Journal of Genetic Disorders & Genetic Reports ISSN: 2327-5790

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Research Article, J Genet Disor Genet Rep Vol: 4 Issue: 1

CGH Array Based Case Report of a Patient Suffering with Amelogenesis Imperfecta, Jalili Syndrome, Situs Inversus and Oligozoospermia

Neetu Singh1*, Dinesh Kumar Sahu2, Parth Purwar3, Sanjeev Gupta4, Anil Kumar Tripathi5, Jaya Dixit3, Ravi Kant6 and Devendra Kumar Gupta1,7
1Advanced Molecular Science Research Center for Advance Research, King George's Medical University, Lucknow, India
2Imperialls Life Sciences Private Limited, Gurgaon, Haryana, India
3Department of Periodontology, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
4Department of Ophthalmology, King George's Medical University, Lucknow, Uttar Pradesh, India
5Department of Clinical Hematology, King George's Medical University, Lucknow, Uttar Pradesh, India
6Department of Surgical Oncology, King George's Medical University, Lucknow, Uttar Pradesh, India
7Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India
Corresponding author : Neetu Singh
Advanced Molecular Science Research Center Center for Advance Research, King George's Medical University, Lucknow-226 003, Uttar Pradesh, India
Tel: +91 5222258988; Fax: +91 5222257539
E-mail: [email protected]
Received: December 21, 2014 Accepted: December 31, 2014 Published: January 02, 2015
Citation: Neetu S, Dinesh KS, Parth P, Sanjeev G, Anil KT, et al. (2015) CGH Array Based Case Report of a Patient Suffering with Amelogenesis Imperfecta, Jalili Syndrome, Situs Inversus and Oligozoospermia. J Genet Disor Genet Rep 4:1. doi:10.4172/2327-5790.1000122

Abstract

CGH Array Based Case Report of a Patient Suffering with Amelogenesis Imperfecta, Jalili Syndrome, Situs Inversus and Oligozoospermia

A 35 year old male patient with family history of dental and ocular features, presented with chief complaint of discolored teeth since childhood and inability to see clearly during daytime reported to the O.P.D of Periodontology, King George’s Medical University, Lucknow, Uttar Pradesh. Henceforth, we suspected the presenting medical condition as syndromic and following investigations were carried out to establish the definitive diagnosis of the case so that appropriate management can be rendered to the patient as well as family members. Routine blood investigations, orthopantomogram (OPG), anterior segment and posterior segment examination was carried out. Advanced macular visualization on spectral domain optical coherence tomography (SDOCT) of retina was also performed. Additionally, chest X-ray and ultrasonography of whole abdomen (USG- abdomen) were also performed. To corroborate the phenotypic disorders with genotype, array based Comparative Genomic Hybridization (aCGH) was performed on the patient’s blood. Through Dental (clinical and radiographic), Opthalmic, X-ray/Ultrasound and Laboratory diagnosis diagnosed the patient as a phenotype with co-morbid occurrence of X-linked Hypoplastic Amelogenesis Imperfecta, Jalili Syndrome, Situs Inversus with oligozoospermia respectively. Further, through aCGH, the identified Copy Number Variations corroborated with the phenotypic features and were also reported in Decipher Database. Hence, the present case with multiple disorders (affecting multiple organs) suggest multi- factorial etiology involving Decipher Database reported MIR4424, NTRK1, UGT2B15 MSR1 PAK6 MIAT, ARHGAP4, XG and novel MIR1256, TMSB15B, H2BFXP, H2BFWT, H2BFM genes.

Keywords: aCGH; Amelogenesis imperfecta; Jalili syndrome; Situs inversus; Oligozoospermia

Keywords

aCGH; Amelogenesis imperfecta; Jalili syndrome; Situs inversus; Oligozoospermia

Introduction

Jalili syndrome (JS), a genetic disorder is the combination of conerod dystrophy of the retina and amelogenesis imperfecta (AI) [1]. Affected individuals commonly suffer from photophobia, nystagmus, achromatopsia, night vision difficulties, optic disc pallor, narrow vessels, macular atrophy with pigment mottling, peripheral deep white dot deposits or retinal pigment epithelium (RPE) alterations in the inferonasal retina, decreased foveal and retinal thickness, attenuation of retinal lamination, hyper-reflectivity in the choroids (due to RPE and choriocapillaris atrophy), impairment of color vision, and progressive loss of vision with advancing age [2-4].
In line with AI, affected patients show yellow-brown and dysplastic teeth, numerous caries, reduced enamel layer, prone to post-eruptive failure and abnormality of dentine morphology [3,4].
The syndrome owes to array of mutations with a linkage at the achromatopsia locus 2q11 on the metal transporter gene, CNNM4. Homozygosity or compound heterozygosity for several different mutations in the CNNM4 gene has been identified [3,4]. Moreover, mutations in the genes AMELX (encodes extracellular matrix proteins of the developing tooth enamel) [5], ENAM (encodes extracellular matrix proteins of the developing tooth enamel) [6], MMP20 (encodes proteases that help degrade organic matter from the enamel matrix during the maturation stage of amelogenesis) [7], KLK-4 (encodes proteases that help degrade organic matter from the enamel matrix during the maturation stage of amelogenesis) [8], FAM83H (plays an important role in the structural development and calcification of tooth enamel) [9], WDR72 (encodes a protein with eight WD-40 repeats) [10], C4orf26 (encodes an extracellular matrix acidic phosphoprotein that has a function in enamel mineralization during amelogenesis) [11,12], SLC24A4 (encodes a calcium transporter that mediates calcium transport to developing enamel during tooth development) [13], LAMB3 (encodes a laminin that belongs to a family of basement membrane proteins) [14] and ITGB6 (encodes a protein that is a member of the integrin super family) [15] have been reported previously. Despite of abundance of literature on the potential mutation sites there is scarcity of literature on the Copy Number Variations (CNVs) involved in the syndromic condition. Situs inversus (SI) is commonly an autosomal recessive genetic condition but occasionally reported as X-linked is characterized by presence of major organs on the opposite side to their normal anatomic position [16,17]. To the best of our knowledge, SI and oligozoospermia has not been reported with JS and AI till now. Although, seven SI cases with different variants have been reported in Decipher Database but none of the CNVs have been reported in the case as described. In the present case, we have attempted to explore the role of CNVs in the combinatorial effect of the disorder.

Materials and Methods

Clinical report
A 35 year old male patient reported to the O.P.D of Periodontology, King George’s Medical University (KGMU), Lucknow, and Uttar- Pradesh with chief complaint of discolored teeth since childhood and inability to see clearly during daytime. The concerned patient did not seek any treatment until now due to lack of dental facility in his rural area. It was only now when he realized that he avoided hard food substances and suffered from progressive loss of vision he was referred to the KGMU. Further questioning revealed that his 3 brothers, 1 sister, grandmother, maternal grandfather and aunt’s male and female child have similar dental and ocular features whereas parents are phenotypically normal (Figure 1). In addition the patient also suffered from oligozoospermia as evident by laboratory investigations (reports attached as supplementary material) and SI as depicted in chest X-ray and USG- abdomen (Figure 2a and b respectively). Other investigations like oral radiographic investigations for dental disorders (Figure 3b) anterior and posterior segment examination segment using slit lamp bio-microscopy and direct and indirect ophthalmoscopy respectively. Advanced macular visualization on SDOCT was also performed to confirm the diagnosis (Figure 4).
Figure 1: Genetic Predisposition of Family: Filled Square: Affected Male, Empty Square: Un- affected Male, Filled Round: Affected Female, Empty Round: Un-affected Female. Patient’s maternal grandfather and his two sisters were suffering from this disease. One of the maternal grandfather’s sisters (suffering from disease) was married to Patient’s grandfather (non-affected). Maternal grandfather and his wife had four sons and three daughters out of which one was Patient’s Mother. Patient’s Mother and father (phenotypically unaffected) had four sons and two sisters out of which three sons (including proband) and one sister was affected. Patient’s Aunt and his husband (phenotypically unaffected) had total seven daughter and two sons out of which one son and one daughter was affected.
Figure 2: (a) X-ray chest and (b) Ultrasound of visceral organs of the patient.
Figure 3: Intra oral examination of patient showing; (a) Photograph of labial surface of maxillary and mandibular teeth, (b) Orthopantomogram (OPG) of concerned patient and, (c) Ground section of extracted tooth showing absence of enamel.
Figure 4: Sections and tomography of eyes from Jalili patient; (a) Corneal optical section taken by slit lamp biomicroscope shows Vogt’s striae in the right eye, (b) Color fundus photograph showing pigmentary changes in fundus with generalized pallor suggestive of rod cone dystrophy with arterial attenuation and disc pallor with marked retinal thinning in macular region in both eyes, (c) Advanced macular visualization acquired by Spectral domain optical coherence tomography depicting marked retinal thinning with hypo reflective inner retinal layer and, (d) single layer maps obtained by spectral domain optical coherence tomography. All the three single layer map show global uneven topography of retina in macular region.
CytoScan 750K array for copy number analysis and gene identification
Genomic DNA was extracted from fresh peripheral blood using a QIAGEN DNA purification kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The Genome-Wide Human CytoScan 750K Array (Affymetrix, CA, USA) was used to analyze to genomic alterations in accordance to the manufacturer's protocol. Briefly, 250 ng of genomic DNA isolated from peripheral blood was digested and ligated with the restriction enzyme NspI and NspI-adapter respectively, followed by PCR amplification using NspIadapter primers. The PCR products were analysed on a 2% TBE gel to assess that the products were between 150 and 2000 bp in length, followed by purification using magnetic beads (Affymetrix). The PCR products were quantified by Quawell spectrophotometer.
Further, the >3 μg products were fragmented using DNase I and the fragmented product (25 to 125 bp) was analysed on 4% TBE agarose gel. The fragmented PCR products were subsequently end-labeled with biotin and hybridized to the array.
Arrays were then washed and stained using a GeneChip® Fluidics Station 450 and scanned using an Affymetrix GeneChip® Scanner 3000 7G. Scanned data files were generated using Affymetrix GeneChip Command Console Software, version 1.2, and analyzed with Affymetrix® Chromosome Analysis Suite v2.1 (ChAS) (Affymetrix Inc., USA) (Figure 5). To calculate copy numbers, thresholds of log2 ratio with default parameters were used to categorize altered regions as copy number gains and losses for amplification and deletions respectively. Amplifications and deletions were analyzed separately.
Figure 5: Karyo-View of the Copy Number Status (Gain/Loss).
To exclude aberrations representing common normal CNVs, all the identified CNVs were compared with those reported in the Database of Genomic Variants (DGV). Further, the genes involved in the CNVs were identified through UCSC database and Ensemble. Gene annotation and gene overlap were determined using the human genome build 19 (hg19) and widely used online databases viz: Ensembl and UCSC.

Results

Dental features
On intraoral examination, teeth were grossly malformed and associated with severe attrition (Figure 3a). Teeth no. 13, 23, 36 and 37 were missing while tooth no. 26 had sole carious lesion involving enamel and dentin.
Radiographic investigations included intra oral radiographs and orthopantomogram (OPG). The OPG illustrated malformation and complete absence of enamel in multiple teeth (Figure 3b). Macroscopically, the teeth surfaces were rough with yellowish brown discoloration. Microscopically, only thin layer of enamel is seen at cervical region of the teeth (Figure 3c). The emergence pattern and timing of teeth eruption was within normal reference range. There was pronounced shift in midline and class-3 malocclusion. Examination of the periodontium revealed the presence of chronic, generalized, marginal, papillary gingivitis with severe staining and unsatisfactory oral hygiene.
Ophthalmic findings
On examination, visual acuity of the patient was observed as perception of hand movements in the right eye and finger counting close to face in the left eye. Direct and consensual pupillary reaction was observed in both the eyes but was ill- sustained in the left eye. Mild simple ptosis was present in the right upper lid. Anterior segment examination was done using slit lamp biomicroscopy and revealed macular grade corneal opacity in the right eye while in the left eye cornea was clear. Anterior keratoconus with Vogt’s striae was present in both the eyes (Figure 4a). The keratometric values K1 and K2 in the right eye was 51.50 and 32.00 respectively and in the left eye was 43.75 and 37.27 respectively. Circular retrolenticular opacity was seen in the left eye. Posterior segment was examined by using direct and indirect ophthalmoscopy along with slit lamp biomicroscopy. Fundus showed generalized loss of pigmentation resulting in retinal pallor suggestive of rod cone dystrophy. There were pigment clumps or bony corpuscles throughout the posterior pole. There was generalized arterial attenuation with mild pallor of neuro retinal rim predominantly in the temporal half of the disc. Localized area of retinal thinning and defects presented inferior to fovea in the macular region of about 2 disc diameter (Figure 4b). Advanced macular visualization by optical coherence tomography showed thinning of retina which is in line to scanning laser ophthalmoscopy (SLO) image for the left eye. It also demonstrated thinned out inner neural retinal layer which was more hypo reflective than normal. The reflectivity of the outer retinal layer was also less than normal. Single layer map showed global uneven topography of retina in macular region and was suggestive of retinal folds. Vision in the right eye was too low for obtaining proper fixation for SDOCT (Figure 4c and d).
CytoScan 750K array for copy number analysis and gene identification
On analysis, we identified gain of MIR4424 (microRNA 4424) an RNA gene and loss of NTRK1 gene a member of the neurotrophic tyrosine kinase receptor (NTKR) family in Ch1. Loss of UGT2B15 (UDP glucuronosyltransferase 2 family, polypeptide B15) in Ch 4, MSR1 (macrophage scavenger receptor 1) in Ch 8, PAK6 (p21 protein (Cdc42/Rac)-activated kinase 6) in Ch15 and MIAT (myocardial infarction associated transcript) an RNA gene in Ch 22 (Figure 5) was also observed.
This is in line with Decipher Database where loss of MIR4424 in patient 248552 with facial asymmetry, ptosis, misalignment of teeth and hypermetropia; gain of NTKR region in patient 251161 with persistent pupillary membrane and lipodystrophy; loss of UGT2B15 in patient 270816 with delayed eruption of teeth, hypermetropia and congenital nystagmus; however gain of MSR1 in patient 263418 with hypospadias, abnormality of face and intellectual disability; gain of PAK6 was observed in gain patient-286150, -250179, -290779 with no phenotypic symptoms; both gain and loss of MIAT in patient 283322 with hypospadias, cryptorchidism and retinal coloboma as well as in patient 4110 with abnormality of the pinna, abnormality of the teeth, cataract, hearing impairment, increased number of teeth, intellectual disability, muscular hypotonia, preauricular pit, proptosis, short phalanx of finger and submucous cleft hard palate have been reported. In Ch X (Figure 5) gain of ARHGAP4 (Rho GTPase activating protein 4) and gain of XG blood group antigen gene was observed which corroborated to Decipher Database with gain of ARHGAP4 in patient 248547 and patient 4571 with cryptorchidism and abnormality of the outer ear/abnormality of liver and foot respectively. Additionally, gain of XG gene has been reported in patient 277458 with azoospermia and patient 285532 with cryptorchidism in Decipher Database (Table 1 and Figure 6).
Figure 6: Identified Copy Number Variations in proband: A) Gain of Ch1-MIR4424, B) loss of Ch1-NTRK1, C) Loss of Ch4-UGT2B15, D) loss of Ch8-MSR1, E) loss of Ch15-PAK6, F) loss of Ch 22-MIAT, G) gain of ChX-XG, H) gain of MIR1256, TMSB15B, H2BFXP, H2BFWT, H2BFM I) gain of Ch X-ARHGAP4, J) loss of ChX-TMLHE.
Table 1: Copy number analysis, gene identification and correlation with Decipher Database.
Novel gain (CN=2.00) of 132 kbp in Ch X (q22.2-q22.2 region) including MIR1256 (microRNA 1256, an RNA gene affiliated with the miRNA class), TMSB15B (thymosin beta 15B, a protein-coding gene playing an important role in the organization of the cytoskeleton), H2BFXP (H2B histone family, member X, a pseudogene), H2BFWT (H2B histone family, member W, testis-specific, a protein-coding gene included in male infertility, and among its related super-pathways), H2BFM (H2B histone family, member M, a protein-coding gene included in systemic lupus erythematosus and among its related superpathways) genes was also observed. Concurrently, novel loss of 31 kbp (CN=0.00), in q28-q28 region including TMLHE (converts trimethyllysine (TML) into hydroxytrimethyllysine (HTML) involved in diseases epsilon-trimethyllysine hydroxylase deficiency, and pervasive developmental disorder, and among its related superpathways) was also identified (Figure 6).

Discussion

The co-occurrence of enamel defects and ocular disease has been documented in oculodentodigital dysplasia (ODD) and Reigers syndrome (RS) but these differ significantly from those reported in present case. Features of ODD includes presence of small teeth with proclivity towards caries, long and thin nose, small eyes, malformation of fourth and fifth finger, glucoma formation, commonly and features of RS showing glucoma, congenital malformation of teeth and face and presence of corneal opacity.
In our case, provisional diagnosis of X-linked Hypoplastic Amelogenesis Imperfecta (AI) was made along with differential diagnosis of enamel and environmental hypoplasia, dentinogenesis imperfecta, dentin dysplasia and regional odontodysplasia. However, the definitive diagnosis of X-linked Hypoplastic AI was made on the basis of typical clinical, radiographic, heterozygous dominant inheritance pattern (phenotypically unaffected mother with affected son and daughter; Figure1)
And previous reports [18-20]. The X-linkage of AI corroborates well with that of Schulze and Lenz report [18] (Schulze and Lenz, 1952). Further, hypoplasticity was characterized on the basis of Witkop [19] classification of AI i.e. AI is classified on the basis of abnormality laid in a reduced amount of enamel (hypoplasia), deficient calcification (hypocalcification), or imperfect maturation of the enamel (hypomaturation), and also recognized the combined defects.
Additionally, the ocular features shows poor direct and consensual pupillary reaction of the left eye, mild simple ptosis of the right upper lid, macular grade corneal opacity of the right eye, anterior keratoconus with Vogt’s striae and high keratometric values in both the eyes, circular retrolenticular opacity in the left eye, rod cone dystrophy, pigment clumps or bony corpuscles in the posterior pole, generalized arterial attenuation with mild pallor of neuro retinal rim predominantly in the temporal half of the disc, localized area of retinal thinning and defects presented inferior to fovea in the macular region of about 2 disc diameter (Figure 4b), retinal thinning with global uneven topography of retina in macular region and retinal folds and low vision of the right eye classified the patient as that of jalili syndrome (JS).
The ocular features reported in the patient corroborated well with the previous reports (Jalili and Smith, 1988; Michaelides, Bloch- Zupan, Holder, Hunt and Moore, 2004; Parry, Mighell, El-Sayed, Shore, Jalili et al., 2009; Polok, Escher, Ambresin, Chouery, Bolay et al., 2009). Based on family history and clinical presentation along with the investigations the patient is more likely to be suffering from JS, with phenotypic variation of situs inversus (SI) and oligozoospermia.
Studies suggest an association of mutations in CNNM4 gene with JS; AMELX, ENAM, MMP20, KLK-4, FAM83H and WDR72 gene with AI; and in lefty genes, nodal genes, and ZIC 3, ACVR2B and Pitxz genes with SI [21]. Regardless of knowing potential mutation sites little is known about the Copy Number Variations involved in JS, AI and SI. Through array based comparative genomic hybridization, we conclude that previously identified variants in Decipher Database corroborated well with our patient’s phenotype i.e. Misalignment of teeth, Ptosis, Congenital nystagmus, Infertility and Azoospermia. However, newly identified variants cannot be categorized into pathologic (inherited) or non-pathologic due to study design limitations. To overcome the above difficulty we have to consider family analysis. Interestingly, the proband has 3 brothers, 1 sister, grandmother, maternal grandfather and aunt’s male and female child with similar dental and ocular features whereas parents are phenotypically normal. Hence, analyzing the whole family for transmission of inherited CNVs will help us to understand the contribution of the CNVs to the associated phenotype and. its pathogenicity.
However, the present case with multiple disorders suggests multifactorial etiology involving Decipher Database reported MIR4424, NTRK1, UGT2B15 MSR1 PAK6 MIAT, ARHGAP4, XG and novel MIR1256, TMSB15B, H2BFXP, H2BFWT, H2BFM genes.

Acknowledgements

This work was supported by Institutional Grant from King George Medical University, Lucknow, India. We are grateful to Mr. Paras Yadav, Imperialls Life Sciences (P) limited, Gurgaon - 122 001, Haryana, India for technical assistance.

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