryptorchidism, severe feeding difficulties, recurrent infections, microcephaly, small and open mouth, ear anomalies, abnormal fingers and toes, and absence of or severely retarded speech [Vasquez et al., 1995; Goodman et al., 1998; Akiyama et al., 2001; Lachlan et al., 2004; Novelli et al., 2004; Sanlaville et al., 2005]. Interestingly, in some neonatal cases, a resemblance to Prader–Willi syndrome has been noted [Goodman et al., 1998; Gecz et al., 1999; Lammer et al., 2001; Lachlan et al., 2004; Sanlaville et al., 2005]. Recently, due to application of array comparative genomic hybridization (array CGH) and real-time quantitative PCR, one female and 45 male patients from 20 families with submicroscopic interstitial duplication, and one male patient with triplication involving Xq28 have been reported. The region of duplication ranged in size from 0.2 to 2.2 Mb and included the MECP2 gene [Ariani et al., 2004; Meins et al., 2005; Van Esch et al., 2005; del Gaudio et al., 2006; Friez et al., 2006; Lugtenberg et al., 2006]. The patients manifested some clinical features overlapping with characteristics described in patients with larger duplications of terminal Xq [Van Esch et al., 2005; del Gaudio et al., 2006]. We describe three unrelated males with MR, absent or delayed speech, and recurrent infections due to the Xq duplications involving MECP2. A detailed clinical evaluation and comparison with similar cases from the literature is presented. CLINICAL REPORT Patient 1 (MT) INTRODUCTION Duplications of any part of the X chromosome except the pseudoautosomal region in XY males lead to functional disomy Grant sponsor: Polish Ministry of Scientific Research and Information Technology; Grant numbers: 2P05A 191-29, 40101731/0307, PBZ/KBN 122/P05/2004/01-9, 2P05A 128 28. *Correspondence to: Prof. E. Bocian, Ph.D., Department of Medical Genetics, Institute of Mother and Child, Kasprzaka 17A, 01-211 Warsaw, Poland. E-mail: email@example.com Received 4 May 2007; Accepted 30 October 2007 DOI 10.1002/ajmg.b.30683 ß 2007 Wiley-Liss, Inc. The proband was an 8-year-old boy, first son of healthy and non-consanguineous parents (a 19-year-old mother and a 25-year-old father). Family history was negative for MR and congenital malformations but the proband’s mother had three early miscarriages. The pregnancy was uneventful until 32 weeks when a maternal urinary tract infection was diagnosed. Cesarean section was performed at 37 weeks because of fetal distress due to maternal health problems: proteinuria, edema of the limbs and high blood pressure. Birth weight was 1750 g (<5th centile), length 45 cm (5th centile), and occipitofrontal circumference (OFC) 29 cm (<5th centile). Apgar scores were 5 and 10 at 1 and 5 min, respectively. At birth, bilateral cryptorchidism was noted. The child had congenital pneumonia and meconium ileus was suspected at this time. Persistent hyperbilirubinemia and secondary metabolic acidosis were observed during his first 2 weeks of life. 800 Smyk et al. Because of recurrent respiratory tract infections and failure to thrive, cystic fibrosis was considered but was excluded at 6 months. Global muscular hypotonia and significantly delayed psychomotor development were noticed from birth. At the age of 8 years, he could sit unsupported but could not stand up. The speech was limited to several unintelligible sounds. CT of brain showed global dilatation of the subarachnoid space, a septum pellucidum cyst and delayed myelination of the white cortex. Anthropometric evaluation showed short stature (<5th centile), microcephaly (OFC <5th centile), and obesity with BMI >97th centile. The following dysmorphic features were noted: small bifrontal diameter, frontal bossing, flat occiput, round and expression-less face, open mouth, large tongue, relatively large, low-set and prominent ears, short nose, bristly hair, short neck with low posterior hairline, camptodactyly, and tapering fingers (Fig. 1a). Axial hypotonia and spasticity of upper and lower limbs were also observed. At the age of 9 years, he developed recurrent tonic-clonic seizures and epilepsy was diagnosed. He died at the age of 11 years because of respiratory insufficiency during a viral infection and status epilepticus. Patient 2 (AS) The proband is a male infant, the second child of healthy and non-consanguineous parents (a 33-year-old mother and a 43-year-old father). His older sibling is healthy. Family history was negative for MR, congenital malformations or exposure to known teratogens. The child was born at 38 weeks gestation after an uneventful pregnancy. The Apgar scores were 8 and 10 at 1 and 5 min, respectively. He showed clinical symptoms of congenital infection and intrauterine growth retardation. His birth weight was 2350 g (<5th centile), length 49 cm (<50th centile), and OFC 31 cm (<5th centile). Feeding difficulties with poor sucking and muscle hypotonia were observed during neonatal period. Brain MRI performed at 3 months showed agenesis of the corpus callosum, severe delay of white cortex myelination and ventricular dilatation. Additionally, gastroesophageal reflux was detected. In the differential diagnosis of unexplained hypotonia, the Prader–Willi/ Angelman syndromes were excluded. Clinical assessment in the Genetic Counseling Unit was performed at 18 months. He demonstrated severe psychomotor retardation with absent speech, axial hypotonia, microcephaly, and bilateral cryp- Fig. 1. torchidism. He was unable to sit or change body position. Brisk tendon reflexes and subtle spasticity were noted in the neurological examination. Recurrent respiratory infections were also reported. Facial dysmorphism with asymmetrical cranium, flat occiput, prominent forehead, round face, full cheeks, open mouth, down-slanting palpebral fissures, epicanthic folds, broad nasal root, short nose, relatively large ears with thickened helix, short neck, and increased subcutaneous skin folds were described. There were no seizures or other neurological symptoms. Patient 3 (TG) The proband, a 3-year-old boy from the second pregnancy of healthy and non-consanguineous parents (a 27-year-old mother and a 28-year-old father), was evaluated because of features of psychomotor retardation. His older sibling is healthy. The proband was born after uneventful pregnancy at the gestational age of 40 weeks. The Apgar scores were 10 at 1 and 5 min. His birth weight was 4100 g (>90th centile), length 50 cm (>50th centile), and OFC 35 cm (50th centile). Bilateral cryptorchidism was revealed after birth. The neonatal period was uncomplicated. Psychomotor retardation was observed since infancy; he walked at the age of 19 months and could speak only few words at the age of 2 years. Storage diseases, especially mucopolysaccharidosis, were suspected at this time because of the facial dysmorphism, but were excluded after obtaining the results of the lysosomal enzymes investigations. No mutation was found in the FMR1 gene. A detailed clinical assessment in the Genetic Counseling Unit was performed at age 17 years. Anthropometric investigation showed proportional body weight, length, and OFC; all parameters were within normal range. Psychological evaluation showed moderate MR with a pleasant personality. IQ was below 50 (in Wechsler’s scale). He could speak only about 20 simple words of one or two syllables. The speech was mumbling. His gait was unsteady with slightly bent knees. Progressive spasticity has been observed from puberty. Seizures have not been noted. However, an abnormal electroencephalography registration was documented. Dysmorphic features with long face (despite round face in early childhood), full lips, open mouth, synophrys, large and prominent simple ears, prognathism, dental caries, and macroorchidism were noted (Fig. 1b). The sister of the Patient 1 (a) and Patient 3 (b). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Functional Disomy and Trisomy of Xq28 in Males proband’s mother has two mentally retarded sons with reportedly similar features. Unfortunately, they were unavailable for further clinical evaluation and investigations. One of them could speak only few simple words of one or two syllables and both had dysmorphic facial features with expression-less face, open mouth, synophrys, large prominent ears, and large testes. Both had epilepsy and one of them demonstrated symptoms of infantile cerebral palsy with spasticity and brisk tendon reflexes. METHODS Cytogenetic and FISH Studies Conventional karyotyping using GTG, CBG-banding, and fluorescent in situ hybridization (FISH) with BAC clones was performed on PHA-stimulated peripheral blood lymphocytes using standard protocols [Shaffer et al., 1997]. BAC clones were identified from the existing physical maps (UCSC genome browser, http://genome.ucsc.edu). 801 RESULTS Patient 1 (MT) GTG-banding revealed a 46,X,der(Y) karyotype with additional material on the short arm (Fig. 2a). Cytogenetic analysis of the patient’s parents showed the rearrangement to be a de novo event. HR-CGH analysis demonstrated that it originated from the Xq27.2-qter region (Fig. 2b). The translocation t(X;Y) was confirmed by FISH with the wcpX probe (Fig. 2c). FISH analysis with X chromosome-specific BAC clones mapped the X chromosome breakpoint to q27.3 between two adjacent BAC clones RP11-433F22 and RP11-1091C11 (Table I). The size of the duplication was estimated as 12 Mb. The breakpoint on chromosome Y was mapped to Yp11.32, between the BAC clones RP11-257O13 and RP11-309M23, identifying a 1– 1.2 Mb deletion involving the SHOX gene. The patient’s karyotype was designated as 46,X,add(Y).ish der(Y)t(X;Y) (q27.3;p11.32)(wcpXþ,RP11-479B17þ,RP11-1091C11þ,RP11433F22,RP11-309M23,RP11-257O13þ). Patient 2 (AS) HR-CGH and Array CGH The probands’ genomic DNAs were isolated from the peripheral blood. HR-CGH was performed as previously described [Kirchhoff et al., 2000]. Array-CGH designed to cover genomic regions of 75 known genomic disorders including Rett syndrome region, all 41 subtelomeric regions, and 43 pericentromeric regions (Baylor College of Medicine, Chromosome Microarray Analysis, V.5, http://www.bcm.edu/cma/assets/ abnormalities.pdf) was used as previously described [Cheung et al., 2005]. XCI Status X-inactivation study was performed by using the androgen receptor gene methylation assay described by Allen et al. . Multiplex Ligation-Dependent Probe Amplification Copy-number analysis of the MECP2 gene and the flanking regions was performed by multiplex ligation-dependent probe amplification (MLPA) (MRC-Holland, Amsterdam, Netherlands). The reactions were performed on GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. Products were separated on ABI 3730 DNA Analyser and the data were analyzed for copy-number differences with the Gene Marker software (Softgenetics, State College, PA). Standard chromosome analysis revealed a 47,XX,der(Y) karyotype with a small submetacentric Y chromosome (Fig. 3a). CBG-banding showed the heterochromatic region on Yq was deleted. The results of cytogenetic analyses of the parents were normal. FISH with DYZ3 and wcpY probes confirmed that the derivative chromosome originated from the Y chromosome and revealed that it had additional material on the long arm. HR-CGH analysis showed a duplication of the Xq27.3qter region (Fig. 3b). The origin of the additional material on Yq was confirmed by FISH with the wcpX probe. The breakpoint on chromosome X was mapped between two adjacent BAC clones RP11-522P6 and RP11-226B15 (Fig. 3c) in a subband q27.3 (Table I). The fluorescent signal for RP11226B15 present on chromosome Y was faint, indicating that the breakpoint maps within this clone. Xq translocated fragment was estimated as 9 Mb in size harboring L1CAM, IRAK1, and MECP2. The breakpoint on chromosome Yq was mapped within the sub-sub-subband Yq11.223, between the BAC clones RP11-106G5 and RP11-539D10, demonstrating a deletion of the AZFc region with the DAZ3 gene (Fig. 3c). The patient’s karyotype was designated as 47,XX,þder(Y).ish der(Y) t(X;Y)(q27.3;q11.223)(wcpYþ,RP11-106G5þ,RP11-539D10, RP11-522P6,RP11-226B15þ, RP11-479B17þ, DYZ3þ). Patient 3 (TG) GTG-banding chromosome analysis on the proband’s blood lymphocytes showed a normal male karyotype. Examination of Fig. 2. The results of investigation in Patient 1. (a) Chromosomes X and der(Y) after GTG-banding. Note the additional material on the short arm of chromosome Y. (b) HR-CGH profile showing a duplication of the Xq27.2-q28 region. (c) The result of FISH with the wcpX probe. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] 802 Smyk et al. TABLE I. Summary of the Array CGH and FISH Results Location XLMR genes (distance from Xpter in Mb) Xq27.1 Xq27.2 Xq27.3 FMR1 (146.8) FMR2 (147.8) IDS (148.3) Xq28 ABCD1 (152.6) SLC6A8 (152.6) L1CAM (152.7) MECP2 (152.9) FLNA (153.2) GDI1 (153.3) IKBKG (153.4) DKC1 (153.6) Clone Patient 1 Patient 2 Distance from Xpter (Mb) FISH signal FISH signal Array CGH combined (log 2 ratio) FISH signal 139.0 141.1 141.9 142.7 142.9 143.0 143.5 143.9 144.2 144.7 144.9 145.3 145.6 145.7 145.8 146.0 146.2 146.4 146.8 147.7 148.8 148.9 148.9 152.5 152.8 153 153 154 154.4 154.7 1x 1x 1x 1x 1x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x n/a n/a n/a n/a n/a n/a 2x n/a n/a n/a n/a n/a 2x 2x 2x 2x 2x 2x 2x 2x 2x 2x 3x 3x 3x 3x 3x 3x 3x 3x n/a n/a n/a n/a n/a n/a 3x n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 0.039 0.483 0.371 0.379 n/a 0.045 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 2x n/a 2x 1x n/a n/a RP11-147G11 RP11-435F11 RP11-179F2 RP11-513H18 RP11-433F22 RP11-1091C11 RP11-99J24 RP11-1145K2 RP11-466E7 RP11-269F10 RP11-387H19 RP11-183K14 RP11-243C2 RP11-691H16 RP11-522P6 RP11-226B15 RP11-42J13 RP11-25E18 RP11-489K19 RP11-62J13 RP11-49C9 RP11-722C13 RP11-716M16 RP11-54I20 RP11-244I10 RP11-119A22 RP4-671D9 RP11-810M4 RP11-218L14 RP11-479B17 Patient 3 Highlighted in bold are results for the clones in excess copy-number. the proband’s DNA using array CGH revealed a duplication of two BAC clones: RP11-244I10, RP11-119A22, and one PAC clone RP4-671D9 (Fig. 4a), involving the entire MECP2 gene. FISH analysis confirmed the duplication. The proximal breakpoint of the duplication was mapped between BAC clones RP11-54I20 and RP11-244I10 and the distal breakpoint between clones RP4-671D9 and RP11-810M4 (Table I). The size of the duplication was estimated as approximately 0.3– 1.3 Mb. FISH examination of the maternal peripheral blood lymphocytes showed that the mother was a carrier of the duplication (Fig. 4b). Analysis of XCI status in the asympto- matic carrier mother showed an extremely non-random pattern with a ratio of 99:1. The karyotype of the patient was designated as 46,XY.arr cgh Xq28(RP11-244I10,RP4-671D9, RP11-119A22) 2 nuc ish dup(X)(q28q28)(RP11-244I10,RP4-671D9) 2 mat. The presence of the microduplication was confirmed also by MLPA (Fig. 4c). In addition to MECP2, the duplication also includes the IRAK1 and L1CAM genes (11 Kb and 160 Kb upstream to MECP2, respectively), and does not contain IDH3G, SLC6A8 (236 Kb and 507 Kb upstream to MECP2, respectively), or SYBL1 (1754 Kb downstream) (Fig. 4c). Fig. 3. The results of investigation in Patient 2. (a) Chromosomes X and der(Y) after GTG-banding. Note the additional material on the long arm of chromosome Y. (b) HR-CGH profile showing a duplication on the Xq27.3q28 region. (c) Metaphase chromosomes after FISH with clones RP11-226B15 (red) and RP11-470K20 (green); note that third red signal present on chromosome Y is faint, indicating that the breakpoint maps within RP11-226B15. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Functional Disomy and Trisomy of Xq28 in Males 803 Fig. 4. The results of investigation in Patient 3. (a) The result of array CGH. This combined profile represents two hybridizations performed simultaneously with dye reversal using reference DNA. There are three clones RP11-244I10, RP11-119A22, RP4-671D9 from Xq28 that showed displacement, indicating a gain of Xq material in the patient versus the reference DNA. (b) Interphase nucleus after FISH with RP4-671D9 (red) and RP11810M4 (green) showed that the mother of the proband is the carrier of the duplication. (c) MLPA raw data of a patient with the duplication (blue peaks) compared with a normal control (red peaks). The amount of PCR product for all MECP2 exons and IRAK1 and L1CAM genes is higher for probes located in the duplicated region. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] DISCUSSION Functional disomies involving chromosome Xq in males are rare and can lead to abnormal phenotype caused by either disruption of a gene at the breakpoint or by the increased dosage of a gene within the duplicated segment. The chromosome region Xq27.3-qter harbors several genes that have been shown to be responsible for syndromic and non-syndromic forms of X-linked MR (XLMR) (e.g., FMR1, IDS, ABCD1, L1CAM, MECP2, FLNA, IKBKG, and DKC1; and FMR2, GDI1, SLC6A8, and MECP2, respectively) [Ropers, 2006]. We report three unrelated male patients with abnormal phenotypes resulting from duplications of distal Xq. The Xq duplicated segments in our patients vary in size; however, in all three cases they encompass the L1CAM and MECP2 genes. Mutations in MECP2 give rise to a wide range of disorders including Rett syndrome (MIM 312750) [Amir et al., 1999; Williamson and Christodoulou, 2006], a progressive neurologic developmental disorder and one of the most common causes of MR in females; severe encephalopathy [Villard et al., 2000]; PPM-X syndrome (MR with psychosis, pyramidal signs, and macroorchidism) [Klauck et al., 2002]; progressive spasticity [Meloni et al., 2000]; non-syndromic XLMR [Orrico et al., 2000], and Angelman syndrome-like [Watson et al., 2001], and Prader–Willi syndrome-like phenotypes [Kleefstra et al., 2002]. Recently, 20 families with cryptic duplication and one case with submicroscopic triplication involving MECP2 have been reported [Ariani et al., 2004; Meins et al., 2005; Van Esch et al., 2005; Friez et al., 2006; Lugtenberg et al., 2006; del Gaudio et al., 2006]. In all cases, the duplicated segment ranged in size between 0.2 and 2.2 Mb. The common phenotypic features in these patients included infantile hypotonia, severe to profound MR, absence of speech development, recurrent infections, seizures, and progressive spasticity [Van Esch et al., 2005; Friez et al., 2006; Lugtenberg et al., 2006]. However, no typical facial dysmorphic features could be assigned to MECP2 duplication. Furthermore, no correlation between the size of the duplication and severity of the phenotype was noted; however, the patient with the triplicated MECP2 gene had the most severe phenotype [del Gaudio et al., 2006]. Except one case, female carriers of duplication were asymptomatic, like the mother of Patient 3 [Friez et al., 2006; del Gaudio et al., 2006]. The phenotypes observed in our patients as well as in two severely affected male cousins of Patient 3 overlap with most of the clinical features reported in patients with MECP2 duplication (Table II) [Van Esch et al., 2005; Friez et al., 2006; Lugtenberg et al., 2006]. In contrast to other MECP2 duplication cases, our Patient 3 had large testes that are also found in patients with the PPM-X syndrome; however, up to now no psychotic symptoms have been observed [Klauck et al., 2002]. Moreover, axial hypotonia, commonly found in patients with MECP2 duplication, was also present in Patients 1 and 2. The motor development was significantly delayed in all patients, especially in Patient 1 who has never walked. In addition, both progressive spasticity and epilepsy were present in Patient 1. However, no epilepsy and only subtle spasticity have been observed in Patient 2 (likely due to his young age). 804 Smyk et al. TABLE II. Comparison of Clinical Symptoms Observed in Our Patients and Previously Reported Cases With Different Sized Duplications of Xq Encompassing MECP2 Clinical features Developmental delay Hypotonia Growth retardation Absent or delayed speech Never walked or limited walking Spasticity Severe feeding difficulties Gastrointestinal reflux Recurrent infections Seizures Obesity Microcephaly Brachycephaly Asymmetric skull Facial hypotonia (expressionless face) Epicanthal folds Large ears Small/open mouth Digital abnormalities Genital abnormalities: including Hs, Cr, LT Duplications Xq27qterb Duplications Xq28qterc Interstitial duplications including MECP2 0.2–2.2 Mb in sized Pt 1 Pt 2 Pt 3 Duplications Xq26.3qtera þ þ þ þ þ þ þ þ þ þ þ þ þ þ 4/4 4/4 4/4 n.r. n.r. 8/8 8/8 8/8 5/7 5/7 5/5 5/5 3/5 4/5 5/5 43/43 26/29 1/1 39/40 20/33 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ n.r. 2/4 n.r. 4/4 1/4 n.r. 4/4 n.r. n.r. n.r. n.r. 4/6 1/3 7/9 1/7 n.r. 8/8 n.r. n.r. 1/2 1/2 2/2 1/2 2/2 3/3 n.r. 5/5 n.r. n.r. n.r. 16/20 15/28 13/16 32/39 22/41 n.r. 5/39 5/7 2/12 18/27 þ þ þ Cr þ þ þ Cr þ þ Cr, LT n.r. 1/3 3/3 3/4 4/4 4/5 4/5 3/3 5/8 6/8 n.r. 2/2 3/5 4/5 3/5 1/7 3/19 4/18 6/19 3/8 Hs, hypospadias; Cr, cryptorchidism; LT, large testes; n.r., not reported. a Akiyama et al. (2001); Vasquez et al. (1995); Kokalj-Vokac et al. (2004). b Goodman et al. (1998); Lammer et al. (2001); Lachlan et al. (2004); Novelli et al. (2004). c Lahn et al. (1994); Sanlaville et al. (2005). d Ariani et al. (2004); Meins et al. (2005); Van Esch et al. (2005); Friez et al. (2006); del Gaudio et al. (2006). Patient 3 has not manifested epilepsy; however, brain encephalography registration was abnormal. Interestingly, both Patients 1 and 2 had delayed brain myelination that was also observed in some of the previous patients with Xq duplications [Sanlaville et al., 2005; Van Esch et al., 2005]. Mutations in L1CAM, another gene located in the duplicated Xq region cause X-linked spastic paraplegia (MIM 312900), MASA syndrome (MIM 303350), and X-linked hydrocephalus due to stenosis of the aqueduct of Sylvius (HSAS; MIM 307000) (Jouet et al., 1994; Vits et al., 1994]. L1CAM has been proposed to be a dosage-sensitive gene that contributes to the MECP2 duplication phenotype; however, recently, two patients with duplications involving MECP2 but not L1CAM had the typical MECP2 duplication phenotype, thus likely disproving this hypothesis [Meins et al., 2004; Friez et al., 2006]. The recurrent infections in patients with Xq duplications have been stressed by many authors. This may result from the increased dosage of the IRAK1 or IKBKG genes contained in the duplicated region. IKBKG encodes the NF-kappa-B essential modulator (NEMO) protein which is a master regulator of proinflammatory responses [Nenci et al., 2007]. Mutations in IKBKG result in immunodeficiency. However, there are no reports on the causative role of duplication of the entire IKBKG gene. Moreover, recurrent infections have been also reported in patients that did not have increased dosage of IKBKG [Van Esch et al., 2005; del Gaudio et al., 2006]. The second candidate gene, IRAK1, is duplicated in all of the hitherto reported cases. IRAK1 encodes interleukin-1 receptor-associated kinase, also involved in proinflammatory cytokine response. This gene is partially responsible for IL1induced upregulation of the transcription factor NF-kappa-B. Interestingly, increased transcript level of Irak1 was reported in a mutant MeCP2-deficient mouse [Jordan et al., 2007]. In addition to MECP2, L1CAM, and IRAK1, the minimal common duplicated Xq region also contains AVPR2, ARHGAP4, ARD1A, RENBP, HCFC1, and TMEM187 genes. Recently, a patient with severe combined immunodeficiency and X-linked nephrogenic diabetes insipidus (NDI) carrying a microdeletion including AVPR2, ARHGAP4, and ARD1A has been described [Broides et al., 2006]. The highly conserved intragenic region between ARHGAP4 and ARD1A was not deleted in other patients with NDI but without immunodeficiency. To date, at least 19 patients with larger-sized duplications involving Xqter have been described [Lahn et al., 1994; Vasquez et al., 1995; Goodman et al., 1998; Lachlan et al., 2004; Novelli et al., 2004; Sanlaville et al., 2005]. In addition to clinical features described in patients with MECP2 duplication, microcephaly, growth retardation, abnormal palate/ maxillar alveolus, facial dysmorphism with a wide face, pointed nose and small mouth, feeding problems, and abnormal genitalia have been reported [Sanlaville et al., 2005]. Both our Patients 1 and 2 also presented with microcephaly, growth retardation, small mouth, cryptorchidism, and severe feeding problems in neonatal period. The duplicated segments in Patients 1 and 2 are, respectively, 12 and 9 Mb in size, and in addition to MECP2, harbor also the GDI1 gene, known to be responsible for the nonsyndromic XLMR [D’Adamo et al., 1998]. Recently, a duplication encompassing the GDI1, FLNA, and EMD genes has been reported in a family with three affected male patients with moderate MR, peculiar facial dysmorphism, and microcephaly and two asymptomatic female carriers [Madrigal et al., 2007]. Interestingly, in all male patients carrying the duplication, a significantly increased level of GDI1 mRNA was noted. Mutations in the FLNA gene cause bilateral periventricular nodular heterotopia (BPNH). This feature was also reported in a boy carrying a 2.25–3.25 Mb inverted duplication Functional Disomy and Trisomy of Xq28 in Males encompassing FLNA [Fink et al., 1997]. However, Lugtenberg et al.  reported duplications involving the GDI1 and FLNA genes in phenotypically normal males, suggesting that these are most likely nonpathogenic copy-number variations (CNVs). Supporting this notion, Patients 1 and 2 with duplication encompassing FLNA do not have BPNH, indicating that this feature may not be associated with the increased dosage of this gene. Patient 1 has also a 1 to 1.2 Mb deletion in Yp11.32, involving the pseudoautosomal homeobox SHOX gene. Haploinsufficiency of SHOX causes short stature and skeletal features in Turner syndrome [Rao et al., 1997] and Leri–Weill dyschondrosteosis (LWD; MIM 127300) [Belin et al., 1998]. This patient does not have any skeletal deformities typical for LWD; however, he has a short stature below 5th centile. Interestingly, the obesity present in Patient 1 was reported in one patient with an interstitial duplication involving Xq27.1q28 [Lachlan et al., 2004] but in none of the cases with terminal duplication. Patient 2 has a deletion on the Y chromosome encompassing the AZFc region with the DAZ3 gene. Deletions removing DAZ3/DAZ4 may have some effects on fertility [Ferlin et al., 2005]. However, this patient will most likely be infertile due to an abnormal sex chromosome complement, XXY. In summary, the increased dosage of MECP2 in our patients is likely responsible for most of their features including severe MR, hypotonia, absent or delayed speech, epilepsy, and spasticity. Recurrent infections can be caused by duplication of IRAK1. Other common abnormalities described in Patients 1 and 2 including microcephaly, growth retardation, facial dysmorphism with a round face and full cheeks in neonatal period, short nose, small mouth, large and prominent simple ears, and feeding problems are not specific for MECP2 and may result from increased dosage of other genes mapping in the duplicated regions. Our data indicate that in each case of MR in males, a possibility of duplication Xq28 has to be considered, especially when hypotonia, absent/delayed speech, and recurrent infections are observed. ACKNOWLEDGMENTS We thank Sandra Peacock for helpful discussion. Supported in part by grants from the Polish Ministry of Scientific Research and Information Technology Grants 2P05A 191-29, 40101731/0307, PBZ/KBN 122/P05/2004/01-9, and 2P05A 128 28. 805 Cheung SW, Shaw CA, Yu W, Li J, Ou Z, Patel A, Yatsenko SA, Cooper ML, Furman P, Stankiewicz P, Lupski JR, Chinault CA, Beaudet AL. 2005. Development and validation of a CGH microarray for clinical cytogenetic diagnosis. Genet Med 7:422–432. D’Adamo P, Menegon A, Lo Nigro C, Grasso M, Gulisano M, Tamanini F, Bienvenu T, Gedeon AK, Oostra B, Wu SK, Tandon A, Valtorta F, Balch WE, Chelly J, Toniolo D. 1998. Mutations in GDI1 are responsible for Xlinked non-specific mental retardation. Nat Genet 19:134–139 (erratum in: Nat Genet 19:303). del Gaudio D, Fang P, Scaglia F, Ward PA, Craigen WJ, Glaze DG, Neul JL, Patel A, Lee JA, Irons M, Berry SA, Pursley AA, Grebe TA, Freedenberg D, Martin RA, Hsich GE, Khera JR, Friedman NR, Zoghbi HY, Eng CM, Lupski JR, Beaudet AL, Cheung SW, Roa BB. 2006. Increased MECP2 gene copy number as the result of genomic duplication in neurodevelopmentally delayed males. Genet Med 8:784–792. Ferlin A, Tessari A, Ganz F, Marchina E, Barlati S, Garolla A, Engl B, Foresta C. 2005. Association of partial AZFc region deletions with spermatogenic impairment and male infertility. J Med Genet 42:209–213. Fink JM, Dobyns WB, Guerrini R, Hirsch BA. 1997. Identification of a duplication of Xq28 associated with bilateral periventricular nodular heterotopia. Am J Hum Genet 61:379–387. Friez MJ, Jones JR, Clarkson K, Lubs H, Abuelo D, Bier JA, Pai S, Simensen R, Williams C, Giampietro PF, Schwartz CE, Stevenson RE. 2006. Recurrent infections, hypotonia, and mental retardation caused by duplication of MECP2 and adjacent region in Xq28. Pediatrics 118: e1687–e1695. Gecz E, Baker E, Donnelly A, Ming JE, McDonald-McGinn DM, Spinner NB, Zackai EH, Sutherland GR, Mulley JC. 1999. Fibroblast growth factor homologous factor 2 (FHF2): Gene structure, expression and mapping to the Börjeson–Forssman–Lehmann syndrome region in Xq26 delineated by a duplication breakpoint in BFLS-like patient. Hum Genet 104:56– 63. Goodman BK, Shaffer LG, Rutberg J, Leppert M, Harum K, Gagos S, Ray JH, Bialer MG, Zhou X, Pletcher BA, Shapira SK, Geraghty MT. 1998. Inherited duplication Xq27-qter at Xp22.3 in severely affected males: Molecular cytogenetic evaluation and clinical description in three unrelated families. Am J Med Genet 80:377–384. Jordan C, Li HH, Kwan HC, Francke U. 2007. Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets. BMC Med Genet 8:36. Jouet M, Rosenthal A, Armstrong G, MacFarlane J, Stevenson R, Paterson J, Metzenberg A, Ionasescu V, Temple K, Kenwrick S. 1994. X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene. Nat Genet 7:402–407. Kirchhoff M, Rose H, Maahr J, Gerdes T, Bugge M, Tommerup N, Tumer Z, Lespinasse J, Jensen PK, Wirth J, Lundsteen C. 2000. High resolution comparative genomic hybridisation analysis reveals imbalances in dyschromosomal patients with normal or apparently balanced conventional karyotypes. Eur J Hum Genet 8:661–668. REFERENCES Klauck SM, Lindsay S, Beyer KS, Splitt M, Burn J, Poustka A. 2002. A mutation hot spot for nonspecific X-linked mental retardation in the MECP2 gene causes the PPM-X syndrome. Am J Hum Genet 70:1034– 1037. Akiyama M, Kawame H, Ohashi H, Tohma T, Ohta H, Shishikura A, Miyata I, Usui N, Eto Y. 2001. Functional disomy for Xq26.3-qter in a boy with an unbalanced t(X;21)(q26.3;p11.2) translocation. Am J Med Genet 99:111–114. Kleefstra T, Yntema HG, Oudakker AR, Romein T, Sistermans E, Nillessen W, van Bokhoven H, de Vries BB, Hamel BC. 2002. De novo MECP2 frameshift mutation in a boy with moderate mental retardation, obesity and gynaecomastia. Clin Genet 61:359–362. Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. 1992. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet 51:1229–1239. Kokalj-Vokac N, Marcun-Varda N, Zagorac A, Erjavec-Skerget A, Zagradisnik B, Todorovic M, Gregoric A. 2004. Subterminal deletion/ duplication event in an affected male due to maternal X chromosome pericentric inversion. Eur J Pediatr 163:658–663. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. 1999. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188. Lachlan KL, Collinson MN, Sandford ROC, van Zyl B, Jacobs PA, Thomas NS. 2004. Functional disomy resulting from duplications of distal Xq in four unrelated patients. Hum Genet 115:399–408. Ariani F, Pescucci C, Longo I, Bruttini M, Meloni I, Hayek G, Rocchi R, Zappella M, Renieri A. 2004. Real-time quantitative PCR as a routine method for screening large rearrangements in Rett syndrome: Report of one case of MECP2 deletion and one case of MECP2 duplication. Hum Mutat 24:172–177. Lahn BT, Ma N, Breg WR, Stratton R, Surti U, Page DC. 1994. Xq-Yq interchange resulting in supernormal X-linked gene expression in severely retarded males with 46, XYq-karyotype. Nat Genet 8:243– 250. Belin V, Cusin V, Viot G, Girlich D, Toutain A, Moncla A, Vekemans M, Le Merrer M, Munnich A, Cormier-Daire V. 1998. SHOX mutations in dyschondrosteosis (Leri–Weill syndrome). Nat Genet 19:67–69. Broides A, Ault BH, Arthus MF, Bichet DG, Conley ME. 2006. Severe combined immunodeficiency associated with nephrogenic diabetes insipidus and a deletion in the Xq28 region. Clin Immunol 120:147–155. Lammer EJ, Punglia DR, Fuchs AE, Rowe AG, Cotter PD. 2001. Inherited duplication of Xq27.2 ! qter: Phenocopy of infantile Prader–Willi syndrome. Clin Dysmorphol 10:141–144. Lugtenberg D, de Brouwer APM, Kleefstra T, Oudakker AR, Frints SGM, Schrander-Stumpel CTRM, Fryns JP, Jensen LR, Chelly J, Moraine C, Turner G, Veltman JA, Hamel BCJ, de Vries BBA, van Bokhoven H, Yntema HG. 2006. Chromosomal copy number changes in patients with 806 Smyk et al. non-syndromic X linked mental retardation detected by array CGH. J Med Genet 43:362–370. Madrigal I, Rodriguez-Revenga L, Badenas C, Sanchez A, Martinez F, Fernandez I, Fernandez-Buriel M, Mila M. 2007. MLPA as first screening method for the detection of microduplications and microdeletions in patients with X-linked mental retardation. Genet Med 9: 117–122. Meins M, Lehmann J, Gerresheim F, Herchenbach J, Hagedorn M, Hameister K, Epplen JT. 2005. Submicroscopic duplication in Xq28 causes increased expression of the MECP2 gene in a boy with severe mental retardation and features of Rett syndrome. J Med Genet 42:e12. Meloni I, Bruttini M, Longo I, Mari F, Rizzolio F, D’Adamo P, Denvriendt K, Fryns JP, Toniolo D, Renieri A. 2000. A mutation in the rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am J Hum Genet 67:982–985. Nenci A, Becker C, Wullaert A, Gareus R, van Loo G, Danese S, Huth M, Nikolaev A, Neufert C, Madison B, Gumucio D, Neurath MF, Pasparakis M. 2007. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446:557–561. Sanlaville D, Prieur M, de Blois M-C, Genevieve D, Lapierre J-M, Ozilou C, Picq M, Gosset P, Morichon-Delvallez N, Munnich A, Cormier-Daire V, Baujat G, Romana S, Vekemans M, Turleau C. 2005. Functional disomy of the Xq28 chromosome region. E J Hum Genet 13:579–585. Schmidt M, Du Sart D, Kalistis P, Leversha M, Dale S, Sheffield L, Toniolo D. 1991. Duplication of the X chromosome in males: Evidence that most parts of the X chromosome can be active in two copies. Hum Genet 86:519–521. Shaffer LG, Kennedy GM, Spikes AS, Lupski JR. 1997. Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: Implications for testing in the cytogenetics laboratory. Am J Med Genet 69:325– 331. Van Esch H, Bauters M, Ignatius J, Jansen M, Raynaud M, Hollanders K, Lugtenberg D, Bienvenu T, Jensen LR, Gécz J, Moraine C, Marynen P, Fryns J-P, Froyen G. 2005. Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males. Am J Hum Genet 77:442–453. Vasquez AI, Rivera H, Bobadilla L, Crolla JA. 1995. A familial Xpþ chromosome, dup (Xq26.3 ! qter). J Med Genet 32:891–893. Novelli A, Bernardini L, Salpietro DC, Briuglia S, Merlino MV, Mingarelli R, Dallapiccola B. 2004. Disomy of distal Xq in males. Am J Med Genet Part A 128A:165–169. Villard L, Kpebe A, Cardoso C, Chelly PJ, Tardieu PM, Fontes M. 2000. Two affected boys in a Rett syndrome family: Clinical and molecular findings. Neurology 55:1188–1193. Orrico A, Lam C, Galli L, Dotti MT, Hayek G, Tong SF, Poon PM, Zappella M, Federico A, Sorrentino V. 2000. MECP2 mutation in male patients with non-specific X-linked mental retardation. FEBS Lett 481:285–288. Vits L, Van Camp G, Coucke P, Fransen E, De Boulle K, Reyniers E, Korn B, Poustka A, Wilson G, Schrander-Stumpel C, Winter RM, Schwartz C, Willems PJ. 1994. MASA syndrome is due to mutations in the neural cell adhesion gene L1CAM. Nat Genet 7:408–413. Rao E, Weiss B, Fukami M, Rump A, Niesler B, Mertz A, Muroya K, Binder G, Kirsch S, Winkelmann M, Nordsiek G, Heinrich U, Breuning MH, Ranke MB, Rosenthal A, Ogata T, Rappold GA. 1997. Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome. Nat Genet 16:54–63. Ropers HH. 2006. X-linked mental retardation: Many genes for a complex disorder. Curr Opin Genet Dev 16:260–269. Watson P, Black G, Ramsden S, Barrow M, Super M, Kerr B, Clayton-Smith J. 2001. Angelman syndrome phenotype associated with mutations in MECP2, a gene encoding a methyl CpG binding protein. J Med Genet 38:224–228. Williamson SL, Christodoulou J. 2006. Rett syndrome: New clinical and molecular insights. Eur J Hum Genet 14:896–903.