How to cite this article: South ST, Bleyl SB, Carey JC. 2007. Two unique patients with novel microdeletions in 4p16.3 that exclude the WHS critical regions: Implications for critical region designation. Am J Med Genet Part A 143A:2137–2142.
Two unique patients with novel microdeletions in 4p16.3 that exclude the WHS critical regions: Implications for critical region designation†
Article first published online: 14 AUG 2007
Copyright © 2007 Wiley-Liss, Inc.
American Journal of Medical Genetics Part A
Volume 143A, Issue 18, pages 2137–2142, 15 September 2007
How to Cite
South, S. T., Bleyl, S. B. and Carey, J. C. (2007), Two unique patients with novel microdeletions in 4p16.3 that exclude the WHS critical regions: Implications for critical region designation. Am. J. Med. Genet., 143A: 2137–2142. doi: 10.1002/ajmg.a.31900
- Issue published online: 17 AUG 2007
- Article first published online: 14 AUG 2007
- Manuscript Accepted: 17 MAY 2007
- Manuscript Received: 29 DEC 2006
- Wolf-Hirschhorn syndrome critical region;
- 4p deletion;
- ring chromosome;
Wolf-Hirschhorn syndrome (WHS) is characterized by growth delay, developmental delay, hypotonia, seizures, feeding difficulties, and characteristic facial features. Deletion of either of two critical regions (WHSCR and WHSCR-2) within chromosome band 4p16.3 has been proposed as necessary for the minimal clinical manifestations of WHS and controversy remains regarding their designation. We describe two patients with novel terminal microdeletions in 4p16.3 who lack the characteristic facial features but do show some of the more nonspecific manifestations of WHS. The first patient had a ring chromosome 4 with an intact 4q subtelomere and a terminal 4p microdeletion of approximately 1.27–1.46 Mb. This deletion was distal to both proposed critical regions. The second patient had a normal karyotype with a terminal 4p microdeletion of approximately 1.78 Mb. This deletion was distal to WHSCR and the breakpoint was near or within the known distal boundary for WHSCR-2. Both patients showed significant postnatal growth delay, mild developmental delays and feeding difficulties. Their facial features were not typical for WHS. The phenotype of the first patient may have been influenced by the presence of a ring chromosome. Seizures were absent in the first patient whereas the second patient had a complex seizure disorder. Characterization of these patients supports the hypothesis that a gene in WHSCR-2, LETM1, plays a direct role in seizure development, and demonstrates that components of the WHS phenotype can be seen with deletions distal to the known boundaries of the two proposed critical regions. These patients also emphasize the difficulty of mapping clinical manifestations common to many aneusomy syndromes. © 2007 Wiley-Liss, Inc.
Wolf-Hirschhorn syndrome (WHS) is a congenital multiple malformation syndrome due to deletions of the distal short arm of chromosome 4 (OMIM # 194190). WHS is considered a contiguous gene syndrome with a critical region in band 4p16.3 and no single gene deletions or intragenic mutations have been found that confer the full WHS phenotype. Identification of a critical region for a contiguous gene syndrome has both diagnostic and prognostic value. Designation of critical regions for WHS has occurred through 4p deletion mapping in patients with and without the minimal WHS phenotype [Estabrooks et al., 1993; White et al., 1995; Altherr et al., 1997; Wright et al., 1997; Rauch et al., 2001; Zollino et al., 2003; Rodriguez et al., 2005]. There are currently two proposed critical regions within 4p16.3, but relationships between specific gene deletions and the different clinical manifestations of WHS remain unknown.
We describe two patients with novel 4p terminal deletions who have growth retardation and developmental delay but lack the typical facial features of WHS. We compare the size and location of their deletions to the two proposed WHS critical regions and to the putative causative genes in the region, and discuss the degree to which this characterization improves our understanding of the phenotype/genotype correlation in WHS.
MATERIALS AND METHODS
Chromosome, Genomic Microarray, and FISH Analysis
G-banded karyotype analysis was performed using standard procedures on PHA-stimulated peripheral blood. Genomic microarray analysis was performed using the Constitutional Chip 3.0 from Spectral Genomics, a subsidiary of Perkin Elmer Corporation (Houston, TX) and following the manufacturer's protocol with the exception that the Cy3-dCTP and Cy5-dCTP were purchased from Amersham Biosciences (Buckingamshire, England). Scanning was performed with Axon's GenePix 400B microarray scanner and the images were analyzed with SpectralWare 2.2 for the preparation of ratio plots.
BAC CTD-2269L21 was identified using the March 2006 assembly of the UCSC Genome Browser (http://www.genome.ucsc.edu) and obtained from Invitrogen Corporation (Carlsbad, CA). Plasmid DNA was isolated using the Ultraclean 6 Minute Mini Plasmid Prep kit from Mo Bio (Carlsbad, CA) according to the manufacturer's protocol. Plasmid DNA was labeled with Spectrum Orange-dUTP using the Nick Translation kit from Vysis (Downers Grove, IL) and FISH hybridization followed the protocol included with the kit. The 4p and 4q subtelomere probes (D4S3359 and D4S2930) and the LSI WHS probe were purchased from Vysis (Downers Grove, IL) and FISH was performed according to the manufacturer's protocol. Slides were viewed with an Olympus BH2 microscope and images analyzed using the CytoVision software package from Applied Imaging Corporation (San Jose, CA).
Quantitative Real-Time PCR (RT-PCR)
Custom TaqMan gene expression probes for exon 13 of the LETM1 gene and for exons 1 and 23 of the PDGFRA gene at 4q12 (used as controls) were designed by Applied Biosystems (Foster City, CA) (probe sequences available on request). One hundred fifty nanograms of genomic DNA was combined with TaqMan probes and Universal master mix and analyzed on an ABI 7900HT RT-PCR system according to manufacturer specifications. Patient and control reactions were done in triplicate and Ct (threshold cycle) measurements were averaged. Relative ratios (RR) were calculated using the ΔΔCt method [Livak and Schmittgen, 2001].
Clinical evaluation of patients was assessed by examination of clinical records and photographs made available by the primary care physicians and specialists and obtained with informed parental consent. Patient #2 was also seen by JC at a WHS national support group meeting.
Patient #1 was a boy born at 34 weeks gestation via cesarean, weighing 1,230 g. Length was 43.2 cm and head was normocephalic at 31.5 cm. The pregnancy was complicated by severe intrauterine growth retardation and significant maternal pregnancy-induced hypertension. Mild hypospadias was noted and is scheduled for surgical repair. In infancy, the patient fed poorly and showed slow weight gain, poor linear growth, and a fall-off with his head circumference. At 24 months the patient weighed 5,953 g (50th centile for a 3-month-old), height was 68.6 cm (50th centile for a 6-month-old) and head circumference was 39.6 cm (50th centile for a 3-month-old). The remainder of his physical examination showed no minor anomalies. He never required a feeding tube. Diagnostic imaging showed a normal upper GI series and small bowel follow-through. IGF-1, IGF-BP3 levels and thyroid function tests were all within normal limits. The patient showed normal to mildly decreased tone and developmental delay. At 14 months of age the patient was beginning to cruise and had only a few syllables without any meaningful speech. By 24 months of age he could say one to two words, point to the things he wanted, and was walking and running well. An echocardiogram and renal ultrasound showed no abnormalities. At age 26 months the patient has not shown any signs of seizures or staring spells.
Review of photographs provided by the family showed a prominent forehead, mild telecanthus, a low nasal root, a thin upper lip and a normal philtral length (Fig. 1A).
Standard G-banded chromosome analysis revealed a ring chromosome 4 with no cytogenetically visible loss of chromosome 4 material (Fig. 1B). FISH analysis of the ring chromosome showed loss of the 4p subtelomere region, retention of the 4q subtelomere region, and retention of a commercially available WHS probe that overlaps the WHSC1 gene on 4p16.3 (data not shown). Genomic microarray analysis demonstrated an apparently terminal deletion on 4p of approximately 1.27–1.46 Mb (Fig. 2). Standard G-banded chromosome analysis of both parents were normal, indicating the chromosome abnormality seen in this patient was de novo.
Patient #2 was a girl born at 41 weeks gestation with growth measurements near the 3rd centile. Birth weight was 2,778 g, length was 45.7 cm, and head circumference was 31 cm. Poor growth continued in infancy and at 30 months she had a weight of 7,881 g (50th centile for a 7-month-old), a height of 71 cm (50th centile for a 10-month-old) and a head circumference of 45 cm (50th centile for a 12-month-old). The patient had a normal echocardiogram, renal ultrasound, hearing screen and eye exam. The patient has a history of gastroesophageal reflux disease with aspiration of thin liquids which improved with therapy. She did not require any tube feeding. The patient had mild hip dysplasia and a marked delayed bone age. Physical examination recorded at a genetics consultation showed no other minor anomalies other than those of the face. The patient exhibited delays in gross motor development. Testing done at a chronologic age of 30 months showed gross motor skills equivalent to 16 months and she was walking without support. At 20 months of age the patient showed near age appropriate scores for speech and language development and at 26 months of age she began to form two to three word sentences.
At age 6 months the patient was evaluated for myoclonic jerking. CT and MRI scans of the brain and an EEG were all normal. The myoclonic jerks increased in frequency and at 8 months of age a 3-day EEG monitoring study showed no epileptiform activity during both daytime or nighttime jerking, and the patient was diagnosed with benign myoclonus of infancy. At 22 months she presented with a single brief generalized tonic clonic seizure. An EEG performed a day later showed left focal spiking, consistent with a lowered threshold for focal onset seizures. Similar results were seen in a follow-up EEG performed 2 months later. The patient was placed on Topamax after the first abnormal EEG. The most recent EEG done at 33 months showed high amplitude multifocal and generalized slow spike and wave discharges consistent with Lennox-Gastaut syndrome. Clinical episodes consistent with generalized tonic–clonic and atypical absence seizures were also recorded.
Facial features include prominent forehead, mild telecanthus and normal philtral length (Fig. 1C,D).
Standard G-banded chromosome analysis was normal. Genomic microarray analysis revealed an apparently terminal deletion on 4p of approximately 1.78 Mb (Fig. 2). FISH analysis of both parents using a probe within the deleted region found no evidence of a deletion or rearrangement in either parent, indicating the deletion seen in this patient was de novo. Because of the proximity of this deletion to genes thought to be directly involved in the WHS phenotype, the boundary of this deletion was confirmed by FISH with BAC CTD-2269L21 which spans between 1.75 and 1.86 Mb from the terminus of 4p. FISH with BAC CTD-2269L21 showed a strong signal on the terminus of each chromosome 4, indicating that at least the majority of this genomic region was not deleted in this patient.
Quantitative real-time (RT) PCR was used to determine if the LETM1 gene, located within BAC CTD-2269L21, was deleted in this patient. An RT-PCR probe was designed to detect copy number changes at exon 13, near the telomeric end of the LETM1 gene, and at two control loci at 4q12. The analysis showed that at least a portion of the LETM1 gene is deleted in this patient (RR = 0.59 and 0.43 for the LETM1 probe vs. each control probe, respectively) (Fig. 3).
Patients with small 4p deletions have been useful in mapping the clinical features associated with WHS and in defining the critical region(s) where hemizygosity results in a phenotype consistent with the minimal diagnostic criteria for WHS. These minimal diagnostic criteria include mild to severe developmental delay, hypotonia, prenatal-onset growth deficiency followed by postnatal growth retardation, and the typical facial features of a broad nasal bridge, high forehead with prominent glabella, ocular hypertelorism, epicanthus, highly arched eyebrows, short philtrum, downturned corners of the mouth, micrognathia, and poorly formed ears with pits or tags. Reports on the occurrence rate of seizures in patients with WHS vary between 50% and 100% [Battaglia et al., 2001]. However, most reports agree that seizures occur in greater than 75% of patients, with an age of onset between 3 and 24 months [Battaglia et al., 1999, 2000; Wieczorek et al., 2000; Zollino et al., 2000]. In our experience, some patients have developed seizures as late as 36 months. Therefore, some of the variation in the seizure occurrence rate may be due to the clinical description of patients in infancy prior to the subsequent development of seizures.
Two critical regions have been proposed. The first region is approximately 165 kb in size and was defined by determining the smallest region of overlap using patients with the WHS phenotype as well as patients with interstitial 4p deletions who lack the WHS phenotype [White et al., 1995; Wright et al., 1997]. This region (WHSCR) exists between markers D4S166 and D4S3327 and is proximal to the FGFR3 and LETM1 genes and encompasses the WHSC2 gene and at least the 3′ end of the WHSC1 gene (Fig. 2). A patient with a partial WHS phenotype (mild growth delay, speech delay, and minor facial anomalies) but lacking mental retardation, microcephaly and seizures, was determined to have a 191.5 kb microdeletion that overlapped this region and removed the WHSC2 gene and disrupted the WHSC1 gene, but left the LETM1 gene intact [Rauch et al., 2001]. This observation lent support to the hypothesis that hemizygosity for gene(s) in this small interval were responsible for the mild growth delay, mild developmental delay, and some minor facial dsymorphisms seen in WHS, as well as the hypothesis that a deletion of the LETM1 gene contributes to seizure development [Endele et al., 1999].
The second critical region was defined following the identification of two patients with the WHS phenotype, including seizures, characteristic facial features, growth retardation, and developmental delay, with terminal 4p deletions distal to WHSCR [Zollino et al., 2003; Rodriguez et al., 2005]. The proximal boundary for the second critical region (WHSCR-2) was defined by marker D4S3327 and the distal boundary was proposed to be between loci D4S98 and D4S168 based on two patients with a full WHS phenotype and interstitial deletions whose distal breakpoints mapped between these two markers [Wright et al., 1996; Fang et al., 1997]. WHSCR-2 contains the LETM1 gene as well as the 5′ end of the WHSC1 gene (Fig. 2). WHSCR-2 may be more definitive since it is based on two patients that meet the all the minimal diagnostic criteria for WHS, whereas the distal boundary of WHSCR was mapped using a patient with typical facial features, growth delay, and moderate psychomotor delay, but did not show any seizure activity by the age of 7.
We are aware of three published reports describing patients with small terminal deletions of 4p that do not have the WHS phenotype. Gandelman et al.  described a patient with a der(4)t(4;7)(p16;q34) whose 4p deletion size was determined to be between 100 and 300 kb from the 4p terminus and whose clinical phenotype was consistent with the 7q duplication seen in this patient. Estabrooks et al.  described two families segregating a satellited 4p presumably due to a translocation between 4p and the short arm of an acrocentric chromosome. Some family members were shown to have a 4p deletion of approximately 150 kb from the terminus of 4p. Some of these individuals were characterized as normal whereas others had borderline developmental delays or growth delays. Still, none of these individuals were characterized as having a WHS phenotype. Van Buggenhout et al.  described a woman analyzed by subtelomere FISH for infertility in which a deletion of the 4p subtelomere region was identified in both her and in her phenotypically normal mother. This deletion was determined to be less than 200 kb from the terminus of 4p by use of a high density microarray. These reports confirm that the gene(s) responsible for the WHS phenotype are proximal to the subtelomeric region of 4p and likely greater than 300 kb from the terminus of 4p.
Our description of two patients with very small terminal 4p deletions helps to delineate genomic regions that may be responsible for certain clinical findings in WHS, yet neither patient meets all of the minimal diagnostic criteria for WHS. The extent of Patient #1's deletion is distal to both critical regions. The extent of Patient #2's deletion has been localized to an interval within BAC CTD-2269L21 (present by FISH) but proximal to exon 13 of the LETM1 gene (deleted by RT-PCR), an interval of less than 85 kb. Therefore, Patient #2's deletion is near or within the known distal boundary for WHSCR-2. Both patients show the characteristic growth retardation, although this finding may be modified by the presence of a ring chromosome in Patient #1, as delayed growth is often reported in individuals with a ring chromosome [Gardner and Sutherland, 2004]. Both patients show facial features that are less distinct compared to those features typically characteristic of the syndrome, yet include some subtle degree of a prominent glabella and apparent wide-spaced eyes. Both patients show mild developmental delay. Patient #1 has thus far shown no signs of seizures (at 26 months) whereas Patient #2 has a complex seizure disorder.
The absence of seizures in Patient #1 provides additional support to the hypothesis that the LETM1 gene is primarily responsible for seizure development in WHS. LETM1 is located between 1.78 and 1.825 Mb from the terminus of 4p and is proximal to Patient #1's deletion breakpoint. However, at least the distal end of the LETM1 gene lies within Patient #2's deletion. Therefore, her seizure presentation is likely due to disruption of normal LETM1 expression.
Taking the extent of these patient's deletions together, it is clear that hemizygosity for 4p16.3 distal to the proposed WHS critical regions can result in a clinical presentation that overlaps WHS and includes growth delay and developmental delay. However, neither patient has the full clinical phenotype of WHS like that seen in the two patients used to define WHSCR-2 [Zollino et al., 2003; Rodriguez et al., 2005]. Therefore, the proximal deletion breakpoints of these patients, particularly Patient #2, may define the distal boundary of the critical region necessary for the full WHS phenotype. Still, since both patients do show some of the manifestations of the WHS phenotype, they highlight the difficulty of mapping phenotypic traits that are common to most aneusomy syndromes such as developmental delay, growth retardation, and facial features.
The authors wish to thank Terese Maxwell, Heidi Whitby, and Emily Aston for technical assistance, Dr. Amy Wirkkala, Dr. George Anadiotis, Dr. Annemarie Sommer, and Matt Pastori for collection of clinical information and the patients and their families for their participation. Funding for this study was provided in part through a grant from the Children's Health Research Center at the University of Utah. Dr. South and Dr. Bleyl are Primary Children's Medical Center Foundation Scholars.
- 1997. Delimiting the Wolf-Hirschhorn syndrome critical region to 750 kilobase pairs. Am J Med Genet 71: 47–53. , , , , .
- 1999. Natural history of Wolf-Hirschhorn syndrome: Experience with 15 cases. Pediatrics 103: 830–836. , , , , , .
- 2000. Wolf-Hirschhorn syndrome (WHS): A history in pictures. Clin Dysmorphol 9: 25–30. , , , , .
- 2001. Wolf-Hirschhorn (4p-) syndrome. Adv Pediatr 48: 75–113. , , .
- 1999. LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients. Genomics 60: 218–225. , , , , .
- 1992. A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype. Am J Hum Genet 51: 971–978. , , , , .
- 1993. Interstitial deletion of distal chromosome 4p in a patient without classical Wolf-Hirschhorn syndrome. Am J Med Genet 45: 97–100. , , .
- 1997. High resolution characterization of an interstitial deletion of less than 1.9 Mb at 4p16.3 associated with Wolf-Hirschhorn syndrome. Am J Med Genet 71: 453–457. , , , , , , , , , .
- 1992. Molecular definition of the smallest region of deletion overlap in the Wolf-Hirschhorn syndrome. Am J Hum Genet 51: 571–578. , , , .
- 2004. Chromosome Abnormalities and Genetic Counseling. New York: Oxford University Press. 577 p. , .
- 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25: 402–408. , .
- 2001. First known microdeletion within the Wolf-Hirschhorn syndrome critical region refines genotype–phenotype correlation. Am J Med Genet 99: 338–342. , , , , , , , .
- 2005. The new Wolf-Hirschhorn syndrome critical region (WHSCR-2): A description of a second case. Am J Med Genet Part A 136A: 175–178. , , , , , , , .
- 2004. Mild Wolf-Hirschhorn syndrome: Micro-array CGH analysis of atypical 4p16.3 deletions enables refinement of the genotype–phenotype map. J Med Genet 41: 691–698. , , , , , , , , , , .
- 1995. Interstitial deletions of the short arm of chromosome 4 in patients with a similar combination of multiple minor anomalies and mental retardation. Am J Med Genet 57: 588–597. , , , , .
- 2000. Unexpected high frequency of de novo unbalanced translocations in patients with Wolf-Hirschhorn syndrome (WHS). J Med Genet 37: 798–804. , , , , , , .
- 1996. High resolution analysis of the Wolf Hirschhorn syndrome region on chromosome 4p16.3. Fourth International Workshop on Human Chromosome 4 Mapping 1996. , , , , .
- 1997. A transcript map of the newly defined 165 kb Wolf-Hirschhorn syndrome critical region. Hum Mol Genet 6: 317–324. , , , , , , , , , , , , .
- 2000. Genotype–phenotype correlations and clinical diagnostic criteria in Wolf-Hirschhorn syndrome. Am J Med Genet 94: 254–261. , , , , , , , , , , , , , , .
- 2003. Mapping the Wolf-Hirschhorn syndrome phenotype outside the currently accepted WHS critical region and defining a new critical region, WHSCR-2. Am J Hum Genet 72: 590–597. , , , , , , , , , .