Bilateral radial agenesis with absent thumbs, complex heart defect, short stature, and facial dysmorphism in a patient with pure distal microduplication of 5q35.2-5q35.3
© Jamsheer et al.; licensee BioMed Central Ltd. 2013
Received: 22 May 2012
Accepted: 18 January 2013
Published: 24 January 2013
A partial duplication of the distal long arm of chromosome 5 (5q35-- > qter) is known to be associated with a distinct phenotype referred to as Hunter-McAlpine syndrome. Clinical spectrum of this disorder mainly consists of mental retardation, microcephaly, short stature, skeletal anomalies, and craniofacial dysmorphism featuring flat facies, micrognathia, large, low-set dysplastic ears, hypertelorism, almond-shaped, down-slanted palpebral fissures, epicanthal folds, small nose, long philtrum, small mouth, and thin upper lip. Less frequent remarkable findings include craniosynostosis, heart defect, hypoplastic phalanges, preaxial polydactyly, hypospadias, cryptorchidism, and inguinal hernia. In most patients with a partial duplication of 5q the aberration occurred due to an inherited unbalanced translocation, therefore the phenotype was not reflective of pure trisomy 5q.
We report on a 9.5-year-old boy with some feature of Hunter-McAlpine syndrome including short stature, complex heart defect (dextrocardia, dextroversion, PFO), bilateral cryptorchidism, hypothyroidism, and craniofacial dysmorphism. Additionally, bilateral radial agenesis with complete absence of Ist digital rays, ulnar hypoplasia with bowing, choroidal and retinal coloboma, abnormal biliary vesicle were identified, which have never been noted in 5q trisomy patients. Karyotype analysis, sequencing and MLPA for TBX5 and SALL4 genes were unremarkable. Array comparative genomic hybridization detected a duplication on 5q35.2-5q35.3, resulting from a de novo chromosomal rearrangement. Our proband carried the smallest of all previously reported pure distal 5q trisomies encompassing terminal 5.4-5.6 Mb and presented with the most severe limb malformation attributed to the increased number of distal 5q copies.
We postulate that a terminal distal trisomy of 5q35.2-5q35.3, which maps 1.1 Mb telomeric to the MSX2 gene is causative for both radial agenesis and complex heart defect in our proband. A potential candidate gene causative for limb malformation in our proband could be FGFR4, which maps relatively in the closest position to the chromosomal breakage site (about 1.3 Mb) from all known 5q duplications. Since the limb malformation as well as the underlying genetic defect are distinct from other 5q trisomy patient we propose that a position effect resulting in altered long-range regulation of the FGFR4 (alternatively MSX2) may be responsible for the limb malformation in our proband.
KeywordsPure distal trisomy 5q Distal 5q duplication Dup (5)(q35.2q35.3) Hunter-McAlpine syndrome MSX2 FGFR4 Radial agenesis Absent thumbs
A partial duplication of the distal long arm of chromosome 5 (5q35-- > qter) is known to be associated with a distinct phenotype referred to as Hunter-McAlpine syndrome . Clinical spectrum of this disorder consists mainly of mental retardation, microcephaly, short stature, skeletal anomalies, and craniofacial dysmorphism featuring flat facies, micrognathia, large, low-set dysplastic ears, hypertelorism, almond-shaped, down-slanted palpebral fissures, epicanthal folds, small nose, long philtrum, small mouth, and thin upper lip. Less frequent remarkable findings include craniosynostosis, heart defect (VSD, ASD, bicuspid aortic valve), hypoplastic phalanges, preaxial polydactyly, hypospadias, cryptorchidism, and inguinal hernia [2–7]. In most patients with a partial duplication of 5q the aberration occurred due to an inherited unbalanced translocation, hence the phenotype was not reflective of pure trisomy 5q. To our knowledge, the smallest pure partial distal duplication of chromosome 5q described to date encompassed about 6.4 Mb of 5q terminus (5q35.2-5q35.3) and was detected in a patient presenting with microcephaly, strabismus, facial dysmorphism, moderate mental retardation, short stature, brachydactyly, and inguinal hernias . Additionally, one more case with a pure gain on distal 5q (an interstitial triplication of 5q35.2-5q35.3) involving 6.56 Mb was identified in a patient manifesting some common features of Hunter-McAlpine syndrome (intrauterine growth retardation, almond-shaped eyes with epicanthal folds, downturned mouth with thin vermillion of the upper lip), as well as other unique findings such as left ventricular noncompaction (LVNC) and absent thumbs .
In our report, we describe a male proband with a pure 5.4-5.6 Mb distal duplication of 5q35.2-- > qter detected by array comparative genomic hybridization (array CGH), resulting from a de novo chromosomal rearrangement. To our knowledge, this is the smallest region of pure distal 5q trisomy. In addition to some features of Hunter-McAlpine syndrome, our patient presented with bilateral radial aplasia with absent thumbs, which seems to be the most severe limb malformation attributed to the increased number of distal 5q copies.
Cytogenetic and Fluorescent In Situ Hybridization (FISH) studies
Chromosomal analysis on the basis of GTG technique at 550 band resolution per haploid genome was performed on peripheral blood lymphocytes of the patient and his parents. FISH was carried out with use of 5p/5q subtelomeric probes (TelVysion probe 5p/5q, Vysis) according to the manufacturer’s protocols.
Sequencing and MLPA
Sequences of the primers used for TBX5 gene (MIM ID*601620; GenBank NM_000192) amplification and sequencing
Exon name (fragment)
Forward primer sequence 5′- 3′
Reverse primer sequence 5′- 3′
Product size (bp)
Sequences of the primers used for SALL4 gene (MIM ID*607343; GenBank NM_020436) amplification and sequencing
Exon name (fragment)
Forward primer sequence 5′- 3′
Reverse primer sequence 5′- 3′
Product size (bp)
Array comparative genomic hybridization (array CGH)
Array comparative genomic hybridization (array CGH) was carried out with the use of two independent microarrays: whole-genome 180 K oligonucleotide array (Agilent) and 135 k NimbleGen oligonucleotide CGX array (Roche NimbleGen) according to standard protocols provided by the manufacturers. In the first case, analysis was performed with Feature Extraction and CGH Analytics software (Agilent), with the following settings used: Aberration Algorithm: ADM-2; Threshold: 6.0; Window Size: 0.2 Mb; Filter: 5 probes, max log2ratio = 0.29. In the second case, analysis was done with NimbleScan (Roche NimbleGen) and Genoglypix® software (Signature Genomics).
Quantitative real-time PCR (qPCR)
To independently test for a number of 5q35.2-5q35.3 copies we developed a qPCR assay. We used a set of five primer pairs, three of which were located within the duplicated region and two lying centromeric against the duplication start point. qPCR was performed in a total volume of 12 μl in each well containing 6 μl of SYBR Green PCR Master Mix (Applied Biosystems), 5 μl of genomic DNA (1 ng/ml), and 1 μl of primers (0.2 mmol each). All samples were run in triplicate in separate wells to allow for the quantification of the target sequences normalized to Albumin (ALB). PCR conditions were as follows: initial denaturation step at 95°C followed by 40 cycles (denaturation at 95°C for 15 s, annealing with elongation at 60°C for 1 min). By use of calibrator DNA derived of a normal healthy control, the gene copy number was measured on the basis of the comparative DDCt method. In addition, we performed a sex determination for the individuals, calculating the Factor VIII (F8) exon 3 relative to our endogenous control Albumin, to assure its reliability. Primer sequences for ALB and F8 were as follows: ALB_F - tgttgcatgagaaaacgcca, ALB_R - gtcgcctgttcaccaaggat; F8_F - gccaagaagcatcctaaaacttg, F8_R - ggcgaggactaagggagcat.
Additional probands with bilateral radial aplasia
To identify further probands and test the frequency of 5q35 duplication we screened a cohort of another 8 unrelated sporadic patients (6 males and 2 females) with bilateral radial agenesis and absent thumbs. All patients were of Polish ethnicity and were seen in our genetic clinic at different ages varying from 3 months to 31 years. Four cases manifested isolated limb malformation, whereas the other four presented with additional features comprising congenital heart defect (3 cases), and mental retardation (1 case). The patients were prescreened by bi-directional sequencing and MLPA for TBX5 and SALL4 mutations and turned out to be negative. Additionally, a patient affected by mental retardation was analyzed by means of GTG banding, which showed normal result.
Sequences of the primers used for quantifying the number of copies of 5q35.2-5q35.3, along with coordinates for each amplified genomic region (HG18), its relative position with regard to the start point of the duplication, and the number of copies detected in the proband
Coordinates of the amplicon (HG18)
No of copies detected in the proband
Position of the amplicon in ref. to the start of the duplication
175182611 - 175182696
- 61 kb
175201237 - 175201316
- 42 kb
175286270 - 175286357
+ 43 kb
176356609 - 176356696
+ 1,12 Mb
179355063 - 179355146
+ 4,12 Mb
Our patient carries the smallest pure distal duplication of chromosome 5q (i.e. terminal part of 5q35.2 band and the entire 5q35.3 band). The phenotype of our patient showed significant overlap with Hunter-McAlpine syndrome, with such common features as short stature, heart defect, cryptorchidism, and craniofacial dysmorphism including prominent widened nasal bridge, almond-shaped eyes, thin vermillion of upper lip, and low-set dysplastic ears. In addition, our patient manifested less frequent symptoms (i.e. hypothyroidism) or even unique findings such as bilateral radial agenesis with absent thumbs, bilateral ulnar hypoplasia, abnormal biliary vesicle, and unilateral choroidal and retinal coloboma. Interestingly, absent thumbs were reported so far only in a single patient with an atypical copy number variation (CNV) on 5q, which was the interstitial triplication of the distal 5q segment encompassing MSX2 (6.56 Mb, coordinates according to HG18: 173897858–180456069). Since absent thumbs were never noted in cases with a distal 5q duplication, the authors hypothesized that the relatively severe limb malformation was due to the increased dosage of 5q copies . Although caused solely by a pure duplication, the skeletal phenotype in our patient was more severe and involved bilateral radial aplasia with completely absent Ist digital rays (thumbs and Ist metacarpals), and ulnar hypoplasia with bowing. The defect seen in our patient as well as in the former case can be both categorized to radial ray deficiency spectrum. We therefore suggest that limb malformation presented by our index was a more severe manifestation of a common defect, namely radial ray deficiency. Of note, the chromosomal microduplication identified in our proband (terminal 5.4-5.6 Mb) did not encompass the MSX2 gene, which is located 1.1 Mb centromeric relative to its beginning. The MSX family, comprises MSX1 and MSX2 homeobox containing genes, which are important developmental regulators involved in the processes of limb, craniofacial, and ectoderm formation in vertebrates . For example, MSX1 is essential for tooth and facial bone development and its mutations lead to Witkop syndrome also known as nail dysplasia with hypodontia . Moreover, duplications of MSX2 cause Boston type craniosynostosis [6, 12], whereas intragenic alterations or gene deletions result in parietal foramina, a disorder of deficient ossification of the skull [13, 14]. It has been therefore postulated that duplications of MSX2 are responsible for craniosynostosis and brachydactyly in Hunter-McAlpine patients. Thus far, 5q distal trisomy has never been associated with the absence of digits. However, based on the observation of a 5q tetrasomy carrying patient, it has been hypothesized that multiple copies of 5q (including MSX2) result in a more severe skeletal anomaly such as absent thumbs. Clinical manifestation of our proband and the underlying genetic defect show that in the case of 5q gain, an extra copy (copies) of MSX2 is not necessary to give rise to a severe limb phenotype involving not only absent thumbs, but also bilateral radial aplasia and hypoplastic ulnae. Importantly, the size of the duplication in our case was smaller than that reported for other trisomy 5q patients and mapped 1.1 Mb telomeric to the MSX2. A plausible candidate gene causative for the limb malformation in our proband could be FGFR4. This gene is duplicated in our patient and maps around 1.3 Mb from the beginning of CNV. FGFR4 encodes for a protein, which is a type 4 receptor for fibroblast growth factors (FGFs). Members of FGF protein family are involved in FGF signalling pathway and play an important role during limb development. FGF4 along with FGF8 is secreted by apical ectodermal ridge (AER) which maintains the FGF10 signal and induces proliferation in the mesoderm [15, 16]. For example, loss of both Fgf4 and Fgf8 in mice is thought to result in a reduction of the proliferation rate in distal mesenchyme, followed by downregulation of Fgf10 and premature degeneration of AER. Hence, in the absence of both Fgf4 and Fgf8, increased mesenchymal cell death results in a reduction in limb bud size . So far there has been no report on radial agenesis and absent thumbs in other patients carrying 5q duplication encompassing FGFR4, suggesting that an extra copy of this gene is not sufficient to give rise to the limb phenotype. Noteworthy, FGFR4 maps about 1.3 Mb from the beginning of the duplication detected in our proband, which is relatively the closest position to the chromosomal breakage site for all known 5q duplications. Since both the limb malformation as well as the underlying genetic defect are unique in our patient we propose that a position effect resulting in altered long-range regulation of the FGFR4 (or possibly MSX2) may be the underlying patomechanism for limb malformation in both our and 5q tetrasomy patient. Alternatively, an extra copy and/or dysregulation of another gene may be responsible for radial agenesis.
The high frequency of congenital heart abnormalities in patients with a 5q trisomy was attributed to the altered dosage of one or two cardiac developmental genes, NKX2-5 and CSX1, both mapping to chromosome 5q34 [18, 19]. Interestingly, patient with the smallest pure distal duplication of 5q (encompassing terminal 6.4 of 5q35.2-q35.3) described to date in the literature, did not show any cardiac abnormality. This pointed to an observation that the direct duplication of 5q35.2-q35.3 may not lead to a cardiac phenotype . In contrast, our patient carried even smaller distal duplication of 5q, however presented with a complex congenital heart defect including dextrocardia, dextroversion, and PFO. This may suggest that not only NKX2-5 or CSX1, but also other genes or regulatory elements located in distal 5q play an important role in the process of embryonic heart formation.
A duplication of the distal arm of chromosome 5q is known to be associated with short stature and microcephaly, and an increased dosage of NSD1 gene was proposed to be responsible for a combination of these two features . Deletions and point mutations of the NSD1 gene cause Sotos syndrome with cerebral gigantism, overgrowth and macrocephaly . It is theoretically possible, as suggested by Chen et al. , that dosage changes (decrease or increase) of NSD1 lead to opposite phenotypes. Nonetheless, our patient had a duplication encompassing NSD1 but although short statured, he did not presented with a microcephaly. This may be explained by the incomplete penetrance of the candidate microcephaly gene.
In conclusion, we postulate that a terminal distal trisomy of 5q35.2-5q35.3, which maps 1.1 Mb telomeric to the MSX2 gene is causative for both radial agenesis and complex heart defect in our proband. Although duplications of MSX2, a highly conserved developmental gene which plays a major role in cardiac and bone morphogenesis were considered responsible for at least some skeletal symptoms (including limb malformations) in 5q trisomy patients, we provide evidence that even more distally located duplications may give rise to a more severe limb phenotype. Based on this observation, we propose that other genes or altered FGFR4 (or possibly MSX2) long range regulation contribute to the development of radial agenesis and absent thumbs.
Finally, we studied a small cohort of 8 unrelated probands manifesting bilateral radial agenesis with or without heart defect, who were negative for TBX5 and SALL4 mutations. None of them had 5q duplication detected upon qPCR, suggesting that this kind of molecular defect is not a common cause of radial agenesis and associated limb phenotype.
Written informed consent was obtained from the patient’s parents to take part in the study as well as for publication of the images (including full-face pictures). A copy of the written consent is available for review by the Editor-in-Chief of this journal. Informed consent was obtained from 8 unrelated probands or their legal guardians to participate, and include details in the manuscript.
Apical ectodermal ridge
Atrial septal defect
Comparative genomic hybridization
Copy number variation
Left ventricular noncompaction
Persistent foramen ovale
Quantitative polymerase chain reaction
Ventricular septal defect.
We are grateful to the patient, his parents, and the referring physicians for participating in this study. This work was supported by a grant from the Polish Ministry of Science and Higher Education (495/N-NIEMCY/2009/0) and a grant from National Science Centre (UMO-2011-03-D-NZ2-06136).
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