Xq21.1q21.31 Duplication in Two Male Siblings

Abstract

Despite the increased use of array comparative genomic hybridisation, duplications of Xq remain rarely reported in the literature. Xq21.1q21.31 duplication has previously been reported only once in a boy with features of Prader Willi syndrome (PWS). We report 2 malesiblings with maternally inherited duplication of Xq21.1q21.31 who demonstrate a variable phenotype. The proband has Prader Willi-like features such as global developmental delay, autism, obesity, short hands, and small genitalia with a history of food seeking behaviour, while his younger brother has isolated speech delay with some autistic features under evaluation. Both siblings have features such as bitemporal narrowing and small hands. It is therefore likely that the phenotype of duplications in this region is broader than PWS phenocopy, and further cases would be required to elucidate this.

Established Facts

• Xq21.1q21.31duplication has only been previously reported in the literature on one occasion associated with a Prader Willi syndrome phenotype.

• Loss of ZNF711 has been associated with intellectual disability in males with Xq21 deletion.

Novel Insights

• There is variability of phenotype with Xq21.1q21.31, but the proband in this case supports the association with a Prader Willi phenotype.

• Duplication of ZNF711 may contribute to the intellectual disability seen in these siblings.

Introduction

Array comparative genomic hybridisation (array CGH) has been a first line genetic test for the investigation of those presenting with developmental delay, intellectual disability or autistic spectrum disorder for over a decade in the UK. It has led to the identification of various disease-causing copy number changes; however, there continues to be a spectrum of deletions and duplications with unclear significance. This can be because of variable expression and reduced penetrance, presence within the healthy population, or absence of similar variants in the medical literature.

Xq21.1q21.31 duplication has previously been reported in the literature by Gabbett et al. [2008] who presented a boy with clinical features of Prader Willi syndrome (PWS) and suggested that duplications of Xq should be considered within the differential diagnosis of PWS. We report 2 male siblings with duplications of this region who demonstrate phenotypic variability.

Case Report

The proband was referred to the Clinical Genetics Service following the identification of an array CGH finding of uncertain significance.

The proband was delivered by elective caesarean section at 38 weeks' gestation due to footling breech presentation. Pregnancy was complicated by obstetric cholestasis. His birth weight was 3,260 g (75th centile) and head circumference was 34 cm (50th centile). He did not require resuscitation. The child initially had poor latch, but this resolved within a few days. As a baby, his mother reported that he would breastfeed excessively and never appeared full. As he got older, he continued to have a large appetite and would become aggressive when denied food. The family had to lock the fridge in view of food-seeking behaviour.

There was a history of developmental delay. He sat at 1 year and walked at 18 months, his speech was delayed. At 15 months of age, he had his first febrile seizure; he had further febrile seizures and subsequently developed idiopathic epilepsy with generalised tonic-clonic seizures. His last seizure was at 8 years of age and at the time of assessment was being weaned off of antiepileptic medication. He had a diagnosis of autism and attention deficit hyperactivity disorder with verbal and physical aggression that was sometimes difficult to manage. He had sleep difficulties requiring melatonin. He had been in mainstream education but due to learning difficulties was moved to a special needs school.

On examination at 10 years of age, his height was 142 cm (<75th centile), weight 50.15 kg (98th–99.6th centile) and head circumference 52.2 cm (2nd–9th centile). He had an elongated face, bitemporal narrowing, full cheeks, large earlobes, and micrognathia (see Fig. 1). He had short hands with short hyperextensible fingers and bilateral 5th finger clinodactyly, as well as a bilateral sandal gap. A further review at 11 years showed developing central obesity and small genitalia with small scrotal size.

Fig. 1.

The physical appearance of the proband at 11 years old demonstrating bitemporal narrowing, elongated face, large earlobes, thin upper lip, and central obesity (a, b). Short hands with short fingers (c), and an increased sandal gap and slightly elongated broad first toes are shown (d). The proband's brother at 6 years old with bitemporal narrowing and depressed nasal bridge (e, f).

The proband's half sibling (maternal) was born by planned caesarean section at 37 weeks with a weight of 3,060 g (75th centile). He was well apart from physiological jaundice in the newborn period. He had normal gross motor developmen. He sat at 7 months and walked at 14 months; however, his speech was delayed.

He had no history of seizures, but his mother had concerns about his behaviour as he had no sense of danger. His paediatrician had referred him for an assessment for autism. His appetite was poor, and he required encouragement to eat.

On examination at 5 years of age his height was 107.5 cm (9th–25th centile), weight 22.55 kg (91st centile) and head circumference 49.5 cm (0.4th centile). He had features of bitemporal narrowing and long philtrum. He had a depressed nasal bridge with full cheeks. His hands and feet had the same features as his brother. He had normal external genitalia.

Their mother had a history of febrile seizures in early childhood. She reported mild learning difficulties at school. She had not had a formal assessment for autism but described autistic traits and hyperactivity. She also described excessive eating and difficulties with weight management from an early age. At the age of 41, she was diagnosed with left ventricular hypertrophy requiring management with Propranolol and Ramipril. She was not dysmorphic. Her weight at age 41 years was 88.5 kg (99.6th centile) and height 164 cm (50th centile) with a body mass index of 33 kg/m².

Array CGH analysis of the proband and his brother was performed using the BlueGnome 8 × 60K v2.0 ISCA platform, with probes mapped to the GRCh37 (hg19) human genome assembly. Data were analysed in BlueFuse Multi. In both brothers, the array CGH showed a duplication of 3.99 Mb within the X chromosome at band q21.1 to q21.31, arr[GRCh37] Xq21.1q21.31(82857739_86848989)×2 mat. This included 10 protein-coding genes (see Fig. 2) 3 of which are recorded as being associated with human disease in the Mendelian Inheritance in Man database (ZNF711, CHM, POF1B). Targeted microarray analysis in the proband's mother confirmed that she has the same duplication in the heterozygous state. X inactivation testing in the mother showed random X inactivation on the DNA extracted from lymphocytes, blood sample, and it did not show skewed inactivation. There were no further copy number imbalances which could be considered clinically significant in the proband or his brother. In view of some of the features seen in the proband, Prader Willi testing was undertaken. Methylation-specific MLPA analysis showed normal methylation and normal dosage of the 15q11q13 region with no evidence of paternal deletion, uniparental disomy or an imprinting defect. The proband was recruited to the 100,000 Genomes Project and 4 panels were applied, these were intellectual disability v2.200, undiagnosed metabolic disorders v1.77, mitochondrial disorders v1.66, and epileptic encephalopathy v1.142. Details of the genes included in these panels can be found on PanelApp (https://panelapp.genomicsengland.co.uk/panels/). The results were normal.

Fig. 2.

Xq21.1q21.31 duplication found in the proband and his brother. Area of interest is shown in the highlighted area, GRCh37 Xq21.1q21.31(82857739_86848989). Genes included in the duplicated segment APOOL, CHM, CYLC1, DACH2, HDX, KLHL4, POF1B, RPS6KA6, SATL1, and ZNF711. Duplicated area reported by Gabbett et al. [2008] highlighted with arrows in red, minimum region 11.14 Mb GRCh37 Xq21.1q21.31(76755905_87898537), maximum region 14.14 Mb GRCh37 Xq21.1q21.31(76158932_90295428).

Discussion

PWS is an imprinting disorder caused by lack of paternally active gene expression at 15q11q13. The commonest cause in 70–75% for this is a deletion of this region, but it can also be caused by maternal uniparental disomy and an imprinting defect of the paternal chromosome [Buiting et al., 2014]. Guidelines are in place for both clinical diagnosis and genetic testing [Holm et al., 1993; Beygo et al., 2019]. The case reported by Gabbett et al. [2008] included many of the clinical features of PWS, and they suggested Xq duplications should be considered in the differential diagnosis of PWS as a potential phenocopy. Their proband did not quite meet the clinical criteria suggested by Holm's Consensus Diagnostic Criteria [Holm et al., 1993] with a score of 7.5 when the threshold is 8. Comparing our patients against these criteria, neither the proband nor his brother would reach the threshold to consider this part of the differential diagnosis, scoring 5 and 1.5, respectively, and this suggests a broader phenotype seen in this duplication.

Some features of our patients are similar to those seen in the previously reported case, see Table 1. These include features which are seen in PWS such as food-seeking behaviour, genital anomalies and obesity, while initial feeding difficulties and hypotonia were absent from our proband and his brother. Autistic features and some facial features appear to be present across the cases. Seizures were seen in our proband, but it is unclear if this is incidental or related to his duplication. Their mother showed random X inactivation and demonstrated some mild features such as mild learning difficulties, some autistic traits, and possible hyperactive disorder.

Table 1.

Summary of clinical features seen in cases with Xq21.1q21.31 duplication

	Gabbett et al. [2008]	Proband	Proband's brother
Gestational age	40	38	37
Sex	Male	Male	Male
Infantile feeding problems	Gavage fed until 3 weeks	Initial poor latch	No
Food-seeking behaviour	Yes	Yes	No
Hypotonia	Yes	No	No
Developmental delay	Yes	Yes	Isolated speech delay
Learning difficulties	Moderate learning difficulties	Moderate learning difficulties	Mild learning difficulties
Genital abnormalities	Yes, genital hypoplasia	Yes, small external genitalia	No, normal external genitalia
Weight	+4 SD	+2 SD	+1.5 SD
Height	75th centile	<75th centile	9th–25th centile
Seizures	No	Yes	No
Autism	Yes	Yes	Autistic features, no formal diagnosis
Attention deficit hyperactivity disorder	Not reported	Yes	Some features
Facial features	Fair complexion, bitemporal narrowing, long almond-shaped palpebral fissures, small downturned mouth	Bitemporal narrowing, elongated face, thin upper lip, and central obesity	Bitemporal narrowing and depressed nasal bridge

The duplication seen in our proband and his sibling is not recorded as a common benign copy number variation in the Database of Genomic Variants (http://dgv.tcag.ca/gb2/gbrowse/dgv2_hg19/?name=chrX%3A82857739-86848989;search=Search). A review of DECIPHER (https://www.https//decipher.sanger.ac.uk/) demonstrates no similar sized duplications but some smaller duplications within the same region of uncertain significance associated with intellectual disability and autism. Xq duplications remain reasonably rare in the literature, and in those previously reported, larger segments are involved [Vejerslev et al., 1985; Thode et al., 1988; Yokoyama et al., 1992; Shapira et al., 1997; Hou 2004; Sismani et al., 2013; Jin et al., 2015; Linhares et al., 2016]. The exceptions to this are a case reported by Pramyothin et al. [2010] of a 20-year-old man with 47,XXY and an additional Xq21.31 duplication who demonstrated features of PWS. They hypothesise that his phenotype was due to a combination of the additional X chromosome and Xq21 duplication. Castro-Gago et al. [2013] reported an Xq21.31 duplication in a 9-year-old without PWS features but with developmental delay, autism, and hyperactive behaviour. However, he had additional features such as hand stereotypes, large ears, synophrys and excessive hair, not seen in our proband or his brother. His body mass index was on the 95th centile. In those with larger duplications, genital abnormalities, developmental delay, hypotonia, and short stature without obesity appear to be common. This variance from our patients likely reflects the larger segments that are duplicated in these cases. ATRX was proposed as the candidate gene in these patients as a cause of obesity; however, this was not duplicated in our proband who demonstrated food-seeking behaviour and obesity.

It is uncertain which genes within the region of duplication are contributing to the phenotype in these patients, particularly because the physiological role of several of these genes remains largely unclear (Table 2).

Table 2.

Genes within the region of duplication

HGNC symbol	HGNC name	Function	Expression Fagerberg [2014]	Disease associations
CYCL1	Cylicin 1	Sperm head cytoskeletal protein	Testis
RPS6KA6	Ribosomal protein S6 kinase A6	Growth factor-regulated serine/ threonine kinase	Broad, high in thyroid
HDX	Highly divergent homeobox	Unknown, putative transcription factor	Broad, low level
APOOL	Apolipoprotein O like	Lipid-binding inner mitochondrial membrane protein	Broad
SATL1	Spermidine/spermine N1-acetyl transferase like 1	Unknown	Broad
ZNF711	Zinc finger protein 711	Transcription factor	Broad, high in testis and brain	Hemizygous loss-of-function variants cause intellectual disability in males [OMIM 314990]
POF1B	POF1B actin binding protein	Actin-binding protein in epithelial cells	Broad, high in skin and gastrointestinal tract	Possible association of biallelic variants with premature ovarian failure in females [OMIM 300604]
CHM	CHM Rab escort protein	Geranylgeranylation of Rab family GTPases	Broad	Loss-of-function variants cause choroideremia, mostly in males [OMIM 300390]
DACH2	Dachsund family transcriptionTranscription factor factor 2	Brain, low level in some other tissues
KLHL4	Kelch like family member 4	Unknown	Broad, high in adrenal gland, brain, ovary in testis

ZNF711 is a good candidate for the neurodevelopmental features. ZNF711 encodes a transcription factor of the zinc finger family that shows high expression in the brain [Rhie et al., 2018]. Hemizygous loss-of-function variants in ZNF711 are associated with mild to moderate intellectual disability in males, with autistic features and mild facial dysmorphisms noted in some patients [Tarpey et al., 2009; Van der Werf et al., 2017]. Loss of ZNF711 is also considered to be causative of the intellectual disability seen in males with Xq21 deletions [Liang et al., 2017]. The duplication of ZNF711 observed in the current patients could potentially lead to dysregulation of ZNF711 target gene expression during development, which could contribute to the neurodevelopmental and behavioural features in patients with Xq21 duplications.

Increased expression of DACH2 might also contribute to the phenotype associated with Xq21 duplication. DACH2 is a transcription factor with strong expression in the brain, although a role for this gene in developmental disorders has not yet been established. A rare missense variant in DACH2 (p.S357C) was reported in 2 brothers with growth retardation, intellectual disability, and difficulty in standing and walking [Zhang et al., 2014]. However, a number of males hemizygous for this variant are recorded in the gnomAD database, which suggests that this may be a benign variant found in the East Asian population. The clinical significance of variation in DACH2 remains uncertain.

Loss-of-function variants and deletions in CHM have been established as a cause of choroideremia, resulting in progressive visual loss in males, with heterozygous females mostly showing only subclinical manifestations [Simunovic et al., 2016]. No ophthalmological abnormalities have yet been detected in association with the whole gene duplication of CHM in the current patients.

Additional potential genes of interest within the duplicated region include RPS6KA6, which functions in signalling downstream of growth factor receptors, and KLHL4, a protein of unknown function with high expression in the brain. Of note, 2 genes within the region, POF1B and CHM, fully or partially escape X inactivation, whereas the flanking genes, ZNF711 and DACH2, are subject to normal patterns of inactivation [Bione et al., 2004].

Conclusion

Duplications of Xq21.1q21.31 remain rarely reported in the literature; the cases we report add to the phenotype from the previously reported cases and demonstrate the likely phenotypic variability associated with this particular duplication, including within the same family. It is likely that further cases will be reported with the widespread use of array CGH, and this will hopefully elucidate the phenotype further in future.

Statement of Ethics

Ethics approval was not required for the publication of case histories. Written informed consent was obtained from the parent of the patients for publication of the details of their medical case and the accompanying images.

Conflict of Interest Statement

The authors declare no conflicts of interest.

Funding Sources

This study had no external sources of funding.

Author Contributions

C.S.: data collection and drafted the manuscript with supervision from S.N. S.N.: conceptualisation, data collection, supervision, and review. A.M.: data collection. S.G. and L.R.: provided details of microarray analysis. All authors have critically reviewed the manuscript.

Data Availability Statement

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.