X chromosome inactivation (XCI) is the epigenetic mechanism through which mammalian cells achieve gene dosage compensation between male (XY) and female cells (XX). Most X-linked genes are therefore expressed in a similar level in females as in males. Functional disomy (FD) is the double expression of X-linked genes compared to their normal level. In males, all structural duplications lead to FD. In females, the X structural imbalances may lead to a variable phenotype depending on the X-inactivation pattern.

X-inactivation of one X chromosome occurs early in female development and is initiated from the X-Inactivation Centre (XIC), located in Xq13 on the proximal part of the long arm, and that contains the XIST gene. As the choice of the inactive X is a random process, each female is a functional mosaic for two cell populations 6. The number of cells that have inactivated the same X chromosome follows a normal distribution around a mean of 50:50. In most studies, skewing and extreme skewing are arbitrarily defined as greater than or equal to 75%, and greater than or equal to 90% of cells expressing the same X chromosome, respectively. The later value is usually retained as significant for morbid manifestations. Structural abnormalities of the X chromosome such as large deletions, duplications, and unbalanced X/autosome translocations result in skewed patterns of inactivation, with the abnormal X chromosome being inactive in most or all cells 7. Conversely, in about 95% of balanced t(X;A), the normal X chromosome is inactivated in all cells 8. This pattern preserves the balance of expression from the X and from the autosome (Figure 2b). In some cases, the derivative containing XIC is inactivated. Consequently, these cells are functionally disomic for a part of the X chromosome. The presence of disomic cells was highly prevalent in translocations with breakpoints at Xp22 and Xq28, even though spreading of X inactivation onto the adjacent autosomal segment was noted in most of these cases 89. This pattern of inactivation is usually associated with an abnormal phenotype. The outcome of the selection process against the functional disomy X seems to be the major determinant of the clinical status in most patients with balanced X-autosome translocations 10.

It is noteworthy that more than 15% of genes on the human X chromosome, including many without functional equivalent on the Y, escape from XCI. They are mainly located in the pseudo-autosomal regions at each end of the X chromosome but a small number of them are found outside these regions 11.

A large number of disease conditions have been associated with the X chromosome because the phenotypic consequences of a recessive mutation are revealed directly in the hemizygous male. The X chromosome contains approximately 1,100 genes and is associated with over 300 Mendelian conditions. Both the X and Y chromosomes have a remarkable enrichment of genes involved in gonadogenesis and gametogenesis. X-linked mental retardation (XLMR) is a common cause of inherited intellectual disability with an estimated prevalence of about 1/1,000 males 12. In a recent review, Chiurazzi et al. listed 215 XLMR conditions including 149 with specific clinical findings of which in 82 the causal MRX gene has been identified. Among these genes, 47 were localised on the long arm 13. Besides the FMR1 gene responsible for the fragile X mental retardation syndrome, the chromosomal region Xq27.3-qter harbours several genes that have been shown to be responsible for syndromic and non-syndromic forms of XLMR (e.g. IDS, ABCD1, L1CAM, MECP2, FLNA, IKBKG, DKC,1 FMR2, GDI1, SLC6A8, and MECP2) 14. This region is at high risk for genomic instability and several of these genes are implicated in genomic disorders due to nonallelic homologous recombination between low copy repeats 15.

Consequences of over-expression of X-linked genes is not well known, with the exception of the PLP1 gene (whose duplication is responsible for the Pelizaeus-Merzbacher syndrome (OMIM 312080)), and, more recently, the MECP2 gene. MECP2 selectively binds to methylated DNA and mutations in the MECP2 gene cause the autism-spectrum neurodevelopmental disorder Rett syndrome (OMIM 312750). Its primary role was thought to form a link between DNA methylation, histone acetylation and co-repressor molecules. Evidence is now accumulating for a more expanded role of MECP2 as a multifunctional protein involved in the modulation of chromatin structure by different mechanisms. The MECP2 gene is expressed in many tissues, but expression is highest in the brain. During development, MECP2 expression is very low or absent in immature neurons, then it increases during neuronal maturation and is highest in post-mitotic post migratory neurons. Mild over-expression of wild-type MECP2 protein induced neurodevelopmental abnormalities in transgenic mice, and duplication of the MECP2 gene causes mental retardation in human males as discussed below.

Distal Xq duplications are the more frequently reported. In particular, the Xq26–q28 chromosome region yields a recognisable phenotype including distinctive facial features, major axial hypotonia, severe developmental delay, severe feeding difficulties, abnormal genitalia and proneness to infections 245161718. In details, these patients present:

- Growth: prenatal and postnatal growth retardation, microcephaly

- Dysmorphism: premature closure of the fontanels or ridged metopic suture, brad face with full cheeks, epicanthal folds, large ears, small and open mouth, ear anomalies, pointed nose, abnormal palate and facial hypotonia are the most common facial features

- Psychomotor retardation: severe mental retardation, absence or severely retarded speech

- Major axial hypotonia

- Spasticity

- Malformations: genitalia malformations including hypoplasic genitalia, hypospadias and/or cryptorchidism are the more frequent malformations

- Abnormal fingers and toes have been reported

- Others frequent clinical manifestations: severe feeding difficulties with gastro oesophageal reflux, excessive drooling, seizures, recurrent infections.

Proximal Xq duplications are more rarely reported. They represent an heterogeneous group of patients with variable breakpoints, most of them studied only by banding karyotyping, with some recent exceptions 119. Patients show craniofacial dysmorphism, brain and neurologic abnormalities such as aberrant brain myelination, hypotonia, mental, psychomotor and growth retardation, feeding issues, hypoplasic genitalia.

Table 1 presents clinical findings found in three groups of patients, namely Xq12q24 duplications, Xq26-qter duplications (or functional disomy), and MECP2 interstitial microduplications.

Several clinical symptoms are common to the three groups, despite different gene content. However, these clinical findings are often non-specific, such as hypotonia, psychomotor delay, feeding problems and genital hypoplasia. Recurrent respiratory infections, especially recurrent pneumonia, help to distinguish Xq28 functional disomy (including MECP2 duplication) from other XLMR-hypotonia syndromes. The recurrent infections might result from the increased dosage of the IRAK1 or IKBKG genes generally present in the duplicated region 20.

Most Xq duplications observed in males are inherited from a mother with normal or near normal phenotype. Less frequently, Xq duplications may be found by karyotyping in manifesting females studied because of mental retardation. The most frequent manifestations found in these patients are short stature, developmental delay, facial dysmorphism and gonadal dysgenesis 2122. Body asymmetry may be present corresponding to functional mosaicism. In the manifesting females, the X-inactivation pattern is usually at random and this explains the clinical manifestations. In rare cases, a favourable skewed X-inactivation is observed. For these cases, other explanations such as local escape from inactivation, expression of recessive genes from the active X, or disruption of a gene by the rearrangement have been suggested to explain the abnormal phenotype 2324. Also a critical region for gonadal dysgenesis has been suggested to be present in Xq13q26 25. For duplications resulting from unbalanced t(X;A), the X-translocated segment separated from its corresponding XIC cannot be inactivated, and abnormal phenotype is fully expressed. In this rare situation, a phenotype as severe as in males is observed in female patients, with microcephaly, seizures and severe mental retardation 5.

Small duplications detected using array comparative genomic hybridization (array CGH) and encompassing only one or a few genes allow description of some more specific phenotypes due to over-expression of individual genes. The most striking example is the MECP2 gene which prove to be the major gene implicated in Xq26-qter duplications.

Recently, array CGH and quantitative polymerase chain reaction (PCR) allowed detection of submicroscopic Xq28 interstitial duplications. These microduplications are variable in size, ranging from 0.2 to 2.2 Mb, but consistently include MECP2 and L1 cell adhesion molecule (L1CAM), as well as intervening genes 26. To date, 47 affected individuals from 24 different families have been reported 20262728293031. The patients manifested severe mental retardation, absent or limited speech, progressive neurologic problems (such as spasticity and seizures), axial and facial hypotonia, mild and non-specific facial dysmorphism, and severe recurrent respiratory infection overlapping with characteristics described in patients with larger Xq27–q28 terminal duplications 2630.

Collective data of previous studies suggest that increased MECP2 gene copy number is mainly responsible for the neurodevelopmental phenotypes in these patients 20262930. Moreover, mice with over-expression of mecp2 have severe motor dysfunction 32 and progressive neurologic decline 33. In addition, Del Gaudio et al. found MECP2 triplication in one patient with a more severe phenotype 30. Additionally, a non-pathogenic duplication of Xq28 that does not include the MECP2 gene has recently been reported 34.

In 2005, Stankiewicz et al. reported a family in which five females presented with short stature, speech and language problems, hearing impairment, and several dysmorphic features associated with a 7.5-Mb duplication of Xq26.2–q27.1 that encompassed or disrupted the SOX3 gene 35. SOX3 gene has previously been suggested as a candidate gene in a female with moderate to severe mental retardation, seizures and hypothyroidism and del(X)(q26.3–q27.3) 36. At the same time, Woods et al. identified a 685 kb submicroscopic duplication of Xq27.1 containing the SOX3 gene in two maternal half males sibs with X-linked panhypopituitarism 37. Brain magnetic resonance imaging (MRI) showed anterior pituitary hypoplasia, ectopic posterior pituitary, and absent infundibulum. They concluded that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily mental retardation.

Recently, using oligonucleotide array CGH, Bedeschi et al. reported a familial chromosome duplication spanning about 800 kb of genomic DNA encompassing the YIPF6, STARD8 and OPHN1 genes. The male proband had microcephaly, a distinct facial appearance, severe mental retardation and language impairment 38. OPHN1 mutations have been reported in patients with moderate to severe mental retardation, myoclonic-astatic epilepsy, ataxia, cerebral hypoplasia strabismus and hypogenitalism 39. The observation of Bedeschi et al., stressed the interest of high resolution array CGH to delineate new clinical phenotype secondary to small chromosome abnormalities, in particular duplication.

Microduplications encompassing the PLP1 gene have also been reported with a clinical phenotype evocative of Pelizaeus-Merzbacher disease (PMD) 40. Pelizaeus-Merzbacher disease (OMIM 312080) is a very rare X-linked recessive inherited leukodystrophy with prevalence estimated at 1 out of 400,000 birth. The disease occurs as an early motor development impairment marked by hypotonia associated with nystagmus, ataxochoreic movements of the axis and limbs, especially during the first 2 years of life. Symptoms often progress slowly until adolescence. PMD is caused by mutation in the gene encoding the proteolipid protein-1 (PLP1) gene. Complete duplication of the PLP1 gene on Xq22 is the cause of 60–70% of PMD cases, whereas deletions of this gene as well as point mutations in coding or splice site regions are involved in most of the remaining cases 41.

In addition, Froyen et al., using array CGH, reported two other small Xq duplications. The first one is a duplication of about 0,8 Mb at Xq22 containing 15 annotated genes. They proposed the NXF5 gene, including in the duplicated region, to explain mental retardation. In fact, increased dosage of NXF5 might disturb storage levels of particular mRNA in neurons. Nevertheless, expression levels of NXF5 mRNA in the patient compared to controls could not be investigated. The second, is a small 0,3 Mb duplication harbouring 12 known genes including the XLMR genes FLNA and GDI1 suggesting a dosage-dependant role for at least one of both genes 42.

Intrachromosomal duplications are the main cause of functional disomy in males. In most cases, they are inherited and transmitted in families through non-manifesting mothers. They are very variable in size, location and, therefore, in gene content. Before the use of array CGH, only cytogenetically visible duplications of at least 5–10 Mb in size were diagnosed. Duplications encompassing the X-inactivation centre are subject to inactivation, as in the rare variant of Klinefelter syndrome with isochromosome Xq 43. An unusual type of intrachromosomal duplication with the Xq26.3 or Xq27 ->qter region translocated to the Xp22.3 band is observed in some cases. It is usually inherited or may derive from a recombination within a maternal pericentric inversion. CGH array allowed detection of far more smaller duplications that may encompass only one or few genes, or even be asymptomatic 42.

In females, intrachromosomal duplications of X chromosome are generally associated with a skewed inactivation pattern biased towards the duplicated X chromosome leading to a normal or near normal phenotype (Figure 1c), and they are detected through abnormal offspring. However, rare cases with a random X inactivation ratio have also been described in female patients with mental retardation/multiple congenital anomalies (MR/MCA) syndromes 44.

Recombination between genomic repeats is a possible mechanism for the origin of the non-recurrent duplications. Giglio et al. described four der(X) chromosomes and proposed recombination between inverted repeat sequences during paternal meiosis as mechanism of origin 45. However, non-recurrent aberrations can result from different non-exclusive recombination-repair mechanisms. The origin and mechanisms underlying some of the Xq duplications have been investigated in cloning their breakpoints. Bauters et al. have analysed the breakpoints of 16 unique microduplications containing MECP2 and shown that none of these duplications are the result of NAHR. They demonstrated non-homologous end joining (NHEJ) as the mechanism in one of them, and a complex two-step rearrangement involving breakage-induced replication with strand invasion of the normal chromatid in another one 46. In Pelizaeus-Merzbacher disease, complex non-recurrent rearrangements are observed that are likely to be caused by a replication mechanism involving template switching 47.

Duplications resulting from an unbalanced form of X-Y [t(X;Y)] or X-autosome translocations [t(X;A)] are more rarely reported, with imbalance limited to the distal portion of Xq.

About six cases of t(X;Y) have been reported in severely retarded males with non-fluorescent Y chromosome and translocation of about 10 Mb of Xqter onto Yq. All cases occurred de novo 52042.

X-autosome translocations [t(X;A)] are rare rearrangements estimated to occur in 1 to 3/10,000 live births 89. Xq duplications resulting from unbalanced t(X;A) have been observed in males and in females for small terminal Xq segments translocated onto an acrocentric short arm, or very distally onto another chromosome arm. In this type of t(X;A), the autosomal imbalance is absent or very limited, and does not impact on the phenotype. In females, the X chromosome segment, separated from the X-inactivation centre in cis, cannot be inactivated and this result in functional disomy whatever the inactivated X. Theoretically, some balanced t(X;A) may result in Xq functional disomy if the derivative X is inactivated. In any case, parental karyotypes are required to eliminate an inherited translocation.

Small X ring chromosomes [r(X)] lacking functional XIC fail to be inactivated. Depending on their gene content, they may be responsible for FD resulting in a more severe phenotype than Turner syndrome, with severe mental retardation 4849.

The patients are usually investigated for mental retardation and a karyotype is performed using banding techniques that detect cytogenetically visible structural chromosome rearrangement such as large duplications, or X ring chromosomes. In most cases, the karyotype resolution is not sufficient, and Xq duplication will be detected applying additional techniques.

DNA techniques such as quantitative PCR (qPCR), multiplex ligation-dependent probe amplification (MLPA) or QM-PSF (quantitative multiplex PCR of short fluorescent) can be used to detect a targeted Xq duplication, in particular for the MECP2 or the PLP1 locus.

FISH (Fluorescence in situ hybridisation) can be used after an initial diagnosis done by karyotype, and selection of a specific probe. Nevertheless, diagnosis of duplication using FISH could be difficult, in particular in prenatal setting. Therefore, quantitative methods are often much better ways of detecting small duplications.

Today, CGH array is rapidly becoming the method of choice to detect small genomic imbalances 50. This technique can detect small duplications (few kb) as well as large duplications (several Mb) allowing description of more specific phenotypes such as MECP2 duplication phenotype.

Since 30% more males than females are diagnosed with mental retardation, full coverage X chromosome CGH-arrays have been developed for the detection of copy number alterations in patients with suspected X-linked mental retardation (XLMR) 51. Froyen at al. recently reported a cohort of 108 patients screened using a tiling X-chromosome-specific genomic array with a theoretical resolution of 80 kb and detected a total of 15 copy number changes in 14 patients (13%) that included two deletions and 13 duplications ranging from 0.1 to 2.7 Mb harbouring the CDKL5, NXF5, MECP2, and GDI1 genes 42. Also, Madrigal et al. detected 26 copy number variants (CNVs) on 52 patients using X-chromosome tiling path array 31. For 8 patients (14,8%) they identified imbalances probably causative of the phenotype observed in the patients. In particular, they reported one patient with Xq12 deletion, one patient with Xq12 duplication and two patients with Xq28 duplication. The genomic size of chromosome rearrangement ranged between 82 to 700 kb.

Whole genome CGH arrays with variable resolution are more generally used to study patients with unexplained MR/MCA and may lead to an increasing number of Xq duplication diagnosis.

X-inactivation studies are required in carrier females. In the prenatal period and in infants, it may help to the prognosis. For small duplications of uncertain significance found by CGH array, a random inactivation in an asymptomatic carrier mother argues against a pathogenic effect of this duplication. X-inactivation studies are most often done using a PCR protocol based on the methylation status of X chromosomes 52. Alternatively, late replication assay by cytogenetic methods using BrdU incorporation distinguishes the two X chromosomes according to their morphology (normal or rearranged).

Prader-Willi syndrome (OMIM 176270) affects 1 out of 25,000 births. It is characterised by two periods: I) neonatal period with severe hypotonia, characteristic facial features, feeding difficulties, hypogenitalism, and small hands and feet; ii) infancy and adulthood period: hyperphagia with a risk of morbid obesity, learning difficulties and behavioural problems. The expert consensus is that diagnosis should be based on clinical criteria (Holm's criteria of 1993, revised in 2001 53). PWS is caused by anomalies involving the critical region of chromosome 15 (15q11–q13). The syndrome results from absence of paternally active gene expression at the 15q11q13 chromosomal region 53. Phenotypic features of PWS have previously been reported in association with distal Xq duplication 21754 and interstitial Xq duplication 19. It has been suggested that proximal Xq duplications can result in features typically seen in children and adults with PWS (obesity and hypogonadism), and duplications distal to Xq25 to the infantile features of PWS in males (severe hypotonia, feeding difficulties and hypogenitalism) 19.

X-linked alpha thalassaemia mental retardation (ATR-X) syndrome (OMIM 301040) presents with many of the clinical features found in patients with Xq duplication: males with profound developmental delay (language is usually very limited), facial dysmorphism, genital abnormalities (in 80% of children, ranging from undescended testes to ambiguous genitalia). In this syndrome alpha thalassaemia is occasionally observed. This syndrome is X-linked recessive and results from mutations in the ATRX gene 55.

The majority of Xq duplications are inherited from a heterozygous mother 56. Most heterozygous females show extreme to complete skewing of X chromosome inactivation and thus are asymptomatic. The risk for siblings depends on the carrier status of the mother.

If the mother is a carrier, the risk of transmitting the duplication is 50% for each pregnancy (Figure 1). All XY sons will be affected, daughters will be usually asymptomatic heterozygous carriers, but may be more or less severely affected depending on their X-inactivation pattern.

If the duplication had arisen de novo, the recurrence risk for siblings is not significantly increased when compared with that of the general population. However, prenatal diagnosis may be counselled because cryptic or germinal mosaicism may be present in the mother.

Here again, the risk for siblings depends on the carrier status of the parents, with a majority of cases occurring de novo. Translocation of a small segment of Xq onto Yq results in all cases in FD and always occurs de novo. X;A translocations are frequently associated with infertility, but may be sometimes present in a balanced form in the mother. In this specific situation, there is a substantial risk of having abnormal offspring due to an unbalanced chromosomal constitution. In unbalanced X;A translocations, X-inactivation of the X segment translocated onto the autosome cannot occur, in females as in males (Figure 2c). The balanced form of the translocation may also be transmitted to daughters with possible detrimental effect due to an unfavourable pattern of inactivation. This requires evaluation of each individual case by a specialist.

Chromosomal rearrangement can be detected by amniocentesis or chorionic villus sampling and cytogenetic testing including FISH and/or DNA quantification as performed in the propositus. The most frequent situation is that of an intrachromosomal duplication inherited from the mother. Prenatal testing may also be done after diagnosis of a de novo case to exclude germinal mosaicism. For girls receiving the duplication, the phenotype depends on X-inactivation pattern. X-inactivation study in prenatal period is possible, with cautious interpretation. For pregnancies in which the mother has been identified as a carrier of an interstitial MECP2 duplication, the usual procedure should be used: determination of foetal sex and testing for the MECP2 duplication in male. For t(X;A), careful examination of individual situation is required.

Large Xq duplications could be diagnosed by chance after foetal karyotyping for any reason. No antenatal ultrasound signs specific to Xq duplications have been described.