Association of structural and numerical anomalies of chromosome 22 in a patient with syndromic intellectual disability

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Association of structural and numerical anomalies of chromosome 22 in a patient with syndromic intellectual disability

Rania Naoufal

^a^,^*

, Marine Legendre

^a^,^b

, Dominique Couet

^a^,^b

,

Brigitte Gilbert-Dussardier

^a^,^b

, Alain Kitzis

^a^,^b

, Frederic Bilan

^a^,^b

, Radu Harbuz

^a^,^b

aService de Genetique, Centre de Reference Anomalies du Developpement Ouest, CHU de Poitiers, France

bEA 3808, Universite de Poitiers, France

a r t i c l e i n f o

Article history:

Received 1 February 2016 Received in revised form 2 June 2016

Accepted 17 July 2016 Available online 21 July 2016 Keywords:

DD/ID CGH

Mosaic trisomy 22

22q13 microdeletion syndrome

a b s t r a c t

Array comparative genomic hybridization (aCGH) is now widely adopted as aﬁrst-tier clinical diagnostic test for patients with developmental delay (DD)/intellectual disability (ID), autism spectrum disorders, and multiple congenital anomalies. Nevertheless, classic karyotyping still has its impact in diagnosing genetic diseases, particularly mosaic cases.

We report on a 30 year old patient with syndromic intellectual disability, a 22q13.2 microdeletion and mosaic trisomy 22. The patient had the following clinical features: intrauterine growth retardation at birth, hypotonia, cryptorchidism, facial asymmetry, enophthalmus, mild prognathism, bifid uvula, hy- poplastic upper limb phalanges, DD including speech delay, and ID. Whole genome aCGH showed ade novo1 Mb interstitial heterozygous deletion in 22q13.2, confirmed byfluorescence in situ hybridization in all cells examined. Moreover, 18% cells had an extra chromosome 22 suggesting a trisomy 22 mosaicism.

Almost all 22q13 deletions published so far have been terminal deletions with variable sizes (100 kb to over 9 Mb). Very few cases of interstitial 22q13.2 deletions were reported. In its mosaic form, trisomy 22 is compatible with life, and there are about 20 reports in the literature. It has a variable clinical pre- sentation: growth restriction, dysmorphic features, cardiovascular abnormalities, hemihyperplasia, genitourinary tract anomalies and ID. Neurodevelopmental outcome ranges from normal to severe DD.

The patient presents clinical features that are common to both the interstitial 22q13 deletion and the mosaic trisomy 22; characteristics related to the interstitial deletion alone and others explained solely by the mosaic trisomy.

Our case points out the role of conventional cytogenetic tools in mosaic cases that could be missed by microarray technology. We therefore suggest the combination of both conventional and molecular karyotyping in the investigation of certain genetic diseases.

1. Introduction

Developmental delay/Intellectual disability (DD/ID) affects about 2e3% of the general population (Shinawi and Cheung, 2008).

We use the term syndromic intellectual disability when it is associated to a certain clinical phenotype, to dysmorphic features and/

or multiple congenital anomalies (MCA). It is reported that up to half the identiﬁed causes of DD/ID are due to a genetic etiology ranging from chromosomal aneuploidies and genetic

rearrangements to monogenic disorders (Srour and Shevell, 2014).

Array comparative genomic hybridization (aCGH) is now widely adopted as aﬁrst-tier diagnostic test in the investigation of isolated as well as syndromic ID (Vulto-van Silfhout et al., 2013). The available platforms have improved tremendously and increased their technical sensitivities in such a way that they henceforth offer a diagnostic yield as high as 20% (Vulto-van Silfhout et al., 2013).

Nevertheless, classic karyotyping still has its impact in diagnosing genetic diseases, particularly mosaic imbalances. In this report, we present a patient with syndromic intellectual disability and two genetic anomalies on chromosome 22, one structural and one numerical.

*Corresponding author.

E-mail address:[email protected](R. Naoufal).

Contents lists available atScienceDirect

European Journal of Medical Genetics

j o u r n a l h o me p a g e :h t t p : / / w w w . e l s e v i e r . c o m/ l o ca t e / e j m g

http://dx.doi.org/10.1016/j.ejmg.2016.07.001

European Journal of Medical Genetics 59 (2016) 483e487

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2. Clinical report

We present a 30-year old male patient with syndromic intellectual disability. His parents are healthy and not consanguineous and he has a younger, healthy sister. The pregnancy leading to his birth was uneventful. He was born at week 41 of gestational age by caesarean section. Height and weight were below the third percentile although no restriction of growth has been noticed during pregnancy; head circumference was normal. The neonatal period was unremarkable and he caught up his growth delay (reaching target height as an adult) and head circumference remained normal. He had repeated upper respiratory tract in- fections, and at the age of 4 years he suffered from an acute otitis complicated by a meningitis after which he lost his hearing. He has not experienced any regression. He underwent surgery during childhood for cryptorchidism. He has no heart defect, no kidney malformation or lymphedema and no seizure disorder. He had hydrocephalus probably related to meningitis on computerized tomography scan of the brain, and no evidence of malformation.

Concerning his developmental milestones, he exhibited hypotonia and delays in sitting up but walked at 18 months of life. Language was delayed. He had learning troubles in school which necessitated a special education. He is a young adult man with persistent intellectual disability, delayed ﬁne motor skills and severe speech impairment (he speaks very few words and uses sign language). He has optic neuropathy and severe hearing loss. He demonstrates dysmorphic features (Fig. 1): facial palcy, biﬁd uvula, enophthalmus, marked supra-orbital ridges, mild prognathism and his upper limb phalanges are hyposplatic. He is shy and solitary, he has inappropriate laughs and a tendency to stereotyped behavior but no obvious autistic behaviors, no aggression, and he does not have sleep disturbances.

After ruling out Fragile X Syndrome, aCGH was prescribed to investigate the patient's syndromic intellectual disability. Whole genome oligonucleotide microarray 105 K (Agilent^®Technologies) was performed on DNA from blood leucocytes using a dye-swap strategy and analysed with the Workbench^® software (adm2 method). The microarray showed ade novo1 Mb interstitial deletion in chromosomal region 22q13.2 (positions

41,864,255e42,865,320 by the GRCh37, hg 19 version) (Fig. 2).

Fluorescence in situ hybridization FISH on cultured blood leuko- cytes using an in-house probe BAC RP11-101F24 with thea^-satellite 14/22 D14Z1/D22Z1 Cytocell^®probe conﬁrmed the microdeletion in all cells of the patient and excluded its inheritance from his parents. Moreover, 18% of the cells examined had three signals with thea-satellite probe in addition to the 22q13.2 deletion (Fig. 3A). R- banded karyotype (Fig. 3B) was done and revealed a trisomy 22 mosaicism. FISH using the DiGeorge/VCFS TUPLE 1 and 22q13.3 Deletion Syndrome Probe Combination (Cytocell^®) further conﬁrmed the mosaic trisomy 22. The patient's karyotype was designated as follows according to ISCN nomenclature:

mos 46,XY.ish del(22)(q13.2q13.2)(RP11-101F24-)[123]dn/

47,XY,þ22.ish del(22)(q13.2q13.2)(RP11-101F24-)[27]dn

Array CGH was performed on DNA extracted from salivary cells using the Oragen-DNA^®kit and the 22q13.2 deletion was present in the saliva as well. Of note, a deletion is represented by an absolute log2 ratio of 1. A log2 ratio of 0.77 was measured in the blood (Fig. 3C, left), mimicking a mosaic deletion. This was in fact due to the hidden mosaic trisomy of an extra, not-deleted, chromosome 22. In contrast, in salivary DNA, the log2ratio was almost equal to 1 (Fig. 3C, right), advocating that the trisomic cell line was not signiﬁcantly present in the salivary tissue.

In an attempt to understand the mechanism behind the pres- ence of two anomalies -one structural and another numerical- on the same chromosome, microsatellite genotyping was performed on the patient and his parents.Fig. 3D shows an informative microsatellite marker localized to the chromosomal region 22q13.2.

The patient's remaining allele in the deleted area was inherited from his father. Thede novomicrodeletion was thus considered to be of maternal origin. None of the markers distributed outside the deletion region on the chromosome 22 were informative within the limits of the technique. Therefore the parental origin of the mosaic trisomy could not be determined.

3. Discussion

The aCGH detected ade novo1 Mb deletion in the 22q13.2 region. The deleted area contains about 20 genes, 3 of which are OMIM genes:TNFRSF13Chas a role in immune cells survival and regulation (Thompson et al., 2001), NAGA codes for a glycoside hydrolase (Sakuraba et al., 2004), and CYP2D6 is a cytochrome involved in the metabolism of endogenous and exogenous mole- cules (Gaedigk, 2013). The vast majority of deletions involving the 22q13 regions are terminal deletions with variable sizes:

100 kbe9.2 Mb (Sarasua et al., 2011), and up to 600 patients have been reported to date (Phelan and McDermid, 2012). The 22q13 deletion syndrome, also known as the Pheland-McDermid syndrome, is characterized by neonatal hypotonia, normal growth, global developmental delay, autistic behavior, absent or severely delayed speech and mild dysmorphic features (Phelan, 2008).

SHANK3was suggested to be the candidate gene responsible for the neurological features of this syndrome (Wilson et al., 2003). The patient described here has an interstitial 22q13 deletion that does not involve theSHANK3gene. To the best of our knowledge, there have been four reports of patients harboring this kind of deletion, with a total of thirteen patients (Disciglio et al., 2014; Romain et al., 1990; Simenson et al., 2014; Wilson et al., 2008). Wislon et al.

postulated that patients with deletions proximal toSHANK3might have a mild phenotype masked by the terminal deletion ofSHANK3.

Common features of patients with interstitial 22q13 deletion not involvingSHANK3gene are developmental delay, absent or severely delayed speech, minor dysmorphic features and hypotonia. The Fig. 1.Photograph of the patient at adult age. Patient has a facial asymmetry due to

facial palsy, prominent supraorbital ridges, enophtalmia, proganathism, high arched palate, biﬁd uvula, and thin upper lip.

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patient reported by Simenson et al. did not have hypotonia but had in addition urticarial rash and an elevated level of immunoglobulin E. The happloinsifficency of TNFRSF13Cand/orNFAM1genes was suggested to be related to his hyper IgE syndrome (Simenson et al., 2014). Our patient had this happloinsufficiency without the suggested related phenotype. Disciglio et al. delineated a new contiguous gene syndrome proximal to the terminal 22q13 deletions not involving theSHANK3gene; and proposed a minimal critical region associated with the phenotypic features (Disciglio et al., 2014). Our patient's deletion overlaped with 5 out of 9 patients' deletions included in Disciglio's series, but did not map in the defined minimal critical region. Thesefindings suggest that there are additional proximal genes yet to be defined that could also be responsible for the clinical features observed in patients with 22q13 interstitial deletions.

Mosaic trisomy 22 is a rare chromosomal disorder, compatible with life, in contrast to non-mosaic trisomy 22 which is quite common in spontaneous abortions but is not associated with survival beyond the neonatal period (Leclercq et al., 2010). In fact, trisomy 22 is an aneuploidy that can only be found in its mosaic form in living humans, and its presumed viability is due to the selection against trisomic cells in certain tissues at certain developmental stages (Biesecker and Spinner, 2013). Mosaic trisomy 22 has a wide clinical spectrum: growth retardation, hypotonia, dysmorphic traits, cranio-facial asymmetry, cardiac, ophthalmic, ear and limb malformations, genital organ anomalies, developmental delay and intellectual disability, as well as normal neurologic development (Leclercq et al., 2010). Our patient has facial asymmetry, which is one of the clinical clues that suggests the diagnosis of cellular mosaicism (Woods et al., 1994); other signs (not described in our patient) include abnormal skin pigmentation and Hypomelanosis of Ito (Hoang et al., 2011). It is highly recommended to test more than one tissue type when mosaicism is suspected. In a review of 21 patients with mosaic trisomy 22, only 30% had evidence of the mosaicism in their blood while the vast majority had mosaic karyotypes in their skinﬁbroblasts (Abdelgadir et al., 2013).

Salivary samples are gaining in popularity as an easy procedure that does not require needles or invasive sampling such as skin biopsies.

The oligonucleotide array done on our patient's salivary DNA as well as blood leucocytes DNA suggested a higher percentage of

trisomic cells in the blood. Thisﬁnding was unexpected considering that analysis of salivary samples offers an increased probability of detecting cellular mosaicism; indeed, several studies reported higher incidence of trisomic cells in the saliva (Papavassiliou et al., 2009; Sdano et al., 2014). Papavassiliou et al. stated that the variable rates of mosaicism seen in distinctive tissues could be explained by variable in vivo selection rates caused by different cellular turnover and/or growth rates; or alternatively by different distributions of mosaic cells across the body during development.

Conventional karyotype analysis on cultured blood lymphocytes could underestimate the percentage of mosaic cells due to selective disadvantages against cells carrying an extra chromosome copy (Ballif et al., 2006; Conlin et al., 2010; Sdano et al., 2014). Array CGH performed on DNA extracted from uncultured blood samples has been advocated to be superior in detecting the level of mosaicism (Ballif et al., 2006). Interestingly in our case, the chromosomal mosaicism was detected by karyotype on cultured cells and not by CGH on unstimulated blood lymphocytes, within the limits of our technique.

Hidden mosaicism as low as 10% was successfully detected by array CGH using speciﬁc analysis parameters and dye-reversed experiments (Hoang et al., 2011). Our routine oligonucleotide array missed the 18% chromosomal mosaicism despite its high 105 K resolution and the dye- swap strategy used. The use of an aCGH technique that combines single nucleotide polymorphism SNP probes to the analysis of copy number variation CNV would likely reveal more mosaicism than would CGH do alone (Sdano et al., 2014).

CNVs mainly occur in the human genome due to the recombination based mechanisms non-allelic homologous recombination NAHR- and non-homologous end joining NHEJ and by retro- transposition (Zhang et al., 2009). Wilson et. Al evaluated 56 patients with 22q13 deletion syndrome and found 69% paternal germline deletions (Wilson et al., 2003). Consistently, a large cohort of patients with ID detected 118 rarede novoCNV with 76% having a paternal origin (Jayne Y Hehir-Kwa, 2011). This was not the case of our patient whosede novodeletion originated from his mother. As for the mosaic trisomies, they occur by meiotic events and the subsequent trisomic zygote rescue which is the loss of the extra chromosome after the conception of a trisomic fetus; or by mitotic Fig. 2.The patient's 22q13 deletion with reported overlapping deletions: localization and gene content; viewed in the UCSC human genome browser, GRCh37, hg 19.

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Fig. 3.Cytogenetic and molecular characterization of the 22q13.2 deletion and the mosaic trisomy 22. A. Dual color FISH with BAC probe RP11-101F24 in red, anda-satellite 14/22 D14Z1/D22Z1 Cytocell^®probe in green showing 3 chromosomes 22 and absence of the red signal on one chromosome 22. B. R-banded karyogram showing a trisomic cell line. C. Oligoarray analysis of the 22q13.2 deletion in blood (left) and in salivary DNA (right). A deletion is generally represented by an absolute log2ratio of 1. The log2ratio of 0.77 seen in the blood mimics a mosaic deletion. This is in fact due to the hidden mosaic trisomy of the chromosome. In contrast, in salivary DNA, the log2ratio is almost equal to 1, suggesting that the trisomic cell line is insigniﬁcantly present in the salivary tissueD. Electropherogram showing the informative microsatellite locus (D22S1178) in the patient (both blood and salivary DNA) and his parents. (For interpretation of the references to color in thisﬁgure legend, the reader is referred to the web version of this article.)

R.Naoufaletal./EuropeanJournalofMedicalGenetics59(2016)483e4876

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events due to non-disjunction or anaphase lag in somatic cells during the development of a normal zygote (Conlin et al., 2010).

To our knowledge, this is theﬁrst report of the co-occurrence of two anomalies, a mosaic trisomy and a microdeletion, on the same pair of chromosomes. Our patient has symptoms that could be attributed to his interstitial 22q13 deletion as well as to his mosaic trisomy 22 (hypotonia, dysmorphic traits and developmental delay). He also presents features that are mainly explained by the 22q13 deletion (speech delay) and others that are mostly seen in mosaic trisomy 22 (growth restriction at birth, facial asymmetry, genital anomaly). It is thus very likely that the 22q13.2 deletion and the mosaic trisomy 22 both contributed synergistically to our patient's phenotype. It was previously shown that the number of CNVs detected in a patient was tightly bound to the severity of the phenotype (Vulto-van Silfhout et al., 2013). Genotype to phenotype correlations should be done with care in view of every patient's unique clinical features, genetic and environmental background.

In conclusion, our case points out the role of conventional cytogenetic tools in mosaic cases that could still be missed by microarray technology. On another note, microarray design should be optimized in order to detect low-level mosaicism: resolution of the array, SNP combined with CNV detection, calling algorithms… With the advances of CGH, new microdeletion and micro- duplication syndromes are being reported. Data is lacking about the interactions of more than one genetic anomaly detected on a same patient.

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