De novo 3q22.1 q24 deletion associated with multiple congenital anomalies, growth retardation and intellectual disability

Gene 517 (2013) 82–88

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De novo 3q22.1 q24 deletion associated with multiple congenital anomalies, growth retardation and intellectual disability Maggie S. Brett a, Ivy S.L. Ng b, Eileen C.P. Lim a, Min Hwee Yong c, Zhihui Li d, Angeline Lai b, Ene-Choo Tan a, e,⁎ a

KK Research Centre, KK Women’s & Children’s Hospital, Singapore Genetics Service, Department of Paediatrics, KK Women's & Children's Hospital, Singapore Cytogenetics Department, KK Women's & Children's Hospital, Singapore d Genomax Technologies, Singapore e Office of Clinical Sciences, Duke-NUS Graduate Medical School, Singapore b c

a r t i c l e

i n f o

Article history: Accepted 19 December 2012 Available online 11 January 2013 Keywords: 3q deletion Array CGH Cleft palate Growth retardation Intellectual disability

a b s t r a c t We describe a boy with a de novo deletion of 15.67 Mb spanning 3q22.1q24. He has bilateral micropthalmia, ptosis, cleft palate, global developmental delay and brain, skeletal and cardiac abnormalities. In addition, he has bilateral inguinal hernia and his right kidney is absent. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. This patient has some phenotypic features that are not shared by the other patients. More cases with smaller deletions defined by high resolution aCGH will enable better genotype–phenotype correlations and prioritizing of candidate genes for the identification of pathways and disease mechanisms. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Deletions on 3q in the published literature can be subdivided into pericentromeric-proximal (involving 3q11.1–q13.33 only), proximal (involving 3q21–q28) and subtelomeric/terminal (involving 3q29 only). Subtelomeric/terminal deletions cause 3q29 microdeletion syndrome. Deletions which include the more proximal region (3q21–q25) have various clinical presentations depending on the size of the deletion

Abbreviations: A4GNT, alpha-1,4-N-acetylglucosaminyltransferase; AGTR1, angiotensin II receptor, type 1; ATR, ataxia telangiectasia and Rad3 related; BAC, bacterial artificial chromosome; BFSP2, beaded filament structural protein 2; BPES, blepharophimosis, ptosis, and epicanthus inversus syndrome; CT, computer tomography; CCD, charge-coupled device; CGH, comparative genomic hybridization; CLDN18, claudin 18; DWM, Dandy– Walker malformation; DZIP1L, DAZ interacting protein 1-like; FOXL2, forkhead box L2; GRCH36/hg18, Genome Reference Consortium Human Reference Build 36; ID, intellectual disability; kg, kilogram; Mb, million bases; MRPS22, mitochondrial ribosomal protein S22; OFC, occipito-frontal circumference; OMIM, Online Mendelian inheritance in man; PCCB, propionyl CoA carboxylase, beta polypeptide; PCOLCE2, procollagen C-endopeptidase enhancer 2; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2; PDA, patent ductus arteriosus; PCR, polymerase chain reaction; qRT-PCR, quantitative real time-polymerase chain reaction; RQ, relative quantitation; SCKL1, Seckel syndrome-1; SLC9A9, solute carrier family 9, subfamily A (NHE9, cation proton antiporter 9), member 9; SNP, single nucleotide polymorphism; SOX14, SRY (sex determining region Y)-box 14; TF, transferrin; VSD, ventricular septal defect; ZIC, Zinc finger protein of the cerebellum. ⁎ Corresponding author at: KK Women's & Children's Hospital, 100 Bukit Timah Road, 229899, Singapore. Tel.: + 65 63943792; fax: + 65 63941618. E-mail address: [email protected] (E.-C. Tan). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.082

and the genes involved. Patients with interstitial deletions can present with manifestations of (i) blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES: OMIM#110100), (ii) Dandy–Walker syndrome (DWS: OMIM%220200) including Dandy–Walker malformation (DWM), (iii) Wisconsin syndrome (Cohen, 1986), (iv) Pierre–Robin sequence (OMIM%602196) and (v) Seckel syndrome-1 (SCKL1: OMIM#210600). The most frequent region involved in interstitial 3q deletions is 3q22–23, accounting for approximately half of the documented cases. These patients have features of BPES which can also be caused by point mutations in the gene. In addition to dysplasia of the eyelids, other common features include microcephaly, skeletal anomalies, congenital heart defects, cranial anomalies, intellectual disability and developmental delay (de Ru et al., 2005; Rea et al., 2010; Willemsen et al., 2010). Unfortunately, the majority of these previously published cases were identified by conventional cytogenetic studies and this has hampered genotype–phenotype correlations and identification of candidate genes for each specific feature. Exceptions are forkhead box L2 (FOXL2) gene mutations/deletions in patients with BPES (Beysen et al., 2009; Crisponi et al., 2001), mutations in the gene encoding ataxiatelangiectasia and RAD3-related protein (ATR) in Seckel syndrome patients (O'Driscoll et al., 2003), and evidence associating the ZIC family member (ZIC) genes, ZIC1 and ZIC4 with Dandy–Walker malformation (Grinberg et al., 2004; Lim et al., 2011; Tohyama et al., 2010). We describe a boy with a de novo deletion of 15.6 Mb spanning 3q22.1q24. He has developmental delay and some features of BPES and

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Seckel syndrome-1. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. 2. Clinical description The patient was the second child of non-consanguineous Chinese parents. Antenatal history was unremarkable and his older brother was well. He was born at term by normal delivery with a birth weight of 2.5 kg, length of 45 cm and occipito-frontal circumference (OFC) of 30 cm. He was noted to be dysmorphic and had a cardiac murmur soon after birth. The child was referred to the Genetics Department at KK Women's & Children's Hospital at 3 weeks of age. On examination, he was noted to be small with microcephaly. At six weeks of age his OFC was 31.9 cm, 2 cm less than the 3rd centile. Dysmorphic features included bilateral micropthalmia, bilateral ptosis and blepharophimosis, micrognathia, midline cleft palate, camptodactyly and prominent ear lobules. He also had rocker bottom feet and bilateral inguinal hernia. Ultrasound examination showed the absence of the right kidney and 2D Echocardiogram showed a moderate sized perimembranous ventricular septal defect (VSD) and a small patent ductus arteriosus (PDA). Cranial CT scan revealed turricephaly and fusion of bilateral lambdoid and coronal sutures. Before age two, the child had fronto-orbital advancement operation for craniosynostosis, cleft palate repair, brow suspension surgery for ptosis, and bilateral herniotomy. Coil-occlusion of the PDA was carried out when he was 4 1/2 years of age. At age 10, he is asymptomatic for his small VSD and closed PDA and no further interventions are planned. He has impaired visual acuity and continues to have problems with his vision. He also shows severe growth retardation with height, weight and OFC below the 3rd centile. He has global developmental delay — he sat unsupported at 10 months, walked at 2 3/4 years and started speaking single words at 3 years. Currently aged 11, he has a learning disability and attends Special School but is able to speak in sentences.

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9 (sodium/hydrogen exchanger), member gene) as the target gene for quantifying gene copy number. Primers were designed using Primer Express (Version 3.0), and the experiment was carried out in triplicate. The patient's and parents' DNA samples were amplified in the same experiment with HBB (hemoglobin beta gene) as the internal reference. Amplification was done using Applied Biosystems StepOnePlus real time PCR system (Applied Biosystems, USA). Results were analyzed using Applied Biosystems StepOne software (version 2.1). 3.4. Network/pathway analysis The coordinates of the minimum deleted region were searched against the Human reference genome (hg18) for known genes in the region. The resulting list of genes was imported into Ingenuity Pathway Analysis (IPA) software using the Entrez ID mapped to the Ingenuity Pathway Knowledge Base identifier. The reference set used was Ingenuity Knowledge Base (Genes only); relationship to include was both direct and indirect. The analysis included endogeneous chemicals and the filter summary was set to consider only relationships where confidence = experimentally observed. The statistical significance for the enrichment of genes of interest in each pathway was evaluated by a Fisher Exact test under the Core Analysis function of IPA. 4. Results 4.1. Molecular karyotyping

3. Materials and methods

Genome-wide analysis of the child from the SNP array showed a minimum loss of 15.67 Mb at 3q22.1q24. The first probe with copy number loss was at 134,366,905 and the last probe with copy number lost was at 150,039,405 [hg18]. The last probe with normal copy number was at 134,362,222 and the first probe with normal copy number is at 150,044,473 (Fig. 1). The maximum size of the deletion is 15.68 Mb.

3.1. Molecular karyotyping

4.2. Fluorescence-in situ-hybridization (FISH)

DNA from the peripheral blood of the patient was used on an Affymetrix SNP 6 array according to the manufacturer's instructions (Affymetrix Inc., USA). Data were processed using the Affymetrix Genotyping Console and further analyzed by the Chromosome Analysis Suite software (Version 1.0.1392(r2426)). Copy number changes were calculated based on hybridization signal intensity data from calculated intensity distributions derived from a reference set from Affymetrix with the setting for marker count at 50, size at 100 kb and confidence at 85.

Parental chromosomes tested using BAC probes RP11-1023P20 (chr3: 134,159,391–134,326,599 [hg18]), RP11-883M5 (chr3: 134,732,494– 134,899,911 [hg18]), RP11-505J9 (chr3: 149,845,223–150,049,901 [hg18]) and RP11-80I8 (chr3: 153,554,710–153,735,639 [hg18]) showed normal hybridization pattern at band 3q22.1 to q25.2 on the two homologues of chromosome 3 for both parents.

3.2. Fluorescence-in situ-hybridization (FISH) Peripheral blood lymphocytes from the child's phenotypically normal parents were cultured using phytohemagglutinin (PHA) stimulation and methotrexate (MTX) synchronization/thymidine release methods. Molecular cytogenetic analysis was performed using probes obtained from The Hospital for Sick Children (Toronto, Canada). Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector Laboratories, Inc., USA) and analyzed by fluorescence microscope Olympus BX51 equipped with a CCD Progressive Scan Video Camera (JAI, Japan). Image analysis was carried out with Cytovision software (version 3.93.2) (Applied Imaging Corp, USA). 3.3. Quantitative real-time polymerase chain reaction (qRT-PCR) Gene copy number was also investigated by relative quantitative real-time-PCR with SYBR Green dye and SLC9A9 (solute carrier family

4.3. Quantitative real-time polymerase chain reaction Quantitative real-time PCR confirmed the copy number loss in the child with the RQ (relative quantitation) at 0.500. The respective RQ values for the father and mother are 1.143 and 1.149 (Fig. 2). 4.4. Network/pathway analysis The top scoring networks from the IPA Core Analysis function are displayed in Table 1. For each biological function identified, the range for level of significance of the genes involved in the pathway is displayed in Table 2. 5. Discussion In addition to the 22 cases of interstitial 3q deletions that Ramieri et al. (2011) cited, there are at least four more cases with deletions limited to 3q13.1–q13.33 (Lawson-Yuen et al., 2006; Shimojima et al., 2009; Simovich et al., 2008), and 19 more cases involving at least part of 3q21–q23 (with some extending to 3q13) (Almenrader et al., 2008; Arai et al., 1982; Brueton et al., 1989; Callier et al., 2009; Croft and

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Fig. 1. Array CGH profile of the patient. Screenshot showing the deletion at 3q22.1q24 from the analysis with Affymetrix Chromosome Analysis Suite software.

Turnpenny, 2008; De Baere et al., 2001, 2005; Engelen et al., 2002; Genuardi et al., 1994; Jenkins et al., 1985; Mackie Ogilvie et al., 1998; McMorrow et al., 1986; Okada et al., 1987; Sargent et al., 1985; Tohyama et al., 2010; Willemsen et al., 2010; Zahanova et al., 2012; Zhou et al., 2010), with a few more microdeletions associated with apparently balanced translocations. In a cohort of BPES patients whose

deletions were mapped by BAC clones, there are also four cases (two microscopic and two submicroscopic) with deletions including FOXL2 and additional genes (Beysen et al., 2009), and another 12 BPES patients with deletions of at least 2.48 Mb (D'Haene et al., 2010). With at least 20 cases for 3q29 microdeletion (Digilio et al., 2009), the number of deletions involving the long arm of chromosome 3 now numbers more

Fig. 2. qRT-PCR confirmation of copy number loss in the patient; referenced against a control sample and the parents.

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Table 1 Networks generated using Ingenuity Pathway Analysis tools. The networks of the 125 genes in the deleted region were generated based on their connectivity and ranked according to the scores based on P values. Genes in the chromosomal region deleted in the patient are in bold. ID Molecules in network

Score Focus Top functions genes

1

43

19

Drug metabolism, nucleic acid metabolism, small molecule biochemistry

34

16

Cellular development, cellular growth and proliferation, cell cycle

27

13

Cell morphology, infectious disease, developmental disorder

16

9

Lipid metabolism, molecular transport, small molecule biochemistry

10

6

Embryonic development, organ development, organismal development

2 2

1 1

Cell-to-cell signaling and interaction, tissue development, cell cycle Cellular assembly and organization, nervous system development and function, cardiovascular system development and function

2

3

4

5

6 7

AGTR1, Akt, Ap1, ATR, CD3, COPB2, EPHB1, ERK1/2, FAIM, FSH, Jnk, MRAS, NCK1, NFkB (complex), P110, P38 MAPK, p85 (pik3r), PI3K (complex), PIK3CB, PLSCR1, PLSCR4, Ras, RASA2, RBP1, RNF7, RYK (includes EG:140585), Shc, Sos, SPSB4, SRPRB, TCR, TFDP2, TOPBP1, TRPC1, Vegf ANAPC13, ATP1B3, BFSP2, CCNB1, CDKN1B, CEP70, DDX31, ELFN1, ELFN2, EP300, FAM108B1, HENMT1, HKDC1, IFNG (includes EG:15978), IL20RB, KY, MRPS22, MSL2, NME9, PLOD2, PLS1, PLSCR2, PPP1CA, PPP1R27, RAB43, SMARCA4, SUMO4, TGFBR2, TMEM132D, TRIM42, UBC, XRN1, YWHAZ, ZBTB38, ZIC1 ANKRD13D, ARHGEF6, ARHGEF17, ARMC8, ARRDC2, C3orf37, CDV3, CLIC2, CUEDC1, DBR1 (includes EG:323746), DZIP1L, EPHB4, GK5, GRK7, KIAA1598, MRPL19, NEDD4, PARP16, PCCB, PRRG1, RAB6B, RFT1, SAAL1, SLC25A36, SP140L, STAG1, TCP11L1, TMEM108, TRIM52, TTYH2, TTYH3, U2SURP, UBC, ZIC4, ZNF598 ACPL2, AMOTL2, APOA1, ATP, beta-estradiol, CEP63, cholesterol linoleate, CLEC2D, CLSTN2, CNN2, COCH, COX6B2, ERK, F8A1 (includes others), FGF1, GJC1, GPR88, HTT, HUNK, Lectin, MAB21L1, MPEG1, MUC8, NMNAT3, PAQR7, PAQR9, PCOLCE2 (includes EG:26577), PCTP, PPARA, prostaglandin E2, RBP2,RBP7, SEZ6, SLCO2A1, ZNF91 12 lipoxygenase, APOC4, CHST2, CHST4, CLDN11, CLDN18, CYP26B1, EGF, EGFR/PDGFR/IGFR, EMP2, FOXA2, FOXE1, FOXF2, FOXL2, FUT3, HS2ST1, IGF1, Ly6, NKD1, NKX2-1, ORM2 (human),PDPN (includes EG:10630), PNLIPRP1, Pp95, PPP2R3A, RHBDF2, RNASE4, SLC12A6, SLC12A7, SOX14, STARD3, TDO2, TF, TNF, ZFP36L2 GNB2L1, SLC9A9 ACAP3, SEMA3F, SLC35G2, WT1

than 80. With many of the reports on new cases including some comparison of regions and phenotypic presentations with past cases and new cases with deletions defined by aCGH, it is likely that we will soon see a list of defining phenotypic features for specific microdeletions of subbands. It might soon be possible to come up with distinct and clinically recognizable microdeletion syndromes for the region. The 15.67 Mb deletion of the patient in this report extended from chr3:134,387,722 to 150,043,915 (hg18). A total of 125 genes were deleted including nine OMIM morbid genes BFSP2, TF, PCCB, FOXL2, MRPS22, ATR, SLC9A9, PLOD2 and AGTR1. Other important genes that are deleted include SOX14, CLDN18, DZIPI1L, A4GNT, PCOLCE2, ZIC1 and ZIC4. Pathway analysis of the deleted genes revealed some interesting results. Within the deleted region were clusters of genes involved in developmental disorders, nervous system development, organ morphology and development. Canonical pathways affected by the deleted genes included the retinoate biosynthesis, and signaling pathways relating to cell growth and development (Table 1). In particular the ZIC1, ZIC4, FOXL2, BFSP2, RBP1, EPHB1, ATR, TRPC1, RYK, and AGTR1 genes were prominently associated with embryonic development, organ morphology and developmental disorders (Table 2).

Table 2 Top biological functions identified by Ingenuity Pathway Analysis of deleted genes. Biological function

P-values

Genes

Developmental disorder Nervous system development Organ morphology Lipid metabolism

2.21E-04– 4.20E-02 6.88E-04– 4.83E-02 6.88E-04– 3.25E-02 6.88E-04– 3.89E-02 6.88E-04– 4.20E-02 6.88E-04– 3.89E-02 3.30E-03– 4.83E-02 3.30E-03– 4.20E-02

ZIC1, ZIC4, FOXL2, BFSP2, PIK3CB, ATR, PCCB, TF, AGTR1 ZIC1, ZIC4, EPHB1, TRPC1, RYK, AGTR1

Molecular transport Small molecule biochemistry Embryonic development Organ development

ZIC1, ZIC4, FOXL2, BFSP2, AGTR1, RBP1, RYK, EPHB1 SLCO2A1, PLSCR1, PIK3CB, PCCB, RBP1, RBP2, AGTR1, TRPC1 SLCO2A1, PIK3CB, RBP1, RBP2, AGTR1, TRPC1,TF SLCO2A1, PLSCR1, PIK3CB, PCCB, RBP1, RBP2, AGTR1, TRPC1, ATR, RYK ZIC1, ZIC4, FOXL2, BFSP2, PIK3CB, ATR, RBP1, RYK, EPHB1 ZIC1, ZIC4, FOXL2, BFSP2, RBP1, RYK, EPHB1, TRPC1

The best characterized disorder in this region is BPES, but the present case could only be classified as BPES-like as he does not have all the four major characteristics of BPES syndrome. Notably his right kidney is also absent which has not been reported in other patients with interstitial 3q deletions. We have compared his phenotype with seven other patients with overlapping and molecularly defined deletions (Table 3, Fig. 3). The seven other cases include a 29.9 Mb deletion described in Patient 250665 in DECIPHER and other published reports of interstitial 3q deletions with array-CGH level resolution (Lim et al., 2011; Rea et al., 2010; Tohyama et al., 2010; Willemsen et al., 2010; Zahanova et al., 2012). Among this group of eight patients whose deletions were mapped by microarray-based methods, the deletion of FOXL2 correlated with patients displaying all the presentations of BPES, or patients displaying some of the major features (BPES-like). The exception was the patient without a FOXL2 deletion. For this patient it was postulated that distant regulation of gene expression could have played a role (Rea et al., 2010). The deletion of ZIC1 and ZIC4 also correlated with the phenotype of brain malformations like DWM, ventriculomegaly and hydrocephalus in the patients where brain imaging was performed. MRI showed a normal brain in Patient 4 who did not have deletion of ZIC1 and ZIC4 (Zahanova et al., 2012). The ZIC family genes are expressed in the central nervous system and have been shown to be essential for cerebellar development. Deletions of ZIC1 and ZIC4 probably play an important role in intellectual disability and developmental delay seen in patients with interstitial 3q deletions. Ingenuity Pathway Analysis identified the two genes as important for the development of the nervous system and organ morphology (Table 2). Microcephaly was noted in the present case and in Patients 5 and 7. Contiguous deletion of the ATR (Ataxia telangiectasia and Rad-3 related) gene has been postulated to cause the non-BPES phenotypes of microcephaly, mild mental retardation and growth retardation seen in a patient with interstitial 3q deletion (de Ru et al., 2005). ATR has been linked to Seckel syndrome-1, an autosomal recessive disorder which presents with facial dysmorphism, microcephaly, growth retardation and intellectual disability (O'Driscoll et al., 2003). This role of ATR was supported by studies which showed that the ATR signaling pathway was sensitive to haploinsufficiency in a cell line derived from a patient with BPES and ATR deletion. For the 8 cases in Table 3, ATR was deleted in all except Patients 7 and 8 while microcephaly was

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Table 3 Clinical features of 8 patients with overlapping interstitial 3q deletions detected by microarray-based methods.

Gender Deletion Start-end Mb (hg18) Size (Mb) Microcephaly Brain malformations Growth retardation Ptosis Blepharophimosis Epicanthus inversus Micropthalmia Abnormal/low set ears Prominent Nose Macrostomia Micrognathia Cleft/high arch palate Digital anomalies Joint anomalies Cardiac anomalies Speech delay Genital anomalies/Hypogonadism Other

Patient 1 this report

Patient 2 DECIPHER 250665

Patient 3 Lim [8]

Patient 4 Zahanova [28]

Patient 5 Rea [3]

Patient 6 Tohyama [9]

Patient 7 Willemsen [4]

Patient 8 Willemsen [4]

Male 3q22.1q24 134.3–150.0 15.67 + +a + + + − + + + − + + +e +h + + − +n

Female 3q22.3q26 139.2–169.1 29.9 NR +b NR NR NR NR NR NR NR NR + NR NR +i + NR NR +o

Male 3q22.3q25.2 139.3–154.2 14.92 NR +c NR + + + + NR NR − NR NR NR NR − + NR +p

Male 3q22.3q23 139.3–144.0 4.7 − − − + + + + + − − NR NR − +j − + + +q

Female 3q22.3q25.1 140.3–153.0 12.7 + +d + + + − + + − − NR − +f +i,k + NR NR −

Female 3q23q25.31 142.4–156.5 14 − +c NR − − − − + + + NR NR NR +j,l − +m − +p,r,s

Female 3q24q25.33 144.6–160.8 16.2 + NR + − − − − + + + NR − − − − NR + −

Female 3q24q26.1 147.2–166.6 19.38 − NR − − − − − + + + NR + +g − − NR + +r

+: present; −:not present; NR: not recorded. a Ventriculomegaly. b Hydrocephalus. c DWM. d Asymmetry of choroid plexus. e Overlapping fingers, camptodactyly. f Bilateral sandal gap. g Recessed 4th toe. h Rocker bottom feet. i Talipes. j Contractures. k Hip dysplasia. l Hip dislocation. m No speech. n Absent right kidney, inguinal hernia. o Small kidneys, esophageal atresia, choanal stenosis, tracheoesophageal fistula, hypertelorism, short palpebral fissures. p Neonatal feeding difficulties. q Spastic diplegia. r Seizures. s Occult spina bifida, scoliosis.

Fig. 3. Overview of 8 overlapping molecularly delineated interstitial 3q deletions; with positions of the OMIM morbid genes and other important genes depicted.

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only reported in Patients 1, 5 and 7. Similarly, growth retardation was only reported in Patients 1, 5 and 7. This suggests that there might be other genes besides ATR associated with microcephaly and growth retardation. ATR is likely to be one of the candidate genes for the developmental delay and intellectual disability that is noted in most of the patients. It has been shown to be important for cell cycle checkpoint signaling and DNA repair and is postulated to impact neuronal development and cell proliferation (O'Driscoll et al., 2007). Interestingly, chromosome instability has been demonstrated for Seckel syndrome patients (Casper et al., 2004; O'Driscoll et al., 2007). In addition to intellectual disability, speech delay has been reported in many patients with interstitial 3q deletions. Patients 1, 3 and 4 presented with speech delay and Patient 6 was reported to have no speech. Among this group of 8 cases, cardiac anomalies were only seen in three patients. The present case and the DECIPHER patient 250665 presented with VSD and PDA while truncus arteriosis was uniquely reported in another patient (Rea et al., 2010). It was postulated that the PCOLCE2 gene might be implicated in cardiac anomalies (Rea et al., 2010). However, cardiac anomalies were not reported in two other patients with deletion of the PCOLCE2 gene. This suggests the presence of other genes involved in the malformation of the cardiovascular system. Our patient presented with overlapping fingers, camptodactyly and rocker bottom feet. Two other patients (2 and 5) had talipes. The gene SRY-related HMG-box 14 (SOX14) has been proposed as a candidate for the limb defects seen in BPES cases (Wilmore et al., 2000). However, SOX14 is deleted only in our patient and not Patient 2 or 5 making it unlikely that SOX14 is solely responsible for limb defects. Other skeletal anomalies were seen in Patient 4 who presented with joint contractures, Patient 5 who had hip dysplasia, and Patient 6 who had joint contractures, hip dislocation, occult spina bifida and scoliosis. First described by John Opitz as a combination of craniosynostosis, ID, upslanted palpebral fissures, small ears and short fourth metatarsals with recessed fourth toes, reports of additional Wisconsin syndrome cases with similar phenotypes suggests that it might be caused by 3q24q25 deletions (Willemsen et al., 2010). Notably, the coarse facial appearance including prominent nose, large mouth, and bushy eyebrows were only seen in Patients 6, 7, and 8. There was no report of coarse facial phenotype in Patient 2 in the DECIPHER database despite the deletion extending into the region. Genital anomalies or hypogonadism were reported in Patients 4, 7 and 8. Patient 4 has a webbed penis and chordee, Patient 8 has a hypoplastic uterus and Patient 7 has primary amenorrhea. A “hypogonadism region” extending from 159,897,607–160,842,453 Mb had been previously proposed (Willemsen et al., 2010). However, the deletion of Patient 4 did not overlap with this hypogonadism region suggesting the involvement of other genes. The present case is the only one in this group with a cleft palate and bilateral inguinal hernia, although Patient 8 is reported to have a high-arched palate. Only our patient and Patient 2 have renal anomalies and micrognathia. In our patient the right kidney was absent and Patient 2 was reported to have small kidneys. As this patient had the most proximal breakpoint among the eight cases, additional cases of interstitial 3q deletions overlapping with the proximal end of his deletion will enable more genotype–phenotype correlations in the 3q22.1–q22.2 band. It appears that apart from the BPES cases, most cases of the deletions are de novo as there are no common breakpoints. Deletion of the ATR gene may be a contributory factor causing sporadic deletions. With the increasing use of array CGH in diagnostic laboratories, we envisage that more patients with 3q2 deletions will be identified. Molecular mapping of deletion breakpoints in additional patients will enable more accurate and extensive genotype–phenotype correlations and lead to the unraveling of more candidate genes and disease mechanisms. Description of more cases such as our patient will add valuable

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information on expected phenotypic presentations which will be useful in the genetic counseling of patients with similar interstitial 3q deletions. In conclusion, we describe a patient who has the most proximal deletion breakpoint compared to other patients whose deletions are defined by aCGH. He shared some features with the seven cases that had overlapping deletions but he also had some phenotypic features that are not reported in others. More cases with smaller deletions defined by high resolution aCGH will enable better prioritizing of candidate genes for specific features. Acknowledgments This work was supported by project BMRC 06/1/50/19/485 from the Agency for Science and Technology and Research, Republic of Singapore. References Almenrader, N., Passariello, M., Coccetti, B., Pietropaoli, P., 2008. Anesthesia for a child with deletion 3q syndrome. Paediatr. Anaesth. 18, 789–790. Arai, K., Matukiyo, H., Takazawa, H., 1982. A case report of partial deletion of the long arm of the no. 3 chromosome. Med. Genet. Res. 4, 1–4. Beysen, D., De Paepe, A., De Baere, E., 2009. FOXL2 mutations and genomic rearrangements in BPES. Hum. Mutat. 30, 158–169. Brueton, L.A., Barber, J.C., Huson, S.M., Winter, R.M., 1989. Partial monosomy 3q in a boy with short stature, developmental delay, and mild dysmorphic features. J. Med. Genet. 26, 729–730. Callier, P., et al., 2009. Detection of an interstitial 3q21.1–q21.3 deletion in a child with multiple congenital abnormalities, mental retardation, pancytopenia, and myelodysplasia. Am. J. Med. Genet. A 149A, 1323–1326. Casper, A.M., Durkin, S.G., Arlt, M.F., Glover, T.W., 2004. Chromosomal instability at common fragile sites in Seckel syndrome. Am. J. Hum. Genet. 75, 654–660. Cohen, M., 1986. Craniosynotosis: Diagnosis, Evaluation and Management. Reven Press, New York. Crisponi, L., et al., 2001. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat. Genet. 27, 159–166. Croft, M.S., Turnpenny, P.D., 2008. Deletion 3q22.1–q23 with blepharophimosis, ptosis and epicanthus inversus and an Albright hereditary osteodystrophy-like brachydactyly phenotype. Clin. Dysmorphol. 17, 189–191. De Baere, E., et al., 2001. Spectrum of FOXL2 gene mutations in blepharophimosis–ptosis– epicanthus inversus (BPES) families demonstrates a genotype–phenotype correlation. Hum. Mol. Genet. 10, 1591–1600. De Baere, E., et al., 2005. Premature ovarian failure and forkhead transcription factor FOXL2: blepharophimosis–ptosis–epicanthus inversus syndrome and ovarian dysfunction. Pediatr. Endocrinol. Rev. 2, 653–660. de Ru, M.H., Gille, J.J., Nieuwint, A.W., Bijlsma, J.B., van der Blij, J.F., van Hagen, J.M., 2005. Interstitial deletion in 3q in a patient with blepharophimosis–ptosis– epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature. Am. J. Med. Genet. A 137, 81–87. D'Haene, B., et al., 2010. FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions. Hum. Mutat. 31, E1332–E1347. Digilio, M.C., et al., 2009. 3q29 Microdeletion: a mental retardation disorder unassociated with a recognizable phenotype in two mother-daughter pairs. Am. J. Med. Genet. A 149A, 1777–1781. Engelen, J.J., De Die-Smulders, C.E., Back, E., 2002. De novo mosaic 46, XX, del(3)(q21q25)/46, XX karyotype in a patient with BPES. Genet. Couns. 13, 359–361. Genuardi, M., Calvieri, F., Tozzi, C., Coslovi, R., Neri, G., 1994. A new case of interstitial deletion of chromosome 3q, del(3q)(q13.12q21.3), with agenesis of the corpus callosum. Clin. Dysmorphol. 3, 292–296. Grinberg, I., Northrup, H., Ardinger, H., Prasad, C., Dobyns, W.B., Millen, K.J., 2004. Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy–Walker malformation. Nat. Genet. 36, 1053–1055. Jenkins, M.B., Stang, H.J., Davis, E., Boyd, L., 1985. Deletion of the proximal long arm of chromosome 3 in an infant with features of Turner syndrome. Ann. Genet. 28, 42–44. Lawson-Yuen, A., Berend, S.A., Soul, J.S., Irons, M., 2006. Patient with novel interstitial deletion of chromosome 3q13.1q13.3 and agenesis of the corpus callosum. Clin. Dysmorphol. 15, 217–220. Lim, B.C., Park, W.Y., Seo, E.J., Kim, K.J., Hwang, Y.S., Chae, J.H., 2011. De novo interstitial deletion of 3q22.3–q25.2 encompassing FOXL2, ATR, ZIC1, and ZIC4 in a patient with blepharophimosis/ptosis/epicanthus inversus syndrome, Dandy–Walker malformation, and global developmental delay. J. Child Neurol. 26, 615–618. Mackie Ogilvie, C., Rooney, S.C., Hodgson, S.V., Berry, A.C., 1998. Deletion of chromosome 3q proximal region gives rise to a variable phenotype. Clin. Genet. 53, 220–222. McMorrow, L., Reid, C., Coleman, J., Medeiros, A., D'Andrea, M., Santucci, T., 1986. A new interstitial deletion of the long arm of choromosome 3. Am. J. Hum. Genet. 39, A124.

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O'Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, P.A., Goodship, J.A., 2003. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat. Genet. 33, 497–501. O'Driscoll, M., Dobyns, W.B., van Hagen, J.M., Jeggo, P.A., 2007. Cellular and clinical impact of haploinsufficiency for genes involved in ATR signaling. Am. J. Hum. Genet. 81, 77–86. Okada, N., Hasegawa, T., Osawa, M., Fukuyama, Y., 1987. A case of de novo interstitial deletion 3q. J. Med. Genet. 24, 305–308. Ramieri, V., et al., 2011. Microdeletion 3q syndrome. J. Craniofac. Surg. 22, 2124–2128. Rea, G., McCullough, S., McNerlan, S., Craig, B., Morrison, P.J., 2010. Delineation of a recognisable phenotype of interstitial deletion 3 (q22.3q25.1) in a case with previously unreported truncus arteriosus. Eur. J. Med. Genet. 53, 162–167. Sargent, C., Burn, J., Baraitser, M., Pembrey, M.E., 1985. Trigonocephaly and the Opitz C syndrome. J. Med. Genet. 22, 39–45. Shimojima, K., Saito, K., Yamamoto, T., 2009. A de novo 1.9-Mb interstitial deletion of 3q13.2q13.31 in a girl with dysmorphic features, muscle hypotonia, and developmental delay. Am. J. Med. Genet. A 149A, 1818–1822.

Simovich, M.J., et al., 2008. Delineation of the proximal 3q microdeletion syndrome. Am. J. Med. Genet. A 146A, 1729–1735. Tohyama, J., et al., 2010. Dandy–Walker malformation associated with heterozygous ZIC1 and ZIC4 deletion: report of a new patient. Am. J. Med. Genet. A 155A, 130–133. Willemsen, M.H., et al., 2010. Further molecular and clinical delineation of the Wisconsin syndrome phenotype associated with interstitial 3q24q25 deletions. Am. J. Med. Genet. A 155A, 106–112. Wilmore, H.P., Smith, M.J., Wilcox, S.A., Bell, K.M., Sinclair, A.H., 2000. SOX14 is a candidate gene for limb defects associated with BPES and Mobius syndrome. Hum. Genet. 106, 269–276. Zahanova, S., Meaney, B., Labieniec, B., Verdin, H., De Baere, E., Nowaczyk, M.J., 2012. Blepharophimosis–ptosis–epicanthus inversus syndrome plus: deletion 3q22.3q23 in a patient with characteristic facial features and with genital anomalies, spastic diplegia, and speech delay. Clin. Dysmorphol. 21, 48–52. Zhou, Z.M., et al., 2010. Deletion and mutation analysis to FOXL2 in blepharophimosis– ptosis–epicanthus inversus syndrome. Zhonghua Yan Ke Za Zhi 46, 532–536.