Year : 2010 | Volume
: 28 | Issue : 2 | Page : 104--109
The role of 9qh+ in phenotypic and genotypic heterogeneity in a Van der Woude syndrome pedigree
GA Moghe1, MS Kaur2, AM Thomas3, T Raseswari4, M Swapna5, L Rao6,
1 Senior Lecturer, Department of Pedodontics and Preventive Dentistry, Panineeya Institute of Dental Sciences and Research Centre, Dilsukhnagar, Hyderabad - 500 060, Andhra Pradesh, India
2 Senior Lecturer, Department of Pedodontics and Preventive Dentistry, BRS Dental College, Panchkula, Haryana, India
3 Professor and Head, Department of Pedodontics and Preventive Dentistry, Christian Dental College, C.M.C, Brown Road, Ludhiana - 141008, Punjab, India
4 Senior Technical Assistant, Centre for Cellular and Molecular Biology, Hyderabad, India
5 Laboratory Assistant, Centre for Cellular and Molecular Biology, Hyderabad, India
6 Scientist, Centre for Cellular and Molecular Biology, Hyderabad, India
G A Moghe
Flat no. 202, Gharonda Sargam Apts., 11-1-269, Sitafalmandi, Secunderabad-500 061
Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited developmental disorder that is characterized by pits and/or sinuses of the lower lip and cleft lip and/or cleft palate. Mutations in the interferon regulatory factor 6 gene (IRF6) have been recently identified in patients with VWS, with more than 60 mutations reported. We report the phenotypic variants of the syndrome in a family and present the application of the multicolor chromosome banding (mBAND) analysis in the identification of complex intrachromosome rearrangements of chromosome 9 in a child with VWS. The authors conclude that increased heterochromatin on chromosome 9 did not have any effect on the phenotypic expression of the syndrome in the family that was studied.
|How to cite this article:|
Moghe G A, Kaur M S, Thomas A M, Raseswari T, Swapna M, Rao L. The role of 9qh+ in phenotypic and genotypic heterogeneity in a Van der Woude syndrome pedigree.J Indian Soc Pedod Prev Dent 2010;28:104-109
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Moghe G A, Kaur M S, Thomas A M, Raseswari T, Swapna M, Rao L. The role of 9qh+ in phenotypic and genotypic heterogeneity in a Van der Woude syndrome pedigree. J Indian Soc Pedod Prev Dent [serial online] 2010 [cited 2019 Nov 14 ];28:104-109
Available from: http://www.jisppd.com/text.asp?2010/28/2/104/66749
Van der Woude syndrome (VWS) is the most common form of syndromic clefting, accounting for 1-2% cases of cleft lip and palate (CLP). With an autosomal dominant mode of inheritance, the prevalence of VWS varies from 1:100,000 to 1:40,000 still born or live births. , No significant difference between sexes has been reported with regard to the prevalence of the syndrome. 
The syndrome shows a varied phenotypic expression. Lower lip pits, cleft lip (CL) with or without cleft palate (CP), and isolated CP are its cardinal signs. Phenotypic expression of the clefts ranges from incomplete unilateral CL, submucous CP, bifid uvula, to complete bilateral CLP. VWS is also associated with Popliteal pterygium syndrome (PPS) and orofacial-digital syndrome type I (OFD-I). 
VWS is of special interest because the phenotype closely resembles nonsyndromic forms of both CL and CP.  One of the unusual features of VWS is that different forms of clefts occur in vertical and horizontal directions in the same pedigree; the average penetrance has been reported to be 96.7%.  In this article, we report one such family which presented with lower lip pits with variable forms of CLP.
The proband, a 6-year-old male child, the first-born of a non-consanguineous marriage, was a case of repaired bilateral CLP, with remaining esthetic defects leading to columella deformity and alar base depression, in addition to lip pits [Figure 1]. He was referred to our department for restoration of carious teeth. Intraoral examination revealed clinically missing maxillary deciduous lateral incisors and carious lesions with respect to deciduous maxillary right central incisor and second molar, and deciduous maxillary left first molar. Bilateral lip pits, circular to oval, were noted on the lower lip at the junction of the wet and dry vermilion, and were paramedian in location and asymptomatic. Complete physical examination did not reveal any other systemic abnormality.
The mother's antenatal history was insignificant. There was no history of any major cardiac illnesses, diabetes, tuberculosis, epilepsy or any teratogenic drug exposure. The birth of the child was by normal vaginal delivery at full term.
Further, family history revealed that the patient's younger sibling, a 2.5-year-old female child, also suffered from congenital lip pits and had undergone corrective surgery for bilateral CLP [Figure 2]. The third pregnancy ended in a miscarriage, details of which could not be elucidated.
No clefts were known to have existed in previous generations or in distant relatives. The children's mother was examined and it was noticed that she had a bifid uvula [Figure 3] and bilaterally symmetrical lower labial fistulae. The patient's father was phenotypically normal. None of the mother's or father's siblings, as reported by them, was affected [Figure 7], but the parents were concerned about the trait being passed on to subsequent generations.
After written, informed consent for further investigations the and institutional review board approval were obtained, radiographs of the proband were taken to investigate the depth and extent of the labial fistulae. A radiograph of the lower lip revealed that the gutta percha points traversed a depth of 6 mm in the right fistula and 5 mm in the left fistula.
Peripheral venous blood samples were collected in vacutainers precoated with heparin and Ethylene Diamine Tetraacetic Acid, and transported to the laboratory within 24 hours for further processing and testing.
All the samples were subjected to Giemsa banding. Additionally, fluorescent in situ hybridization (FISH) and multicolor chromosome banding analysis (mBAND) were performed using the proband's blood sample .
In this family, karyotyping of the samples done using the Giemsa banding technique. Interestingly, an increase in heterochromatin in the long arm of chromosome 9 of father [Figure 4], the younger sibling [Figure 5], and the proband [Figure 6] was noted, heterozygous in all cases. Since many authors have confirmed the occurrence of microdeletions on chromosome 1 in VWS, no further attempt was made to characterize these deletions. Rather, special emphasis was laid on chromosome 9 which may have acted as a modifier locus leading to complete phenotypic expression of the syndrome in the children. The mBAND confirmed an increase in heterochromatin in the long arm of chromosome 9, read as 9qh+.[Figure 6]
Nonparametric linkage analysis (NPL) was done using a Unix-based version of GENEHUNTER and a NPL Logarithm of the odds (LOD) score of 0.48 was obtained. Chi-square value obtained was 2.20, which was not statistically significant.
Congenital lip pits can be a sole developmental abnormality or may be syndromic. The pits are usually found at the junction of the wet and dry vermilion, at a distance of 5-25 mm from each other,  and they traverse the orbicularis oris. The lip pits form canals lined by labial mucosa, and are between 1 and 25 mm long. The canals have blind ends in/near minor salivary glands. Lip pits are usually asymptomatic; the only symptom might be the intermittent drainage of salivary/watery secretions.  Taylor and Lane have suggested that a peristaltic projection of the mucous secretion may occur upon contraction of the orbicularis oris fibers. 
The genetic basis of VWS can be traced back to the study of Bocian et al. who reported a patient with lip pits and a deletion in 1q32-q41, and subsequently to a study of Murray et al. who found linkage between VWS and markers from the same region.
Futher studies  narrowed the region to a 1.6-cM region between the flanking markers D1S491 and D1S205. In this region, there are at least 15 confirmed genes, 9 putative genes and 3 pseudogenes. 
The identification of deletion mutations in three independent cases of VWS ,, suggests that VWS is caused by haploinsufficiency of a gene at the VWS locus. A second locus (VWS2) has been mapped to 1p34.  Kondo,  Matsuzawa  and Gatta  have reported missense mutations in the IRF6 gene. IRF6 is a member of a gene family of transcription factors characterized by the presence of an highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain termed Smad-interferon regulatory factor binding domain (SMIR), required to form homo and heterodimers. 
For those with VWS, the risk of transmitting a cleft is between 11 and 22%. , The relative risk of transmitting lower lip pits only, or being nonpenetrant, is from 24.7 to 42.7%. , Study of the reported family reveals a combination of cleft of soft palate and lip pits in the mother. Both the siblings, however, presented with a complete bilateral cleft of the lip and palate along with lip pits. Thus, the expressivity was calculated to be 100% [Figure 7].
Explanations for the difference in expressivity in this family may be (1) the development of clefts in persons carrying a "lip pit" major gene may be influenced by modifying genes at other loci; (2) a mutant allele may produce lip pits with only occasional clefts; (3) a different mutant allele (at the same or a different locus) may frequently lead to clefts in addition to lip pits.
On the simple assumption that everybody with the disease carries a mutant allele at the affected locus/loci, if the disease is dominant,one may conclude they will share at least one parental haplotype.
Because sib-pair analysis is model free, it can be performed without making any assumption about the genetics of the disease. By convention, an LOD score greater than 3.0 is considered as an evidence for linkage. But NPL analysis revealed an LOD score of 0.48 which clearly indicated that increased heterochromatin on chromosome 9 did not have any effect on the phenotypic expression of the syndrome in the proband. This is in agreement with several literature reports which state that 9qh+ is a normal variant among populations.
Among Asiatic population groups, the frequency of 9qh+ varies from total absence among both the sexes of newborns of Delhi city (Bhasin et al.)  to 8.30% among Indian normal individuals. 
Holbek et al. observed a high frequency of 9qh+ in parents of chromosomally abnormal abortions, whereas Hemming and Burns  did not encounter a significant difference in the 9qh+ regions between aborting and nonaborting couples. Kunze and Mau  reported high frequency of 9qh+ heteromorphism in patients with multiple congenital malformations. Nielsen and Sillesen  observed 9qh+ heteromorphism in 8% of the members of children with de novo major chromosomal aberrations, whereas the incidence of 9qh+ was 0.04% among newborns. 9qh+ mutations have also been reported in patients with schizophrenia, fragile X syndrome, and Elllis van Creveld syndrome. Contrary to this, Madan and Bobrow did not observe any adverse effect of 9qh+ in their study. 
Further linkage analysis studies need to be performed to narrow the critical region responsible in this family.
A case of VWS with familial occurrence has been presented. However, it should also be noted that congenital lip pits could also be a part of other syndromes of great heterogeneity. Careful physical examination of family members should be done, in all sporadic cases, to confirm the diagnosis in those presenting only minor manifestations and to identify less severely affected relatives of those with full expression. VWS patients may rarely show clefts without pits; these cases represent a small group of cleft patients with a high recurrence risk and underline the need for specific questions and examination for lip pits, including microforms, in relatives of cleft patients. The importance of genetic counseling should be emphasized at all times along with multidisciplinary treatment. Further research should be done to detect the genetic basis of the developmental abnormality, since VWS is the most common form of syndromic clefting. Any other modifier locus whose alteration may lead to a variable phenotypic expression still needs to be established.
The authors gratefully acknowledge the contribution of Dr. Vijay Obed , Dr. Deepak J. Bhatti (Department of Plastic and Microvascular Surgery, CMC, Ludhiana, India), and Dr. Viraj S. Tambwekar (Plastic Surgeon, Seven Hills Hospital, Mumbai, India) for their support. We also thank Dr. Lalji Singh, Ex-Director, Centre for Cellular and Molecular Biology, Hyderabad, India, for allowing us to use the laboratory facilities at the institute.
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