Human Genetics

, Volume 126, Issue 4, pp 589–602

Redefined genomic architecture in 15q24 directed by patient deletion/duplication breakpoint mapping

Authors

  • Ayman W. El-Hattab
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • Teresa A. Smolarek
    • Division of Human GeneticsCincinnati Children’s Hospital Medical Center
  • Martha E. Walker
    • Division of Human GeneticsCincinnati Children’s Hospital Medical Center
  • Elizabeth K. Schorry
    • Division of Human GeneticsCincinnati Children’s Hospital Medical Center
  • LaDonna L. Immken
    • Clinical Genetics, Specially for Children
  • Gayle Patel
    • Clinical Genetics, Specially for Children
  • Mary-Alice Abbott
    • Clinical GeneticsBaystate Medical Center
  • Brendan C. Lanpher
    • Division of Genetics and Genomic MedicineVanderbilt University
  • Zhishuo Ou
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • Sung-Hae L. Kang
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • Ankita Patel
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • Fernando Scaglia
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • James R. Lupski
    • Department of Molecular and Human GeneticsBaylor College of Medicine
  • Sau Wai Cheung
    • Department of Molecular and Human GeneticsBaylor College of Medicine
    • Department of Molecular and Human GeneticsBaylor College of Medicine
    • Department of Medical GeneticsInstitute of Mother and Child
Original Investigation

DOI: 10.1007/s00439-009-0706-x

Cite this article as:
El-Hattab, A.W., Smolarek, T.A., Walker, M.E. et al. Hum Genet (2009) 126: 589. doi:10.1007/s00439-009-0706-x

Abstract

We report four new patients with a submicroscopic deletion in 15q24 manifesting developmental delay, short stature, hypotonia, digital abnormalities, joint laxity, genital abnormalities, and characteristic facial features. These clinical features are shared with six recently reported patients with a 15q24 microdeletion, supporting the notion that this is a recognizable syndrome. We describe a case of an ~2.6 Mb microduplication involving a portion of the minimal deletion critical region in a 15-year-old male with short stature, mild mental retardation, attention deficit hyperactivity disorder, Asperger syndrome, decreased joint mobility, digital abnormalities, and characteristic facial features. Some of these features are shared with a recently reported case with a 15q24 microduplication involving the minimal deletion critical region. We also report two siblings and their mother with duplication adjacent and distal to this region exhibiting mild developmental delay, hypotonia, tapering fingers, characteristic facial features, and prominent ears. The deletion and duplication breakpoints were mapped by array comparative genomic hybridization and the genomic structure in 15q24 was analyzed further. Surprisingly, in addition to the previously recognized three low-copy repeat clusters (BP1, BP2, and BP3), we identified two other paralogous low-copy repeat clusters that likely mediated the formation of alternative sized 15q24 genomic rearrangements via non-allelic homologous recombination.

Introduction

The recent expanded use of high resolution genome analysis by array comparative genomic hybridization (array CGH) has led to the identification of several novel microdeletion and microduplication syndromes (Slavotinek 2008; Stankiewicz and Beaudet 2007). These rearrangements can be recurrent, resulting from non-allelic homologous recombination (NAHR) between homologous low-copy repeats (LCRs), and lead to loss or gain of a dosage-sensitive gene or genes (Stankiewicz and Lupski 2002) like 15q13.3 deletion (Sharp et al. 2008), 15q24 deletion (Sharp et al. 2007), 17p11.2 deletion (Smith–Magenis syndrome) and duplication (Potocki–Lupski syndrome) (Bi et al. 2003; Potocki et al. 2007), and 17q21.31 deletion and duplication (Kirchhoff et al. 2007; Koolen et al. 2006; Sharp et al. 2006; Shaw-Smith et al. 2006). Recently, a number of non-recurrent rearrangements have been shown also to result from genomic architectural features that may stimulate their formation through the proposed fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR) mechanism (Lee et al. 2007), e.g., MECP2 duplication (Carvalho et al. 2009), deletions and duplications in 17p13.3 (Bi et al. 2009; Nagamani et al. 2009), and deletions and duplications in 17p11.2 (Zhang et al. 2009; for review see Gu et al. 2008).

With the exception of the common recurrent 15q11.2q12 deletions which, depending on parental origin, result in either Prader–Willi syndrome or Angelman syndrome, and deletions and duplications in 15q13.3 (Sharp et al. 2008; Ben-Shachar et al. 2009), rearrangements of chromosome 15q are relatively rare (Cushman et al. 2005); those involving the 15q24 region were described in patients with growth deficiency, psychomotor retardation, and birth defects (Bettelheim et al. 1998; Clark 1984; Formiga et al. 1988; Spruijt et al. 2004). Cushman et al. (2005) reported three patients with a 15q24 deletion and reviewed the previously reported cases with cytogenetically visible deletions involving the 15q22q24 region. They concluded that the majority of these cases shared developmental delay, growth deficiency, hypotonia, skeletal abnormalities, urogenital system defects, and similar facial features, including epicanthal folds, ear abnormalities, and arched palate, and it was suggested that these cases represent a new cytogenetic deletion syndrome.

Sharp et al. (2007) identified overlapping submicroscopic 15q24 deletions in four patients with developmental delay, growth deficiency, digital abnormalities, hypospadias, loose connective tissue, and characteristic facial features, including high anterior hair line, broad medial eyebrows, hypertelorism, down-slanting palpebral fissures, long smooth philtrum, and full lower lip. The deletion breakpoints mapped to homologous LCR clusters, designated as BP1, BP2, and BP3, with a minimal deletion critical region of ~1.7 Mb spanning the genomic interval from BP1 to BP2. Subsequently, Klopocki et al. (2008) reported another patient who shared common clinical features with the previously reported patients and had a 15q24 microdeletion with breakpoints mapping to BP1 and BP3. Recently, Van Esch et al. (2009) described an additional patient with a 15q24 microdeletion, who presented with features common to the previously reported patients, including developmental delay, loose connective tissue, digital and genital anomalies, and distinct facial features; in addition, this patient has a congenital diaphragmatic hernia. The proximal deletion breakpoint mapped to an LCR cluster located proximal to BP1, whereas the distal breakpoint coincided with BP2.

Six cytogenetically visible interstitial duplications involving 15q24 have been reported in patients with growth deficiency, psychomotor retardation, birth defects, and characteristic facial features, including facial asymmetry, ear malformations, micrognathia and palatal clefting (Browne et al. 2000; Dhaliwal et al. 1990; Han et al. 1999; Roggenbuck et al. 2004). Pectus excavatum was found to be associated with a duplication in 15q23–q26 (Brewer et al. 1999). Recently, an ~1.7 Mb microduplication in 15q24, extending from BP1 to BP2, has been described in a 2-year-old boy with global developmental delay, characteristic facial features, digital and genital abnormalities. This duplication is apparently reciprocal to the minimal deletion critical region for the 15q24 deletion syndrome and was inherited from his healthy father (Kiholm Lund et al. 2008).

We report four new patients with an overlapping 15q24 microdeletion (cases 1–4) and further delineate the 15q24 deletion syndrome. In addition, we describe four patients with 15q24 microduplications: one patient (case 5) with duplication involving part of the minimal deletion critical region, and two siblings (cases 6 and 7) and their mother, who have duplication adjacent and distal to the minimal deletion critical region. We also redefine the genomic structure of this chromosomal region guided by breakpoint mapping of the above patients that revealed additional LCR clusters.

Subjects and methods

DNA samples

Patients 1–5 were referred to the Medical Genetics Laboratories of Baylor College of Medicine for clinical array-CGH analysis. Patients 6 and 7 were referred to the Cytogenetic Laboratory of Cincinnati Children’s Hospital Medical Center for SNP array analysis. DNA samples were obtained from the probands and their family members after informed consents approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine or Cincinnati Children’s Hospital Medical Center. DNA was extracted from whole blood using the Puregene DNA extraction kit (Gentra, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Array CGH

The four 15q24 deletion cases 1–4 and the duplication case 5 were identified through screening the database of Baylor College of Medicine Medical Genetics Laboratories of array CGH studies performed in over 9,000 patients using oligonucleotide Chromosomal Microarray Analysis (CMA Versions 6 and 7 OLIGO) (Cheung et al. 2005; Ou et al. 2008). Of these patients, approximately 4,700 patients were tested using V6 OLIGO (44 K array) and over 4,000 patients using V7 OLIGO (105 K array).

The oligonucleotide based chromosomal microarray version 6 (CMA V6 OLIGO) consists of 44 K targeted oligonucleotides emulating genomic regions contained within BAC clones arranged in the BAC V6 CMA. It contains an average of 28–30 oligos per genomic region interrogated by single BAC clones with a minimum of approximately 10–15 oligos whenever possible (Ou et al. 2008). This array was used for patients 2, 3, and 4.

The oligonucleotide based chromosomal microarray version 7 (CMA V7 OLIGO) utilizes array-based comparative genomic hybridization with approximately 105,000 oligos covering the whole genome at an average resolution of 30 kb with increased coverage at known disease loci. Included in CMA are probes for all the known microdeletion/microduplication syndromes (over 270 genetic syndromes), 41 unique subtelomeric regions, all 43 unique pericentromeric regions and the mitochondrial genome. In addition to these targeted regions, the entire genome (between disease regions) is covered with an average resolution of 30 kb, excluding repetitive sequences through a combination of bioinformatics, computation, and empirical studies. The array also includes six regions of known polymorphic variants: the Amylase (AMY1A) gene cluster in 1p21.2, the UDP glucuronosyltransferase 2 (UGT2B17) gene in 4q13.2, the chemokine CC motif ligand 3 and 4 (CCL3, CCL4) genes in 17q12, the defensin beta 107B (DEFB107B) gene family in 8p23.1, the opsin 1 medium-wave-sensitive 2 (OPN1MW2) gene in Xq28, and exon 2 of the Apolipoprotien (LPA) gene in 6q26 to serve as internal controls. This array was performed in patients 1 and 5. Patient 2 was also analyzed with CMA V.7 OLIGO to better delineate the breakpoints (Fig. 1a, b).
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Fig. 1

a, b The oligonucleotide based chromosomal microarray version 7 (CMA V7 OLIGO) profiles for patients 1 and 2. The averaged combined data of hybridizations performed using reference DNA. Oligonucleotides presented with red color and displaced down, indicating a loss of chromosome 15 material in the patient versus the reference DNA. c Results of Agilent 244 K array-based oligonucleotide CGH performed on DNA samples for patients 3 showing the deleted region 15q24 (70.750–73.856 Mb), patient 4 showing the deleted region (70.780–73.856 Mb), and patient 5 showing the duplicated region 15q24 (70.708–73.332 Mb). A deviation of the dots to the left of the central axis indicates a loss in copy number (represented with green dots), and a deviation to the right of the central axis indicates a gain in copy number (represented by red dots). d Chromosome 15q24 duplication in patients 6 and 7 using the Illumina DNA Beadarray (SNP) 370 K chip (region indicated by arrows and shaded). The upper plot shows the B allele frequency and the lower plot shows the Log R ratio (DNA dosage). SNP region is showing duplication between rs9672824 and rs4886534. Linear position on chromosome 15:73858160–75969880, with the total size of 2.11 Mb, and including 208 markers. e Results of the FISH analysis in patient 4. The two green fluorescence signals represent chromosome 15 centromeric FISH probe, and the single red signal represents probe specific to the deleted region (indicated by arrow), confirming the 15q24 deletion

The rearrangements in three patients (cases 3–5) were further investigated using a high-resolution Agilent 244 K Whole Human Genome Oligo Microarray (Agilent Technologies, Inc., Santa Clara, CA), which contains 238,459 probes, representing a compiled view of the human genome at a 8.9 kb overall median probe spacing (7.4 kb in Refseq genes) (http://www.chem.agilent.com) (Fig. 1c). The procedures for DNA digestion, labeling, and hybridization were performed according to the manufacturer’s instructions with some modifications (Probst et al. 2007).

The slides were scanned into image files using a GenePix Model 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA) or an Agilent G2565 laser scanner. Microarray image files of oligo arrays were quantified using Agilent Feature extraction software (v9.0), and text file outputs from the quantitation analysis were imported to our in-house analysis package for copy number analysis, as described (Ou et al. 2008).

SNP microarray

SNP microarray studies were performed in patients 6 and 7 using the commercially available Illumina 370 K DNA Beadchip. DNA was processed according to manufacturer’s specifications and chips processed according to the protocol.

Cytogenetic and FISH analyses

Confirmatory chromosome and FISH analyses with BAC clones were performed on peripheral blood lymphocytes using standard procedures after detection of copy number changes observed in the CMA as described by Shchelochkov et al. (2008).

Bioinformatics and in silico sequence analysis

Genomic sequence based on the oligonucleotide coordinates from the array CGH experiment was downloaded from UCSC genome browser (Build 36, UCSC genome browser, March 2006). Interspersed repeat sequences were analyzed by RepeatMasker (http://www.repeatmasker.org). Regional assemblies for known LCR15q24s (BP1, BP2, and BP3) and a search for additional LCRs were assembled using NCBI BLAST 2 and the Sequencher software (Gene Codes). We eliminated small insertions/deletions from the calculation of the DNA sequence identity and took only base pair changes into account.

Results

Clinical findings associated with 15q24 deletion and duplication

We identified overlapping 15q24 microdeletions involving the minimal critical region and ranging in size from 3.10 to 3.95 Mb, in four unrelated patients (cases 1–4). These individuals share many features, including developmental delay, short stature, hypotonia, digital anomalies, joint laxity, genital abnormalities, and characteristic facial features, including a long smooth philtrum and ear malformations (Table 1; Fig. 2a, b). The deletions are de novo in patients 1, 3, and 4. The mother of patient 2 was reported to be healthy, but limited data are available about the father, and both parents were not tested.
Table 1

The deleted regions and the clinical manifestations in patients with the 15q24 deletion

 

Case 1

Case 2

Case 3

Case 4

Van Esch et al. (2009)

Klopocki et al. (2008)

Sharp et al. (2007) IMR349

Sharp et al. (2007) C45/06

Sharp et al. (2007) ID204

Sharp et al. (2007) IMR371

del 15q24 (length)

72.252–75.937 Mb (3.686 Mb)

72.130–76.080 Mb (3.950 Mb)

70.750–73.856 Mb (3.106 Mb)

70.708–73.856 Mb (3.148 Mb)

70.6–73.7 Mb (3.1 Mb)

72.2–75.9 Mb (3.7 Mb)

72.15–76.01 Mb (3.86 Mb)

72.15–76.01 Mb (3.86 Mb)

72.15–73.85 Mb (1.7 Mb)

70.40–74.21 Mb (3.72 Mb)

Inheritance

De novo

Unknown

De novo

De novo

De novo

De novo

De novo

De novo

Unknown

De novo

Age and gender

33 months

5 months

14 years

9 years

33 years

10 years

14 years

14 years

33 years

15 years

Male

Female

Male

Male

Male

Male

Male

Male

Male

Male

Growth

Short stature

Short stature

Normal growth

Obesity

Obese

Obesity

Short stature

Short stature

Short stature

Normal growth

    

Macrocephaly

 

Microcephaly

Microcephaly

Microcephaly

 
      

IUGR

IUGR

IUGR

 

Development

Mild delay

MR

Delayed

Moderate MR

Severe MR

Mild MR

Mild MR

Mild MR

Mild MR

Mild MR

Face

High anterior hair line

Frontal bossing

Normal appearance

Asymmetry

High anterior hair line

High anterior hair line

High anterior hair line

High anterior hair line

High anterior hair line

High anterior hair line

Broad forehead

Brachycephaly

 

Round face

Long face

 

Long narrow face

Asymmetry

Asymmetry

Asymmetry

    

Small maxilla large mandible

  

Happy facial expression

Happy facial expression

Happy facial expression

         

Long narrow face

Eye

Sparse eye brows

  

Normal appearance

Broad medial eyebrows

Broad medial eyebrows

Broad medial eyebrows

Broad medial eyebrows

Broad medial eyebrows

Broad medial eyebrows

 

Hypertelorism

  

Hypertelorism

 

Hypertelorism

Hypertelorism

Hypertelorism

Hypertelorism

  

Down-slanting palpebral fissures

 

Down-slanting palpebral fissures

Down-slanting palpebral fissures

Down-slanting palpebral fissures

Down-slanting palpebral fissures

Down-slanting palpebral fissures

 

Epicanthus

Epicanthus

Epicanthus

 

Strabismus

Strabismus

 

Strabismus

Strabismus

 
        

Epicanthus

 

Nose

Depressed nasal bridge

Broad upturned nasal tip

Normal appearance

Normal appearance

Broad nasal base

Hypoplastic nostrils

Broad nasal base

Broad nasal base

Small nose

Broad nasal base

      

Flaring alae nasi

Flaring alae nasi

Hypoplastic alae nasi

Flaring alae nasi

Mouth

Long smooth philtrum

Small mouth

Small mouth

Smooth philtrum

Long smooth philtrum

Long smooth philtrum

Long philtrum

Long smooth philtrum

Long smooth philtrum

Long philtrum

Full lower lip

   

High palate

 

Full lower lip

Full lower lip

Full lower lip

Full lower lip

    

Bifid uvula

     

Ear

Ear lobe pit

Normal appearance

Thick, small ears

Cup-shaped protruding ears

Large ears

NA

Ear abnormalities

Ear abnormalities

Small everted ears

Hearing loss

  

Ear lobe pit

   

Hearing loss

   

Nervous system

Hypotonia

Myelo-meningocele Hydro-cephalus

Hypotonia

Hypotonia

Hypotonia

Hypotonia

Wide basal cisterna on brain MRI

Hypotonia

NA

NA

       

Dysplastic corpus callosum on brain MRI

  
       

Seizure

  

Genital

Normal

 Normal

Hypospadias

Microphallus

Microphallus

Microphallus

Normal

Hypospadias

Hypospadias

Hypospadias

    

Cryptorchidism

     

Skeletal

No abnormalities

Clubfeet

Joint laxity

Joint laxity

Scoliosis

Joint laxity

Joint laxity

Scoliosis

Joint laxity

Joint laxity

  

Pectus carinatum

Pes planus

Clubfeet

Lumbar lordosis

Scoliosis

  

Narrow chest

     

Genua valga

   

Pes cavus

Digital anomalies

Small hands

Overriding second toes

Long first toes and short second toes

Brachymeso-palangy II and V

Long slender fingers with distal tapering

Broad thumb

Long slender fingers

Proximally implanted thumbs

Small hands

Hypoplastic right thumb

 

Clinodactyly

 

Clinodactyly

 

Brachydactyly

Proximally implanted thumbs

 

Brachydactyly

Contractures of fingers

      

Clinodactyly

    

Respiratory

No symptoms

No symptoms

Recurrent ear infections

Recurrent ear infections

High pitched voice

Nasal speech

Recurrent ear infections

Low tone voice

Recurrent upper airway infections

Recurrent chest infections

  

Velo-pharyngeal insufficiency

  

Hoarse voice

   

Asthma

  

Soft nasal speech

       

Other

Acute lymphoblastic leukemia

Tetralogy of Fallot

Café au lait spots

Aggressiveness

Hyperactivity Aggressiveness

Inguinal and umbilical hernia

Attention deficit hyperactivity disorder

Edema of extremities

Feeding difficulties as child

Diaphragmatic hernia

Feeding difficulties

 

Feeding difficulties

Cafe-au-lait spots

Diaphragmatic hernia

Skin laxity

Autistic features

GH deficiency

 

Inguinal hernia

Hepatomegaly

  

Acanthosis nigricans

Inguinal hernia

Splenomegaly

GH deficiency

Hypogonado-tropic hypogonadism

  
      

Delayed puberty

Bowel atresia

  
      

Bowel atresia

   

NA not reported, MR mental retardation, IUGR intrauterine growth restriction

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Fig. 2

a Patient 1: Facial features, including high anterior hair line, broad forehead, sparse eye brows, epicanthal fold, depressed nasal bridge, long smooth philtrum, and full lower lip. b Patient 4: Facial features, including round face, facial asymmetry with left-sided smaller, cup-shaped protruding ears, and smooth philtrum. c Patient 5: Facial features, including long face, epicanthal folds, down-slanting palpebral fissures, ptosis, full puffy hooded eyelids, high nasal bridge, smooth philtrum, thin upper lip, and full lower lip. d Patient 6: Facial features, including rounded face, hypertelorism, flattened nasal bridge, and prominent ears

We also identified four patients with a 15q24 microduplication, one patient (case 5) has a duplication involving a portion of the minimal deletion critical region, whereas two siblings (case 6–7) and their mother have a duplication adjacent and distal to the minimal deletion critical region. Patient 5 is a 15-year-old male with short stature, mild mental retardation (IQ 71), behavioral problems, decreased range of motion of elbows, wrist and fingers, increased tone, and characteristic facial features (Table 2; Fig. 2c). Serum amino acids and urine organic acid analysis, karyotype, fragile X syndrome DNA testing, brain MRI, and EEG were all normal. Chromosomal microarray analysis revealed an ~2.62 Mb duplication in 15q24 (70.708–73.332 Mb, build 36). The mother was reported to have depression. She was tested by FISH for the duplication with normal results. The father was reported to have schizophrenia; he was not available for either examination or testing, but the mother reported that the patient’s facial features do not resemble his father’s facies.
Table 2

The duplicated regions with the clinical manifestations in patients with the 15q24 duplication and the summary of the clinical features of the 10 patients with the 15q24 deletion

 

15q24 deletion

15q24 duplications involving the minimal deletion critical region

15q24 duplications not involving the minimal critical region

Case 5

Kiholm Lund et al. (2008)

Case 6

Case 7

Dup 15q24 (length)

 

70.708–73.332 Mb (2.62 Mb)

72.15–73.85 Mb (1.7 Mb)

73.858–75.969 Mb (2.11 Mb)

73.858–75.969 Mb (2.11 Mb)

Inheritance

 

Unknown

Inherited from the father

Inherited from the mother

Inherited from the mother

Age and gender

 

15 years, male

2 years, male

3.5 years, male

2 years, female

 Growth

  Short stature

5/10

Short stature

Normal growth

Normal growth

Normal growth

  Microcephaly

3/10

  IUGR

3/10

  Obesity

3/10

Development delay

10/10

Mild MR

Delayed

Mildly delayed

Mildly delayed

Face

 High anterior hair line

7/10

Normal appearance

 Facial asymmetry

4/10

 Long face

3/10

Long face

Round face

Round face

    

Flat occiput

 
    

Plagiocephaly

 

Eye

 Broad medial eyebrows

6/10

 Down-slanting palpebral fissures

6/10

Down-slanting palpebral fissures

Down-slanting palpebral fissures

 Hypertelorism

6/10

Hypertelorism

Hypertelorism

Hypertelorism

 Epicanthus

4/10

Epicanthus

Full puffy hooded eyelids

Epicanthus

Epicanthus

 Strabismus

4/10

Strabismus

  

Ptosis

Thick upper eyelid

  
   

High arched eyebrows

  

Nose

 Broad nasal base

4/10

High nasal base

Broad nasal bridge

Flat nasal bridge

Normal appearance

Mouth

 Long/smooth philtrum

8/10

Smooth philtrum

Smooth philtrum

Normal appearance

Normal appearance

 Full lower lip

5/10

Full lower lip

Full lower lip

  
  

High arched palate

Triangular mouth

  
  

Crowded teeth

   
  

Retrognathia

   

Ear

 Malformation

7/10

Normal appearance

Low set posteriorly rotated ears

Prominent ears

Prominent ears

    

Ear tag

 

Nervous system

 Hypotonia

6/10

Hypertonia

Hypertonia

Truncal hypotonia

Truncal hypotonia

   

Agenesis of corpus callosum on brain MRI

Lower extremities hypertonia

 

Genital

7/10

Normal

Hypospadias

Normal

Normal

Skeletal

 Joint laxity

6/10

Decreased joint range of motion

Joint contractures

No abnormalities

No abnormalities

Digital

10/10

Broad thumbs

Overlapping fingers

Tapering fingers

Tapering fingers

 

Blunt finger tips

Hypoplastic nails

  
 

Hyperconvex nails

Broad finger pads

  
 

Broad feet

   

Other

 

Attention deficit hyperactivity disorder

Oligohydramnios

Recurrent sinusitis, bronchitis, and otitis

Gastro-esophageal disease

  

Asperger syndrome

Low posterior hairline

  

Patient 6 is a 3.5-year-old boy with mild global developmental delay, truncal hypotonia, lower extremity hypertonia, and characteristic facial features (Table 2; Fig. 2d). Renal ultrasound, brain MRI, hearing evaluation, fragile X syndrome DNA testing, and karyotype all were normal. A SNP microarray revealed an ~2.11 Mb duplication of chromosome 15q24 (73858160–75969880, build 36). His 2-year-old sister (case 7) has developmental delay, truncal hypotonia, and characteristic facial features (Table 2). SNP microarray revealed the same ~2.11 Mb duplication in chromosome 15q24. Both children were adopted. The biological mother, who has been described as a slow learner and attended special education classes in school, was tested by FISH and found to have the same duplication; she was unavailable for physical examination, and limited data were available about her medical and mental history (Table 2). Both the sister (case 7) and the mother were also found to have gain of 15 clones at Xp22.31 that was interpreted as a benign copy number change.

Additional complexity to 15q24 genomic architecture

Using Blast2, we analyzed the DNA sequence in the 15q24 region between 70.600 and 76.600 Mb that harbors all the genomic rearrangements, and searched for alignments more than 300 bp in length and greater than 90% DNA sequence identity. Unexpectedly, in addition to the three previously identified LCR clusters (BP1, BP2, and BP3) (Sharp et al. 2007), we identified two more paralogous LCR clusters that we designated LCR15q24A, spanning ~65 kb, and located proximal to BP1, and LCR15q24C, spanning ~124 kb, and located between BP1 and BP2. For consistency, we elected to change the nomenclature for the previously identified LCR clusters as follows: LCR15q24B for BP1 (spanning ~54 kb), LCR15q24D for BP2 (spanning ~128 kb), and LCR15q24E for BP3 (spanning ~93 kb). We constructed a physical map for this region where LCR subunits with identity greater than 90% are represented with the similar-colored arrows with arrowheads indicating the LCR subunit orientations and arrows lengths, the subunit sizes. Most of these subunits have DNA sequence identity greater than 95% and are represented in darker colors (e.g. red), whereas some subunits have sequence identity between 90 and 94%, and those subunits are shown in lighter colors (e.g. light red) (Fig. 3; Table 3). Interestingly, the homologous subunits located in LCR15q24A and LCR15q24C are in direct orientation, whereas the subunits located in LCR15q24B (BP1) and LCR15q24E (BP3) are in the opposite orientation to the subunits located in LCR15q24A and LCR15q24C. In contrast, LCR15q24D (BP2) has a complex structure with most of the subunits being directly oriented with reference to LCR15q24A and LCR15q24C, whereas two small subunits (~3 kb subunit in light green color and ~7 kb subunit in light brown color as shown in Fig. 3) are directly oriented to subunits in LCR15q24B and LCR15q24E.
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Fig. 3

Summary of the results of the computational analysis of the genomic architecture in 15q24. The five LCR clusters: ~65 kb LCR14q24A (70.685–70.750 Mb), ~54 kb LCR14q24B (72.134–72.188 Mb), ~124 kb LCR14q24C (73.295–73.419 Mb), ~128 kb LCR14q24D (73.760–73.888 Mb), and ~93 kb LCR15q24E (75.948–76.041 Mb). LCRs subunits with sequence identity greater than 90% are shown as arrows with similar colors. The subunits with sequence identity more than 95% are represented in darker colors (e.g. red), whereas, the subunits with sequence identity between 90 and 94% are shown in lighter colors (e.g. light red). Arrowheads indicate LCR subunits orientation. The numbers inside the arrow represent the length of the area in kb

Table 3

The coordinates, length, and orientation of LCR subunits for the five LCR clusters, LCR15q24A, LCR15q24B, LCR15q24C, LCR15q24D, and LCR15q24E

 

Start

End

Length (kb)

Orientation

Subunit color

LCR14q24A

70,685,541

70,691,956

6,415

+

Light red

70,707,347

70,719,598

12,251

+

Blue

70,719,670

70,721,200

1,530

+

Green

70,721,201

70,728,580

7,379

+

Brown

70,729,074

70,750,593

21,519

+

Red

LCR14q24B

72,134,535

72,144,840

10,305

Violet

72145146

72,166,963

21,817

Red

72,167,460

72,174,829

7,369

Brown

72,174,830

72,176,359

1,529

Green

72,176,431

72,188,813

12,382

Blue

LCR14q24C

73,295,360

73,303,732

8,372

+

Light brown

73,304,189

73,307,393

3,204

+

Light green

73,328,277

73,330,953

2,676

+

White

73,332,931

73,354,116

21,185

+

Red

73,354,117

73,356,715

2, 598

+

Brown

73,357,211

73,378,995

21,784

+

Red

73,379,350

73,386,517

7,167

+

Violet

73,387,968

73,393,463

5,495

+

Pink

73,406,153

73,418,809

12,656

+

Light blue

73,463,299

73,466,244

2,945

+

White

LCR14q24D

73,760,220

73,772,788

12,568

+

Yellow

73,777,284

73,796,657

19,373

+

Black

73,807,737

73,815,337

7,600

+

Tan

73,828,848

73,838,186

9,338

+

Light blue

73,839,617

73,842,915

3,298

Light green

73,842,941

73,849,886

6,945

Light brown

73,849,902

73,870,477

20,575

+

Light red

73,870,478

73,881,040

10,562

+

Light purple

73,882,218

73,888,387

6,169

+

Pink

LCR14q24E

75,948,172

75,954,860

6,688

Tan

75,955,173

75,973,569

18,396

Black

75,973,925

75,985,550

11,625

Yellow

75,987,909

76,011,121

23,212

Red

76,011,796

76,019,176

7,380

Light brown

76,019,184

76,020,691

1,507

Light green

76,020,763

76,033,111

12,348

Light blue

76,039,907

76,041,935

2,028

Light violet

The last column shows the colors of the subunits as presented in Fig. 3. In bold are highlighted the newly identified LCR15q24A and LCR15q24C

Rearrangement breakpoint analysis supports genomic complexity

The breakpoints for the deleted and the duplicated regions were determined using either array CGH or SNP arrays. The proximal and distal deletion breakpoints in patients 1 and 2 map to LCR15q24B (BP1) and LCR15q24E (BP3), respectively. The proximal breakpoints for the deletions in patients 3 and 4, and the duplication in patient 5, map to LCR15q24A. The distal breakpoints for deletions in patients 3 and 4 map to LCR15q24D (BP2); however, the distal breakpoint for duplication in patient 5 maps to the novel LCR15q24C. The proximal and distal breakpoints for duplications in patients 6 and 7 map to LCR15q24D (BP2) and LCR15q24E (BP3), respectively (Fig. 4). The findings of breakpoints mapping to LCRs support that NAHR is the mechanism for these alterations.
https://static-content.springer.com/image/art%3A10.1007%2Fs00439-009-0706-x/MediaObjects/439_2009_706_Fig4_HTML.gif
Fig. 4

Schematic of the deleted/duplicated regions in chromosome 15q24: Case 1 (15q24del: 72.252–75.937 Mb), Case 2 (15q24del: 72.130–76.080 Mb), Case 3 (15q24del: 70.750–73.856 Mb), Case 4 (15q24del: 70.708–73.856 Mb), Case 5 (15q24dup: 70.708–73.332 Mb), and Cases 6 and 7 (15q24dup: 73.858–75.969 Mb), and the previously cases reported by Sharp et al. (2007), ID204 (15q24del: 72.15–73.85 Mb), IMR349 (15q24del: 72.15–76.01 Mb), C45/06 (15q24del: 72.15–76.01 Mb), and IMR371 (15q24del: 70.40–74.21 Mb), Klopocki et al. (2008), (15q24del: 72.2–75.9 Mb), Van Esch et al. (2009) (15q24del: 70.6–73.7 Mb), and Kiholm Lund et al. (2008) (15q24dup: 72.14–73.85 Mb). The LCRs are represented as arrows, LCR15q24A, LCR15q24B, LCR15q24C, LCR15q24D, and LCR15q24E. The minimal critical deletion region of ~1.75 Mb extends between LCR15q24B and LCR15q24D (72.134–73.888 kb) and the suggested minimal critical region for 15q24 duplication of ~1.33 Mb extending from LCR15q24B to LCR15q24C (72.134–73.419 kb). Selected genes in this region are shown: STRA6, CYP11A1, SEMA7A, CSK, CPLX3, MPI, and SIN3A

Discussion

By comparing the clinical features in our four 15q24 microdeletion patients (cases 1–4) with the other six previously reported patients with a 15q24 microdeletion (Klopocki et al. 2008; Sharp et al. 2007; Van Esch et al. 2009), we observed many common features, supporting the notion that subjects harboring 15q24 deletion manifest a distinct syndrome (Tables 1, 2). These features include developmental delay that varies from mild to severe, hypotonia, short stature, digital anomalies, joint laxity, genital anomalies, and characteristic facial features, including a high anterior hair line, facial asymmetry, ear malformations, broad medial eyebrows, down-slanted palpebral fissures, hypertelorism, epicanthal folds, strabismus, long smooth philtrum, full lower lip, and broad nasal base. The distal extremity malformations observed in all reported patients consist of thumb anomalies, small hands with brachydactyly, clinodactyly, and foot-ankle deformities. Less common features are microcephaly, IUGR, feeding difficulties, low tone or nasal speech, and obesity. Diaphragmatic hernia was observed in two patients (Sharp et al. 2007; Van Esch et al. 2009); tetralogy of Fallot and myelomeningocele were observed in one patient (case 2), and acute lymphoblastic leukemia was also found in one patient (case 1). All patients with a 15q24 microdeletion share the same ~1.75 Mb minimal deletion critical region located between LCR15q24B (BP1) and LCR15q24D (BP2) (Fig. 4).

Recently, a microduplication in 15q24 involving the minimal critical deletion region has been described with proximal and distal breakpoints mapping to LCR15q24B (BP1) and LCR15q24D (BP2), respectively (Kiholm Lund et al. 2008). We identified another case of a 15q24 microduplication involving a portion of the minimal deletion critical region, mapping from LCR15q24A to LCR15q24C (case 5) (Fig. 4). These two patients share some clinical features, including developmental delay, hypertonia, joints limitation, digital abnormalities, and certain facial features, including down-slanting palpebral fissures, epicanthus, full eyelids, smooth philtrum, and full lower lip. The previously reported patient with a 15q24 microduplication had hypospadias, agenesis of corpus callosum, low set ears, and normal growth parameters; however, our patient has normal genitalia, normal brain MRI, normally set ears, short stature, and behavioral problems (Table 2). These two patients with a 15q24 duplication involving the minimal critical deletion region share some overlapping features with patients with a 15q24 deletion, including developmental delay, short stature, digital anomalies, genital anomalies and some facial features (epicanthal folds, down-slanting palpebral fissures, hypertelorism, ear malformation, smooth philtrum, and full lower lip). Most patients with a 15q24 deletion have hypotonia and joint laxity, whereas the two patients with duplication exhibit increased tone and joints limitations. Patient 5 exhibits behavioral disorders that were not reported in patients with the 15q24 deletion syndrome (Table 2). The patient reported by Kiholm Lund et al. (2008) inherited the duplication from a healthy father; the father of patient 5 was unavailable for testing, so it is unclear whether the duplications are responsible for the observed phenotype. However, the similarity in the phenotype between these two patients and the overlapping features with patients with 15q24 deletion support the contention that this duplication could be responsible for the observed phenotype, suggesting that a 15q24 duplication may represent a clinical syndrome with minimal critical region of 1.33 Mb spanning between LCR15q24B (BP1) and LCR15q24C (Fig. 4). Nevertheless, more subjects with similar duplications need to be studied to support or refute this hypothesis.

We also report two siblings and their mother with a 15q24 microduplication distal and adjacent to the minimal deletion critical region with breakpoints mapping to LCR15q24D (BP2) and LCR15q24E (BP3) (cases 6 and 7) (Fig. 4). These siblings manifest a milder phenotype with mild developmental delay, hypotonia, tapering fingers, and characteristic facial features, including round face, hypertelorism, and prominent ears (Table 2). The overlapping features between these siblings and patients with a 15q24 deletion are less obvious and include only developmental delay, hypotonia, and hypertelorism. The two siblings inherited the duplication from their mother; thus it is also unclear whether this duplication is responsible for the phenotype. However, the fact that their mother has learning disabilities and the similarity in the phenotype between the siblings indicate that this duplication could be responsible for the observed phenotype; nevertheless, more subjects with similar duplication need to be studied.

The finding of 15q24 deletion and duplication breakpoints clustering in LCR regions supports the notion that NAHR is the most likely mechanism for these genomic rearrangements (Stankiewicz and Lupski 2002). LCR15q24B (BP1) and LCR15q24E (BP3) contain multiple homologous subunits that are directly oriented and they harbor breakpoints for the deletions in patients 1 and 2. In addition, LCR15q24A and LCR15q24C contain multiple homologous subunits that are directly oriented and they encompass breakpoints of the duplication in patient 5. The proximal breakpoints for duplications in patients 6 and 7, and the distal breakpoints for deletions in patients 3 and 4 map to LCR15q24D (BP2) that has a complex structure with most subunits being directly oriented to LCR15q24A. The latter repeat harbors the proximal breakpoints for deletions in patients 3 and 4. LCR15q24D (BP2) has two small subunits (~3 kb subunit in light green color and ~7 kb subunit in light brown color) directly oriented to subunits in LCR15q24E (BP3), that harbors the distal breakpoints for duplication in patients 6 and 7. These two small subunits are also directly oriented to the subunits in LCR15q24B (BP1). We therefore believe that these two small subunits mediated the NAHR in patients 6 and 7 and in patient ID204 (Sharp et al. 2007), who has a deletion with breakpoints mapping to 15q24B (BP1) and 15q24D (BP2) (Figs. 3, 4).

The four 15q24 deletion patients (cases 1–4) share the same 1.7 Mb minimal deletion region extending from LCR15q24B (BP1) to LCR15q24D (BP2). The deleted regions extend more distally in patients 1 and 2, the cases IMR349 and C45/06 (Sharp et al. 2007), and the case reported by Klopocki et al. (2008); compared to patients 3 and 4, case IMR371 (Sharp et al. 2007), and the case reported by Van Esch et al. (2009) with deletions extend more proximally (Fig. 4). Although all these patients share many similar features, they also differ in several aspects, e.g., growth deficiency and obesity (Table 1), and thus it is difficult to find a specific genomotype–phenotype (Bi et al. 2009) correlation with this limited set of patients for each specific deletion.

The critical region contains the STRA6 gene that recently has been shown to be associated with syndromic microphthalmia, alveolar capillary dysplasia, and other malformations (Pasutto et al. 2007), the cholesterol side chain cleavage enzyme gene (CYP11A1) associated with lipoid congenital adrenal hyperplasia (Tajima et al. 2001), and the mannose phosphate isomerase gene (MPI) that is associated with congenital disorder of glycosylation type Ib (Niehues et al. 1998). All these disorders are autosomal recessive. It was suggested that the haploinsufficiency of cholesterol side chain cleavage enzyme might contribute to the genital abnormalities (Klopocki et al. 2008). There are two other candidate genes located in this region, which are related to the nervous system and their loss may contribute to the developmental delay. Semaphorin 7A (SEMA7A) enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development (Pasterkamp et al. 2007), and complexin 3 (CPLX3) is a positive regulator of neurotransmitter release in mouse hippocampal neurons (Reim et al. 2005) (Fig. 4).

Patient 1 developed acute lymphoblastic leukemia. The loss of two genes, SIN3A and CSK, may lead to an increased risk of developing neoplasm. SIN3A encodes a protein that participates in the formation of the histone deacetylase complex, which in turn interacts with tumor suppressor proteins like P53 and pRb (Fleischer et al. 2003). Cytoplasmic-src tyrosine kinase (CSK) down-regulates tyrosine kinase activity of the SRC oncoprotein and might function as an antioncogene (Armstrong et al. 1993) (Fig. 4). Larger numbers of patients with 15q24 deletion need to be studied before drawing any conclusions about neoplasm predisposition.

In conclusion, patients with a 15q24 deletion sharing the minimal deletion critical region between 15q24B (BP1) and 15q24D (BP2) have many common clinical features, supporting the notion that 15q24 deletion leads to a recognizable syndrome. Based on two patients with overlapping duplications, we better defined the phenotype of 15q24 duplication syndrome and narrowed its minimal critical region to ~1.3 Mb spanning between LCR15q24B (BP1) and the identified LCR15q24C. The breakpoints for the deleted and duplicated regions (except the case IMR371 (Sharp et al. 2007)) map to LCR clusters, indicating that NAHR is the likely mechanism of their formation.

Acknowledgments

We would like to thank our patients and their families. P.S. was supported in part by Grant R13-0005-04/2008 from the Polish Ministry of Science and Higher Education.

Copyright information

© Springer-Verlag 2009