Conflict of interest: none.
Exome Analysis in Clinical Practice: Expanding the Phenotype of Bartsocas–Papas Syndrome†
Article first published online: 22 APR 2013
Copyright © 2013 Wiley Periodicals, Inc.
American Journal of Medical Genetics Part A
Volume 161, Issue 5, pages 1058–1063, May 2013
How to Cite
2013. Exome analysis in clinical practice: Expanding the phenotype of Bartsocas–Papas syndrome. Am J Med Genet Part A 161A:1058–1063., , .
- Issue published online: 22 APR 2013
- Article first published online: 22 APR 2013
- Manuscript Accepted: 3 FEB 2013
- Manuscript Received: 31 DEC 2012
- cleft lip/palate;
- ankyloblepharon–ectodermal defects–cleft lip/palate syndrome;
- Bartsocas–Papas syndrome;
- autosomal recessive popliteal pterygium syndrome;
Exome analysis has had a dramatic impact on genetic research. We present the application of such newly generated information to patient care. The patient was a female, born with normal growth parameters to nonconsanguineous parents after an uneventful pregnancy. She had bilateral cleft lip/palate and ankyloblepharon. Sparse hair, dysplastic nails and hypohidrosis were subsequently noted. With exception of speech related issues, her development was normal. A clinical diagnosis of ankyloblepharon–ectodermal defects-cleft lip/palate or Hay–Wells syndrome resulted in TP63 sequence analysis. TP63 sequence and deletion/duplication analysis of all coding exons had a normal result, as did chromosome and SNP array analysis. Diagnostic exome analysis revealed a heterozygous nonsense mutation in KRT83 categorized as deleterious and associated with monilethrix. In addition, a homozygous missense variant of unknown clinical significance was reported in RIPK4. Using research based exome analysis, RIPK4 had just a few months prior been identified as pathogenic for Bartsocas–Papas syndrome. While the clinical diagnostic report implied the KRT83 mutation as a more likely cause for the patient's phenotype, clinical correlation, literature review and use of computerized mutation analysis programs allowed us to identify the homozygous RIPK4 (c.488G > A; p.Gly163Asp) mutation as the underlying pathogenic change. Consequently, we expand the phenotype of Bartsocas–Papas syndrome to an attenuated presentation resembling Hay–Wells syndrome, lacking lethality and pterygia. In contrast to the autosomal dominant Hay–Wells syndrome, Bartsocas–Papas syndrome is autosomal recessive, implying a 25% recurrence risk. © 2013 Wiley Periodicals, Inc.
Whole exome sequencing (WES) is a powerful research tool as it allows identification of novel disease genes, even in the absence of linkage data. Here we report on the clinical diagnostic use of WES for a proposita with a presumed diagnosis of ankyloblepharon–ectodermal defects–cleft lip/palate (AEC) syndrome. The proband presented with bilateral cleft lip and palate, ankyloblepharon and ectodermal dysplasia with slow growing hair, dysplastic nails and hypohidrosis, consistent with a clinical diagnosis of ankyloblepharon–ectodermal defects–cleft lip/palate or Hay–Wells syndrome [Sutton et al., 2009]. Hay–Wells syndrome is caused by heterozygous mutations in exons 13 and 14 of TP63 [Rinne et al., 2006]. Despite exhaustive molecular testing, no TP63 mutation was identified in the proband. Subsequently performed diagnostic WES revealed a homozygous sequence variation in RIPK4.
Based on the prior report of RIPK4 as the disease gene for Bartsocas–Papas syndrome [Kalay et al., 2012; Mitchell et al., 2012], the proposita's homozygous mutation could be interpreted as clinically relevant. While the proposita lacked the pterygia considered characteristic for this recessive form of popliteal pterygium syndrome [Bartsocas and Papas, 1972], her findings were consistent with an attenuated phenotype. Consequently, we expand the phenotype of Bartsocas–Papas syndrome.
MATERIALS AND METHODS
The female proposita was born after induction due to pre-eclampsia at 34 weeks gestation. The pregnancy was otherwise uncomplicated, and there was no prenatal exposure. Her 25-year-old mother was of English, Spanish, and Irish ancestry, and the nonconsanguineous 30-year-old father was of Irish and other European ancestry. Two maternal half-siblings were in good health. A maternal great-uncle had a unilateral cleft lip; there was no other family history of clefting or pregnancy loss. Birth weight was 2.02 kg (50th centile), length 43 cm (25th centile), and OFC 31 cm (50th centile). She had bilateral cleft lip and palate with associated nasal deformity (Fig. 1). Ankyloblepharon of her left lid was noted and surgically released. Lids and eye lashes were otherwise normal. Fifth toes were described as slightly short. The patient required phototherapy for hyperbilirubinemia as a neonate. She underwent numerous surgical procedures for repair of the facial clefting, ear tube placement, and dental restoration. Her sparse hair grew slowly (Fig. 1), and her primary dentition was normal with exception of the maxillary teeth in the areas affected by clefting. Her fingernails were dysplastic and her toenails did not require cutting. She had no webbing or lip pits. Development and speech were age appropriate, with exception of a concern for possible oromotor speech disorder and articulation problems, for which she received speech therapy. At the age of 5 years, the patient attended a regular kindergarten and her performance and language were age appropriate. At age 5 2/12 years her height was 114 cm (75–90th centile), weight 16.9 kg (10–25th centile), and OFC 50.5 cm (50th centile).
Laboratory Study Results
Karyotype was 46,XX. BAC and SNP arrays had normal results, and no increased homozygosity was noted using the Affymetrix 1.8 million SNP platform. TP63 sequence and deletion/duplication analysis of all coding exons had normal results. WES performed in a CLIA-certified laboratory (Medical Genetics Laboratories at Baylor College of Medicine) revealed several sequence changes, with a paternally inherited nonsense mutation in KRT83 reported in the category “deleterious mutations in disease genes related to clinical phenotype,” and a homozygous missense mutation, inherited from each parent, in RIPK4 (c.488G > A; p.Gly163Asp) reported in the category “variants of unknown clinical significance in disease genes related to clinical phenotype.” This sequence change was a novel variant. No other variant was reported in either diagnostic category.
The patient's bilateral cleft lip and palate, ankyloblepharon and ectodermal dysplasia with slow growing hair, dysplastic nails and reported hypohidrosis were consistent with a clinical diagnosis of ankyloblepharon–ectodermal defects–cleft lip/palate or Hay–Wells syndrome [Sutton et al., 2009]. Hay–Wells syndrome is caused by heterozygous mutations in exons 13 and 14 of TP63 [Rinne et al., 2006]. Ectodermal dysplasia-ectrodactyly-clefting (EEC), limb-mammary and ADULT syndrome, amongst others, are allelic to AEC [for review, see Sutton et al., 2010]. In the proposita, TP63 mutation analysis did not reveal an abnormality and genomic studies, including SNP array, were non-diagnostic. Whole exome analysis was performed in order to identify the underlying genetic cause for the patient's apparent AEC syndrome, and the report focused on genes associated with the phenotype. Interestingly, only one mutation was listed as “deleterious,” a KRT83 nonsense mutation. This was based on a single previously reported missense mutation in this gene, thought to cause the autosomal dominant hair disorder monolitherix [OMIM 158000]. Since the patient's KRT83 variant was paternally inherited and her father had normal hair, we did not consider this mutation clinically relevant. The homozygous RIPK4 mutation was reported as a variant of unknown clinical significance requiring clinical correlation. Heterozygosity for this mutation in both parents is consistent with the presumed autosomal recessive inheritance pattern of the associated Bartsocas–Papas syndrome. While there was no increased homozygosity in the proposita's SNP analysis and no reported consanguinity between her parents, their shared European ancestry suggests a distant common ancestor underlying the homozygous RIPK4 sequence change.
Amongst the syndromic forms of orofacial clefting, those with pterygia are well recognized. The autosomal dominant popliteal pterygium syndrome [OMIM 607199] results from IRF6 mutations, and is allelic with orofacial clefting and lip pits without webbing or van der Woude syndrome 1 [MIM 119300]. The rarer and often lethal autosomal recessive popliteal pterygium syndrome is also known as Bartsocas–Papas syndrome [OMIM 263650], after its original report [Bartsocas and Papas, 1972]. This condition is characterized by severe orofacial clefting, ankyloblepharon, filiform bands between the jaws, syndactyly and severe popliteal webbing affecting the genitalia. Hair, eyebrows, lashes and nails are typically absent. Synostosis of the hands and feet, talipes equinovarus and hypoplasia of the pelvic bones and scapula may occur. Most published cases were born to consanguineous families from Mediterranean and Middle Eastern countries; however, Dutch [Veenstra-Knol et al., 2003] and Gambian [Shanske et al., 2004] patients were reported. While most affected individuals did not survive the neonatal or early childhood period, patients alive at ages 1.5 and 13 years [Kalay et al., 2012]; 5 years [Maganzini et al., 2006]; and 6 years [Mitchell et al., 2012] have been described. Based on the definition of a popliteal pterygium syndrome, to the best of our knowledge, all clinically reported patients had pterygia (Table I).
|Ankyloblepharon–ectodermal defects–cleft lip/palate||Patient presented here||Bartsocas–Papas syndrome|
|Hypotrichosis of brows and lashes||+||+(mild)||+|
|Oligodontia||+||+(primary dentition, in area of clefting)||+(single report)|
|Dysplastic or hypoplastic nails||+||+||+|
|Lethal in early childhood||−||−||+|
RIPK4 Mutations in Bartsocas–Papas Syndrome
Mitchell et al.  utilized exome sequencing on a research basis to identify a homozygous RIPK4 mutation (Table II) in a patient with findings typical for Bartsocas–Papas syndrome, and pathogenicity was supported by a different RIPK4 mutation in a second family. Synchronously, Kalay et al.  reported additional RIPK4 mutations in Bartsocas–Papas syndrome (Table II). Interestingly, Mitchell et al.  showed through functional studies that p63 is a direct transcriptional activator of RIPK4, setting the stage for an overlap of the phenotypic spectrum resulting from mutations in either gene. RIPK4 is member of the receptor-interacting protein (RIP) kinase gene family and encodes a 784 amino acid protein. Its serine/threonine kinase domain is comprised of amino acids 22–283. Thus, the glycine in position 163, altered to aspartic acid in our proband, is located within this active domain. This glycine residue is highly conserved (Fig. 2). Further, using the protein variation effect analyzer v.1.1 (PROVEAN; available at www.http://provean.jcvi.org), with a default threshold for being deleterious at −2.5, this mutation scored −4.395 and was considered deleterious with >90% specificity. While only functional studies can prove the pathogenicity of this missense mutation, the evidence in favor of this mutation being disease causing appears overwhelming.
|RIPK4 change||p.Ser376X||p. Ile81Asn||p.Ile121Asn||p.Thr184Ile||p.Arg260ThrfsX14||p.Gly163Asp|
|Oral synechia||+||+||+||+||(No oral opening)||−|
|Alopecia||Sparse||Sparse hair, normal lashes, brows||+||+||+||Sparse hair and brows, normal lashes|
|Dentition||N/A||Oligodontia, cone-shaped maxillary incisors||N/A||N/A||N/A||Primary dentition affected by clefting|
|Development||Normal||Normal||No intellectual disability||N/A||N/A||Normal|
|Number of patients||1||2 (1 alive)||6 (2 alive)||1||1||1|
|Oldest living patient||6 years||5 years||13 years||Died at 57 days||Died at 3.5 months||5 years|
|References||Mitchell et al. ||Mitchell et al. , Maganzini et al. , Shanske et al. ||Kalay et al. , Aslan et al. ||Kalay et al. ||Kalay et al. ||Present report|
Expanding the Phenotypic Spectrum of Bartsocas–Papas Syndrome
Realizing that the proband's phenotype resulted from the homozygous RIPK4 mutation, a diagnosis of Bartsocas–Papas syndrome follows. As this patient lacked the characteristic pterygia and syndactyly, her phenotype can be considered attenuated. It is noteworthy that she is alive and well at age 5 years. While Bartsocas–Papas syndrome is typically considered a lethal condition, several individuals surviving to later childhood have been reported (Table II). Importantly, older individuals do not appear to have significant cognitive impairment. Their phenotype may resemble AEC syndrome, as in our proposita. In hindsight, the absence of dermal erosions, erythroderma and pigmentary changes, which are very common in AEC, may have suggested a diagnosis other than AEC. Using this information, future patients presenting with an attenuated Bartsocas–Papas phenotype may be recognized clinically, and molecular testing may consist of RIPK4 Sanger sequencing, rather than WES. Further, for laboratories performing WES, awareness for this attenuated phenotype may result in accurate interpretation of RIPK4 mutations.
Impact of WES on Recognized Mendelian Disorders
WES is a powerful research tool, allowing identification of novel disease genes, even in the absence of linkage data. This newly generated information can rapidly be used for clinical purposes, for example, by subsequently sequencing only the identified disease gene, as reported in the patient with suspected talipes—ASD–Robin sequence—persistent left superior vena cava (TARP) syndrome, in whom identification of a pathogenic RBM10 mutation confirmed the diagnosis [Gripp et al., 2011]. Here we report a different use of novel WES research-derived information, resulting in a change of the clinical diagnosis based on mutations in a gene recently identified as causally related to a different syndrome not previously considered for this patient. Identification of pathogenic mutations allowed for recognition of an expanded phenotype in Bartsocas–Papas syndrome, similar to the expanded phenotype including long-term survival in TARP syndrome described above. Similar expansion of recognized clinical phenotypes will likely occur in numerous Mendelian disorders. While this furthers our understanding of genetic conditions, the critical evaluation of WES data is necessary in order to avoid misinterpretation. For example, an inexperienced reader may misunderstand the KRT83 sequence variation reported as “deleterious mutations in disease genes related to clinical phenotype” to be disease causing, and may erroneously conclude that the previously associated phenotype monilethrix should be expanded to encompass facial clefting.
Impact of WES on Clinical Care and Counseling
The value of an accurate diagnosis cannot be overstated. In addition to allowing for appropriate medical care and screening studies for associated medical issues and complications, an accurate diagnosis allows for appropriate recurrence risk counseling. For the family reported here, the originally diagnosed autosomal dominant AEC syndrome presumably resulted from a de novo mutation. In contrast, Bartsocas–Papas syndrome is an autosomal-recessive condition, entailing a 25% recurrence risk for offspring born to these parents. The recurrence risk for offspring born to the proband would have been 50% for an autosomal dominant condition, but is much lower for an autosomal recessive condition. Of note, while the proband reported here is relatively mildly affected, a compound heterozygote offspring may present with a more classical severe Bartsocas–Papas phenotype. Lastly, identification of the familial pathogenic mutation or mutations through WES provides a tool for prenatal and pre-pregnancy testing, and thus affects and possibly benefits the extended family.
Exome analysis is a valuable research tool, which rapidly informs diagnostic WES. Clinical correlation of WES results in turn informs interpretation of novel sequence variations. While all previously reported Bartsocas–Papas syndrome patients had homozygous RIPK4 mutations, compound heterozygote patients are to be expected. Mutations in RIPK4 are associated with Bartsocas–Papas syndrome, and the phenotype is more variable than previously reported. Clinical recognition of the attenuated phenotype can facilitate molecular diagnosis through sequencing, and should result in timely identification of the 25% recurrence risk.
We thank the patient and her family for allowing us to share this information. We are grateful to Dr. Louis E. Bartoshesky for clinical information.
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