A SRI LANKAN FAMILY WITH CEREBELLAR HEMANGIOBLASTOMA DUE TO A HETEROZYGOUS NONSENSE MUTATION IN THE VON HIPPEL-LINDAU TUMOR SUPPRESSOR , E 3 UBIQUITIN PROTEIN LIGASE ( VHL ) GENE

Mutations in the von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase (VHL) gene cause a variety of phenotypes including von Hippel-Lindau (VHL) disease. This report describes a Sri Lankan family with three siblings with cerebellar haemangioblastoma due to a nonsense mutation in the VHL gene. A heterozygous nucleotide substitution in exon 3 was identified in all three siblings resulting in a stop codon at amino acid position 175 leading to a truncated non-functional VHL protein[NM_000551.3(VHL):c.525C>G;p.Tyr175Ter;rs5030835C>G]. Patients with rare tumours characteristic of VHL should undergo clinical and genetic evaluation for VHL. The Sri Lanka Journal of Surgery 2015; 33(1): 30-32


ACKNOWLEDGEMENT
I would like to express my utmost gratitude towards Prof. Vajira Dissanayake and Prof.
Rohan Jayasekare for their invaluable guidance throughout the M.Sc.programme.I would also like to thank the Norwegian Center for International Cooperation in Education for funding the M.Sc.programme with NOMA grant and the Department of Medical Genetics at Oslo University Hospital for support in conducting the M.Sc.programme in collaboration with Human Genetics Unit at University of Colombo.My Special thanks go to Dr. Kalum Wettasinghe, Ms.Vindya Udalamatta, Ms.Imalki Kariyawasam and Dr. Nirmala Sirisena for their supervision, guidance and help during the research work and manuscript preparations.I would like to thank Dr. Asantha Jayawardane for his support during the preparation of molecular cytogenetic report and my friend Mr. Navoda Palpola for his continuous support during my research.I thank all my colleagues reading for masters in Genetic Diagnostic and Clinical Genetics, all the staff members at Human Genetics Unit, scientists at Asiri Center for Genomics and Regenerative medicine and my family for their invaluable support given during my studies and research.

Mutations in the VHL tumor suppresser gene cause a variety of phenotypes including von
Hippel-Lindau disease (VHL), familial phaeochromocytoma and inherited polycythaemia [2].VHL is an autosomal dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumors [3] such as haemangioblastomas of the cerebellum, spinal cord, brainstem and retina, clear cell renal carcinomas, pheochromocytomas, endolymphatic sac tumours, pancreatic islet cell tumours, haemangiomas of the adrenals, liver and lungs, epididymal and broad ligament papillary cyst adenomas as well as visceral cysts in the kidneys and pancreas [4].A germline mutation of the VHL gene is the basis of familial inheritance of VHL syndrome.According to Knudson's ("Two Hit") hypothesis, both alleles of a tumor suppresser gene need to be mutated in order for a tumour to develop, therefore a patient who manifests a tumour, inherits one mutation from a parent, and develops the second mutation in the same gene in the affected organ as a somatic mutation, at which point the tumour begins to manifest [5].
To date, more than 300 mutations have been identified in families with VHL disease, consisting of partial and whole gene deletions, frameshift, nonsense, missense, and splice site mutations [6].About 20% of cases are due to de novo mutations.This report describes a Sri Lankan family with 3 siblings with cerebellar haemangioblastoma due to a heterozygous nonsense mutation in the VHL gene.

The Family
A 28 year old female that was clinically diagnosed with cerebellar hemangioblastoma was referred to the Human Genetics Unit for genetic evaluation.The patient was clinically diagnosed with cerebellar haemangioblastoma at the age of 13 years, since then, she had undergone four surgeries for removal of the recurrent tumour in the posterior cranial fossa.In addition, a tumor arising from the fourth ventricle of the brain was also surgically removed.
Two of her male siblings were also diagnosed with cerebellar hemangioblastoma.The CT scan of one of the brothers showed dilatation and a cystic mass in the lateral third ventricle as well as a renal cyst.Figure 1 shows the pedigree of the family with VHL disease.

Materials and Methods
The VHL gene was sequenced in the patient and her 2 siblings after obtaining their written informed consent.DNA was extracted from peripheral blood using QIAamp blood DNA midi kit from Qiagen.All 3 exons and flanking intronic regions of the VHL gene were sequenced using an ABI PRISM 3130 Genetic Analyzer.The published human VHL gene Reference Sequence file obtained from GenBank (http://www.ncbi.nlm.nih.gov) was used for comparison of the nucleotide sequences generated from the patients and to confirm the presence of any mutations.

Results
A heterozygous nonsense mutation was identified in all 3 individuals in exon 3 of the VHL gene.A single nucleotide substitution at position 13214 (NG_008212.3.g13214C>G)replaced the codon for amino acid tyrosine (UAC) in transcript variant 1 (NM_000551.3.c525C>G) to a stop codon (UAG) resulting in premature termination of the VHL protein at amino acid position 175 (NP_000542.1.pTyr175Ter).This mutation has previously been reported in other families and documented in the dbSNP database and assigned the SNPID rs5030835 (http://www.ncbi.nlm.nih.gov/projects/SNP/rs=5030835).

Discussion
This report describes a Sri Lankan family with 3 siblings with cerebellar haemangioblastoma due to a heterozygous nonsense mutation in the VHL gene.VHL mutations are associated with various benign and malignant tumours resulting in high morbidity and mortality rates.
Mutations in the VHL gene are known to cause haemangioblastomas of the central nervous system (CNS) in 60-80% of VHL patients [6,7].
A study conducted by van der Harst et al. in 1998 reported that 8 out 68 patients with pheochromocytoma had mutations in the VHL gene.Among these patients, two were relatives and had a familial mutation [8].Familial mutations in the VHL gene have also been reported in VHL families presenting with clear cell renal cell carcinoma.Recent advances in understanding the genetic basis of VHL disease has resulted in improved diagnosis of VHL disease and provided greater insights into the molecular pathogenesis of the disease [1].The prognosis can be improved through early screening, diagnosis and surveillance [9].Molecular genetic testing coupled with genetic counseling is now considered standard for the evaluation of patients and families with suspected VHL [10].

Introduction
In the human population there is a vast variability to drug response among different individuals which is always connected with drug safety and efficacy.Different reasons such as environmental factors, physiologic factors, drug-drug interactions and genetic factors play an important role in this phenomenon.These genetic factors are involved in polymorphism in genes related to drug metabolizers, drug transporters and drug receptors [1].
"Pharmocogenetics" is the study of how different genetic variants like Single Nucleotide Polymorphisms and Copy Number Variants(SNPs &CNVs) in particular gene effects the drug response [2].
CYP2D6 gene is located on chromosome 22q13.1 and consists of 4.2 kb region [1].
Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic gene, which is responsible for the metabolism of several important endogenous substrates and other xenobiotics [3].Human cytochrome P450 is a gene supper family which contains 57 genes and 58 pseudogenes.Out of which, CYP2D6, CYP2C19, and CYP2C9 play a crutial role in pharmocogenomics since in the present day, 80% of the prescribed drugs are being metabolized by these enzymes [2].
In this study we have selected CYP2D6 gene since it metabolizes around 25% of the currently used drugs worldwide [4].This gene contains around 82 allelic variants important in the context of pharmacogenomics [3].There is a wide difference in the distribution among different ethnic groups.CYP2D6 is responsible for metabolizing antidepressants, antipsychotics, antiarrhythmics, antiemetics, beta-adrenoceptor antagonists (beta-blockers) and opioids.The presence of SNPs alters the CYP2D6 enzymatic activity with effects ranging significantly within a population and includes individuals with ultrarapid (UM), extensive (EM), intermediate (IM), and poor (PM) metabolizer status [3].The response to the drug may vary depending on the fact that whether the enzyme converts the drug in to the active form or whether the enzyme converts the active form of the drug in to the inactive form [4].
Most of the times CYP2D6 genotypes are depicted by using a star (*) eg: -CYP2D6 * 1.Each allele / haplotype can be identified using a specific combination of SNPs and/or other sequence variants (deletions, duplications) within the CYP2D6 gene [5].More than 130 SNPs have been identified within the CYP2D6 gene [3].In this study we use 9 SNPs (Table .1),which will allow us to identify CYP2D6 alleles in our population [1].
Currently, different molecular genetic techniques have been employed to genotype CYP2D6 gene variants, including Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and allele specific PCR methods.Multiplex single base primer extension technique is a well-documented method developed for genotyping CYP2D6 gene [1].In this study we attempted to develop a comparatively less expensive new multiplex allele specific PCR method to genotype the above given variants.

Materials and methods
Genomic DNA was obtained from an already existing population-based DNA collection maintained in our unit for studies of this nature with the approval of the Ethics Review Committee of the Faculty of Medicine, University of Colombo.Eighteen allele specific PCR primers for normal alleles (CYP2D6NPs) and mutated alleles (CYP2D6MPs) of all nine SNPs with another nine common reveres primers (CYP2D6RPs) were designed.In the majority of the allele specific primers, additional mismatches were introduced at the 3rd base from the 3' end of the primer to increase their efficacy.
Initially, the allele specific primers were separated into two master mixtures (MN and MM).
MN mixture contained all the normal allele while MM mixture contained all the mutated allele specific primers.In the final 25 µL reaction tube, concentration of each primer was 0.2 µM, 1X PCR buffer, 3mM MgCl2,50µM each dNTPs and 0.04U/µL Taq DNA polymerase.

Final extension -72 o C -7mins
The PCR products were visualized in a 3% agarose gel.However, it was hard to distinguish between 1846G>A (203bp) and 1661G>C (193bp) due to the poor band separation.
Therefore, primers were separated in to four master mixtures (MN1, MM1, MN2, MM2).The relevant reverse primers were also added to the master mixtures respectively (MN1- The final 25 µL reaction mixture consisted of 0.5 µM of each primer, 1X PCR buffer, 3mM MgCl2,50µM each dNTPs and 0.04U/µL Taq DNA polymerase.2 µL of the genomic DNA was added each reaction mixtures separately.PCR tubes were labeled as N1, M1, N2, and M2 accordingly.Amplification of desired product were carried out in applied bio system 2720 thermal cycler with following conditions, Initial denaturation -94 C -5 min.
The PCR products were separated by electrophoresis on a 3% agarose gel impregnated with ethidium bromide for 1 hour at 65V and visualized under UV light.

Interpretations
CYP2D6 plays a crucial role in metabolizing antidepressant drugs.Therefore, pharmocogenomic testing should be appropriately combined with the clinical practice in order to get better results.Furthermore, phromocogenomic data should be utilized at the correct moment to improve the patient outcome.Thus, the dosage of the drug should be determined accordingly [7].
There are several very efficient high throughput SNP genotyping methods currently in use, nevertheless, these methods require specialized probes, chemicals and instrument which are highly expensive hence, unaffordable to the public.These reasons lead to the limitation of the use of these methods in clinical diagnosis.Concurrently, traditional PCR-RFLP methods are laborious and time consuming [8].Therefore, a solution for above predicament was to design a multiplex allele specific PCR assay.
Multiplex allele specific PCR technique provides an opportunity to amplify many targeted product simultaneously, with several drawbacks .Thus, a complex optimization process is required to enhance the assay.Additionally, the increase number of primer sets reduces the efficiency and flexibility of amplification of desired products [9].
Therefore, the current assay was optimized by a very complex and tedious optimization process.The only limitation with this method is its low consistency.

Conclusion
Multiplex Allele specific PCR method is a comparatively cost effective method for genotyping CYP2D6 variants nevertheless, this method require further optimization to increase the consistency and reproducibility.

Reasons for Referral:
Evaluating Genetic factors affecting drug metabolism for patients taking drugs metabolized by CYP2D6.(Ex: antidepressant drugs, Tamoxifen)

Limitations:
Not all variants with known impact on enzyme expression and activity are tested in this assay.
Testing Methodology: DNA amplification is done by allele specific multiplex PCR and the analyzing of PCR products is done by agarose gel electrophoresis.

MOLECULAR-CYTOGENETIC ANALYSIS OF A RING CHROMOSOME 18 IN A SRI LANKAN FEMALE CHILD WITH CONGENITAL MALFORMATION, HEART DEFECTS AND GLOBAL DEVELOPMENT DELAY Introduction
Ring chromosome 18, (r18) is a rare chromosomal disorder [1].It can be formed when a part of one or both ends of chromosome 18 has been deleted and joined together [2].As a result, clinical features of patients with ring chromosome 18 depends on the extent of the deleted region at the chromosomal ends [3].Clinical features of ring chromosome 18 may include facial dysmorphism, blepharoptosis, hypotonia, development delay, short stature, microcephaly, mental retardation, heart defect and IgA deficiency [1,3,4].Association of global development delay, congenital malformation and heart defects with ring chromosome 18 has been reported in previous studies [4] Ring chromosome 18 can also arise without any deletions due to dysfunctional telomeres [5].This may lead to asymptomatic carriers of ring chromosome 18.
We report cytogenetic and molecular-cytogenetic findings of a Sri Lankan female child with congenital malformation, heart defects and global development delay.

Case presentation
A two year old girl with congenital malformation, heart Defects and global development delay was referred to the Human Genetics Unit for karyotyping.She is the second child born to a non consanguineous healthy couple at term by a normal delivery following an uneventful pregnancy.The first child of this family is a healthy boy and the second pregnancy ended up in a spontaneous miscarriage at 10 weeks of gestation.
She had a birth weight of 2.2kg,a head circumference of 32 cm and length of 50 cm.All her birth parameters were below the third centile.She had a complete left side cleft lip and cleft palate which was severe enough to make breast feeding impossible.She developed a cyanotic episode in early neonatal period and was referred to a Paediatric cardiology unit.Initial 2D echocardiogram revealed double outlet right ventricle, large permembranous ventricular septal defect and moderate subpulmonic pulmonary stenosis.Further echocardiograms and cardiac catheterization identified single opening in mitral valve with mitral stenosis.Surgical correction of the cardiac defect had been ruled out due to high risk and she was managed conservatively.
She had severe developmental delay and hypotonia.Only partial head control has been achieved at the time of presentation.Social smile was present.She had severe growth failure.
At the age of 2 years her weight was 4.5 kg, head circumference was 39.5 cm and the length was 59 cm.All parameters were well below the 3 rd centile.Apart from dysmorphic features such as microcephaly, flat occiput, mid face hypoplasia and micrognathia were also present.
She had telecanthus, epicanthic folds and slightly up slanted palpebral fissures.Her nasal bridge was flat and ears were marginally low set.Except for long tapering fingers there was no other deformity in the upper limbs but in lower limbs bilateral talipes equinovarus deformity was noted.Appearance of her external genitalia was normal and she did not have any thoracic or spinal abnormalities.Furthermore, ultra sound scan of the abdomen was found to be normal and CT scan of the brain did not identify any structural malformation of the brain.

Materials and Methods
Ethical clearance for the study was obtained from the Ethics review committee of Faculty of Medicine, University of Colombo.Patient was recruited for this study after obtaining the written informed consent from the parents.

Cytogenetic analysis
Peripheral blood of the patient and her parents were cultured in PB-MAX karyotyping media separately for 72 hour.Peripheral blood lymphocytes were harvested and chromosomal staining was carried out with GTL banding technique according to standard protocols.
Metaphase chromosome spreads were analyzed under Olympus BX61 microscope and analyzed using Cytovision 3.1.

FISH analysis
Fish probes RP11-317F19 (specific for GLAR1 gene at 18q23 position) and RP11-1082M21 (specific for TGIF 1 gene at 18p11.3) were ordered from Empire Genomics, USA after studying about the probable genes causing the phenotype.Slide was cleaned with 70% ethanol.Slide preparation and hybridization of probes were carried out according to standard FISH protocol of Empire Genomics (http://www.empiregenomics.com/resources/genomic_procedures).
Slide preparation from harvested cell suspension carried out with 50% relative humidity.
After air drying the slide, it was kept at 45 0 C in the incubator and then 10 µL of probe mixture (2 µL of the probe and 8 µL of hybrid buffer) was added to the slide.Cover slip was applied on the slide and sealed with rubber cement.Slide was placed in the Thermobrite.
Denatured at 73 0 C for 2 minutes and hybridized for 16 hours.After hybridization cover slip was removed and the slide was washed by agitating with prewarmed WS1 (wash solution 1) at 73 0 C for 10 seconds and incubated for 2minutes in the solution.Slide was transferred to WS2 at room temperature for 1minutes then it was dried in the dark and 10 µL of DAPPI was added.Again another cover slip was applied on the slide.After 30 minutes fluorescent signals were observed under Olympus BX61 and analysis was done using Applied Spectral Imaging system software.Above procedures were carried separately for both probes.

Results
Twenty six metaphase chromosomal spreads of the patient were analyzed and six of these spreads were karyotyped.Ring chromosome 18 was observed in all the spreads analyzed.
Karyotypes of the parents were found to be normal.
When showing the TGIF1 gene has not been disrupted.Figure 3 shows FISH images of metaphase spreads.

Discussion
The patients with ring chromosome 18 show a wide range of phenotypic features [1].Majority of the patients reported with ring chromosome 18 shows symptoms of 18q-syndrome while the others show symptoms of 18p-syndrome symptoms or both 18q-and 18psyndromes [4].Mental retardation, development delay, heart defects and facial dysmorphism was reported with many cases of ring chromosome 18 [4,6].
Stankiewicz et al carried out a clinical and molecular cytogenetic study in 7 patients with ring chromosome 18.In that study they even found a patient mosaic for ring chromosome 18 and another patient with a duplication in 18p arm with mild phenotypes [4].Table 1 compares the prominent phenotypes of 5 patients of the above study with our patient excluding the patient with mosaic ring chromosome 18.Previous studies had suggested that growth hormone insufficiency is associated with the haploinsufficiency at 18q23 position and GLAR 1 gene(also known as GALNR1) is known to play a role in this phenomena [7].This explains the deletion of GLAR1 gene in the ring chromosome 18 of our patient FISH probe RP11-1082M21 which is specific for TGIF 1 gene at 18p11.3, had signals in both normal and ring chromosome 18.This result shows the presence of TGIF 1 gene in both ring and normal chromosome, but it does not exclude any possibilities of a deletion distal to the location of TGIF gene.
TGIF 1 Gene (also known as HPE4) at 18p11.3 is found to be associated with holoprosencephaly which is associated with abnormal development of the forebrain and midface [8].Holoprosencephaly is also characterized by hypotelorism (http://www.ncbi.nlm.nih.gov/books/NBK1530/)but our patient showed telecanthus.The CT scan report of the patient did not identify any structural malformation of the brain.From the FISH results it was found that TGIF 1 gene was not deleted in ring chromosome 18.Presence of TGIF 1 gene in both ring and normal chromosome may explain the above phenomena.
Further FISH analysis with more probes and molecular studies can be employed to determine the exact break points of ring chromosome 18 of this patient.

Figure 2
Figure 2 shows the partial electropherogram with the point mutation at position 13214 of the

Figure 1 :
Figure 1: Pedigree of the family with VHL disease showing the familial mutation in the three siblings.

Figure 2 :
Figure 2: Partial electrophoragram of the patient showing the heterozygous nonsense mutation in the VHL gene of results were achieved by the presents or absence of the bands on the gel.The bands which represent the PCR product of N1 and N2 mixtures (mixtures with normal allele specific primers) in the gel indicates the presence of normal alleles and the bands which represent M1 and M2 PCR products indicates the presence of mutated allele.If both bands (mutated and normal) were present, the sample was considered as heterozygous for the respective SNP.

Figure1.
Figure1.Facial dysmorphic features of the patient.This view shows complete left sided cleft lip and palate, telecanthus, epicanthic folds and slightly up slanted palpebral fissures , flat nasal bridge and upsweep of the frontal hairline.
FISH probe RP11-317F19 (specific for GLAR1 gene at 18q23) position was used, Green fluorescent signals were only seen in normal chromosome 18 and no signal was observed in ring chromosome of all the analyzed spreads.Absence of fluorescent signals on ring chromosome 18 indicates that the GLAR1 gene is deleted from the18q23 region.When FISH probe RP11-1082M21(specific for TGIF 1 gene at 18p11.3) was used red fluorescent signals were observed in both normal 18 and ring chromosome 18 of all the analyzed spreads,

Figure 3 :Figure 4 :
Figure 3: FISH image of probe RP11-317F19 showing green fluorescent signals only in normal chromosome 18.Ring chromosome 18 is indicate by an arrow fi

Table 1 :
List of primers and their sequences used in the study.

CYP) 2D6 gene metabolizes around 25% of current drugs used worldwide. CYP2D6 is responsible in metabolizing different drugs such as tricyclic antidepressants, antipsychotics not only that but also antiarrhythmics, antiemetics, beta- adrenoceptor antagonists (beta- blockers) and opioids. The presence of SNPs alters the CYP2D6 enzymatic activity with effects ranging significantly within a population and includes individuals with ultrarapid (UM), extensive (EM), intermediate (IM), and poor (PM) metabolizer status. The response to the drug may vary depending on the fact that weather the enzyme converts the drug in to the active form or the inactive form.
Run 10µl of PCR product at 65v in 2% gel for 30 min. Date:-………………

Confidential Molecular Genetic Laboratory Test Report Date : DD/MM/YY Patient Identification:
These 9 variants would not cover all the pharmocogenomically important variants in CYP2D6 gene.