FREQUENCY OF HAEMOGL OF SRI LANKAN PATIENT DESIGN AND IMPLEMEN AND 719A>G POLYMORPH A MATERNALLY INHER TRISOMY 15 (pterq22) IN A C PRANIDH DIS THE UNIV IN PARTIAL FULF MASTER OF GLOBIN BETA GENE (HBB) MUTATIONS I TS REFERRED FOR β THALASSAEMIA S ~ NTATION OF A NEW ASSAY TO GENOTY HISMS IN THE THIOPURINE S-METHYLT (TPMT) GENE ~ RITED PARTIAL TRISOMY 1 (q44qter) AND CHILD WITH SILVER RUSSELL & PART 15q SYNDROME BY HEE BHAGYA JEERASINGHE (B.Sc) FMC/GD/02/2012/02 SSERTATION SUBMITTED TO VERSITY OF COLOMBO, SRI LANKA FILMENT OF THE REQUIREMENTS OF T F SCIENCE IN GENETIC DIAGNOSTICS AUGUST 2014 IN A COHORT SCREENING YPE 460G>A TRANSFERASE D PARTIAL TIAL TRISOMY THE
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FREQUENCY OF HAEMOGLOBIN BETA GENE (HBB) MUTATIONS IN A COHORT
OF SRI LANKAN PATIENTS REFERRED FOR β THALASSAEMIA SCREENING
~DESIGN AND IMPLEMENTATION OF A NEW ASSAY TO GENOTYPE 460G>A
AND 719A>G POLYMORPHISMS IN THE THIOPURINE S-METHYLTRANSFERASE
(TPMT) GENE
~A MATERNALLY INHERITED PARTIAL TRISOMY 1 (q44qter) AND PARTIAL
TRISOMY 15 (pterq22) IN A CHILD WITH SILVER RUSSELL & PARTIAL TRISOMY
15q SYNDROME
BY
PRANIDHEE BHAGYA JEERASINGHE (B.Sc)
FMC/GD/02/2012/02
DISSERTATION SUBMITTED TO
THE UNIVERSITY OF COLOMBO, SRI LANKA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
MASTER OF SCIENCE IN GENETIC DIAGNOSTICS
AUGUST 2014
FREQUENCY OF HAEMOGLOBIN BETA GENE (HBB) MUTATIONS IN A COHORT
OF SRI LANKAN PATIENTS REFERRED FOR β THALASSAEMIA SCREENING
~DESIGN AND IMPLEMENTATION OF A NEW ASSAY TO GENOTYPE 460G>A
AND 719A>G POLYMORPHISMS IN THE THIOPURINE S-METHYLTRANSFERASE
(TPMT) GENE
~A MATERNALLY INHERITED PARTIAL TRISOMY 1 (q44qter) AND PARTIAL
TRISOMY 15 (pterq22) IN A CHILD WITH SILVER RUSSELL & PARTIAL TRISOMY
15q SYNDROME
BY
PRANIDHEE BHAGYA JEERASINGHE (B.Sc)
FMC/GD/02/2012/02
DISSERTATION SUBMITTED TO
THE UNIVERSITY OF COLOMBO, SRI LANKA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
MASTER OF SCIENCE IN GENETIC DIAGNOSTICS
AUGUST 2014
FREQUENCY OF HAEMOGLOBIN BETA GENE (HBB) MUTATIONS IN A COHORT
OF SRI LANKAN PATIENTS REFERRED FOR β THALASSAEMIA SCREENING
~DESIGN AND IMPLEMENTATION OF A NEW ASSAY TO GENOTYPE 460G>A
AND 719A>G POLYMORPHISMS IN THE THIOPURINE S-METHYLTRANSFERASE
(TPMT) GENE
~A MATERNALLY INHERITED PARTIAL TRISOMY 1 (q44qter) AND PARTIAL
TRISOMY 15 (pterq22) IN A CHILD WITH SILVER RUSSELL & PARTIAL TRISOMY
15q SYNDROME
BY
PRANIDHEE BHAGYA JEERASINGHE (B.Sc)
FMC/GD/02/2012/02
DISSERTATION SUBMITTED TO
THE UNIVERSITY OF COLOMBO, SRI LANKA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
MASTER OF SCIENCE IN GENETIC DIAGNOSTICS
AUGUST 2014
CERTIFICATION
I certify that the contents of this dissertation are my own work and that I have acknowledged the
sources where relevant.
…………………………………………
Signature of the candidate
This is to certify that the contents of this dissertation were supervised by the followingsupervisors:
MUTATION REPORT
……………………………. …………………………….…Dr. U.N.D. Sirisena Prof. V.H.W. Dissanayake
PHARMACOGENOMICS REPORT
……………………………. …………………………….…Dr. K.T. Wettasinghe Prof. V.H.W. Dissanayake
MOLECULAR CYTOGENETICS REPORT
……………………………. …………………………….…Ms. I. Kariyawasam Ms. V. Udalamaththa
…………………………….Prof. V.H.W. Dissanayake
ACKNOWLEDGEMENT
The writing of this dissertation has been one of the significant academic challenges I have faced.
Without the support, patience and guidance of the following people, this dissertation would not
have been completed. It is to them that I owe my deepest gratitude.
I am indebted to Prof Vajira Dissanayake for his continuous encouragement and guidance. He
sets high standards for his students and he encourages and guides them to meet those standards.
Furthermore, Dr. U.N.D. Sirisena, Dr. K.T. Wettasinghe, Ms. I. Kariyawasam, Ms. V.
Udalamaththa who undertook to act as my supervisors for their guidance, wisdom, knowledge
and constant supervision as well as for providing necessary information regarding the
dissertation.
I am also grateful to the molecular laboratory staff at Asiri Centre for Genomics & Regenerative
medicine, Colombo, Sri Lanka for assistance with DNA sequencing and helping me to find
patient data.
I would like to thank all my colleagues for their kind co-operation and encouragement which
helped me throughout in completing this dissertation.
Most importantly, none of this would have been possible without the love and patience of my
family to whom this dissertation is dedicated to. They have been a constant source of love,
concern, support and strength all these years.
MUTATION REPORT
FREQUENCY OF HAEMOGLOBIN BETA
GENE (HBB) MUTATIONS IN A COHORT
OF SRI LANKAN PATIENTS REFERRED
FOR β THALASSAEMIA SCREENING
1
ABSTRACT
Introduction: Thalassaemia is an autosomal recessive disorder commonly seen in the Sri
Lankan population. Majority of mutations seen in the HBB gene leading to β thalassaemia are
single nucleotide substitutions, deletions or insertions of oligonucleotides leading to frame-shift
mutations. Some of these mutations cause an absence of β-chain production and the resulting
disease is called β-zero (B0) thalassaemia, whereas others result in a reduced output of β chains,
β-plus (B+) thalassaemia.
This report presents the frequency of different HBB gene mutations in a cohort of Sri Lankan
patients referred for β thalassaemia screening.
Methods: The mutations that were screened using direct sequencing method in the present study
includes IVS1–5 (G>C), IVS 1–1 (G>A), CD 26 (G>A), CD 6 (A>T), CD15(G>A), IVS1-130
Table 4. Data on HBB gene mutations found with the presence of three single nucleotide
polymorphisms in 12 patients
HBB gene mutation HBB Gene SNPs GeneticIVSII 16
C>GIVSII 74
A>CCD2A>C
Diagnosis
rs10768683
rs7480526 rs713040
CD26 G>A + IVS I-5 G>C CG AA AC HbE/ β thalassemia
IVSI-5 G>C Homozygous CC AA AA βthalassaemia Major
IVSI-5 G>C Homozygous GG AA AA βthalassaemia Major
IVSI-5 G>C Heterozygous CG AC AC Carrier
IVSI-5 G>C Heterozygous CG AC AC Carrier
CD15 G>A Heterozygous CG AC AC Carrier
IVSI-5 G>C + CD15 G>A CC AA AA β thalassaemia Major
∆619bp Homozygous
deletion
CC AA AA β thalassemia Major
IVSI-1 G>A Heterozygous CG AA AC Carrier
CD6 A>T + IVS1-130 G>A CG AA AC HbS/β thalassemia
IVSI-5 G>C Heterozygous CG AC AC Carrier
IVSI-5 G>C + IVSI-1 G>A CC AA AA β thalassemia Major
v
Figure 1: IVS 1-1 (G>A) heterozygous mutation in a patient with Beta Thalassaemia
v
Figure 1: IVS 1-1 (G>A) heterozygous mutation in a patient with Beta Thalassaemia
v
Figure 1: IVS 1-1 (G>A) heterozygous mutation in a patient with Beta Thalassaemia
vi
Figure 2: IVS1-5 (G>C) heterozygous mutation in a patient with Beta Thalassaemia
vi
Figure 2: IVS1-5 (G>C) heterozygous mutation in a patient with Beta Thalassaemia
vi
Figure 2: IVS1-5 (G>C) heterozygous mutation in a patient with Beta Thalassaemia
vii
Figure 3: IVS1-5 (G>C) homozygous mutation in a patient with Beta Thalassaemia
IVSI-5 G>C
vii
Figure 3: IVS1-5 (G>C) homozygous mutation in a patient with Beta Thalassaemia
IVSI-5 G>C
vii
Figure 3: IVS1-5 (G>C) homozygous mutation in a patient with Beta Thalassaemia
IVSI-5 G>C
viii
Figure 4: Compound heterozygosity for CD 26 (G>A) and IVS1-5 (G>C) mutations in a
patient with HbE/β-Thalassaemia
IVS1-5 G>CCD26 G>A
viii
Figure 4: Compound heterozygosity for CD 26 (G>A) and IVS1-5 (G>C) mutations in a
patient with HbE/β-Thalassaemia
IVS1-5 G>CCD26 G>A
viii
Figure 4: Compound heterozygosity for CD 26 (G>A) and IVS1-5 (G>C) mutations in a
patient with HbE/β-Thalassaemia
IVS1-5 G>CCD26 G>A
ix
Figure 5: Homozygous deletion for ∆619bp in a patient with β-Thalassaemia
Blank
PHARMACOGENOMICS REPORT
DESIGN AND IMPLEMENTATION OF A
NEW ASSAY TO GENOTYPE 460G>A
AND 719A>G POLYMORPHISMS IN THE
THIOPURINE S-METHYLTRANSFERASE
(TPMT) GENE
1
ABSTRACT
Introduction: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme which catalyses
the S-methylation of thiopurine drugs that are commonly used to treat a wide range of
conditions. Variation in sensitivity to these drugs among patients has been detected due to the
presence of point mutations in the TPMT gene. Studies have shown that TPMT*3C is known to be
the predominant mutant allele reported in Asian and African populations whereas TPMT*3A is
the predominant mutant allele found in Caucasians. The tetra-primer Amplification Refractory
Mutation System ARMS assay described here provides genotyping for two common TPMT
mutations 460G>A, and 719A>G seen in the South Asian region, and offers a simple, cost
effective, precise and rapid option for screening of patients in clinical settings. The aim of this
study was to design and implement new assay to genotype 460G>A, and 719A>G
polymorphisms in the TPMT gene.
Method: We designed a tetra-primer ARMS-Polymerase Chain Reaction PCR to genotype
polymorphisms in the TPMT gene.
Results: Out of the 30 samples used, none were found to be heterozygous or homozygous for
both mutations. All were of the wild type genotype (TPMT*3C – AA and TPMT*3B – GG).
Conclusion: This assay can be used to detect and analyze more variants of the TPMT gene by
designing tetra primers for each polymorphism in order to identify patients who are at risk of
developing hematotoxicity in response to treatment with thiopourine drugs.
Keywords: Thiopurine S-methyltransferase gene; Drug metabolism; Pharmacogenetics
2
Introduction
Thiopurinemethyltransferase or thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme in
humans that is encoded by the TPMT gene located on chromosome 6p22.3 and is approximately
27Kb in size consisting of 9 exons (1). TPMT enzyme is involved in S-methylation of aromatic
and heterocyclic sulfhydryl compounds including the anticancer agents, 6-mercaptopurine, 6-
thioguanine and the immunosuppressant, azathioprine (2). Such thiopurine drugs are prescribed
for the treatment of many diseases including hematologic malignancies, rheumatoid arthritis,
inflammatory bowel disease (IBD) and as immunosuppressants in solid organ transplants (3).
These thiopurine drugs are metabolized to nucleotide intermediates by hypoxanthine-guanine
phosphoribosyl transferase and further metabolized to thioguanine nucleotides (TGN).
Alternatively, TPMT enzyme metabolizes thiopurine to inactive S-methylated metabolites and 6-
thiouric acid by xanthine oxidase (XO). This causes decrease in the amount of drug available for
activation to TGN. Negative correlation between the activity of TPMT in erythrocytes and
intracellular concentration of TGN has been reported (1). Thus, people who are having
intermediate or low activity of TPMT enzyme shows an increased risk of hematopoietic toxicity
when treated with conventional doses of drugs which are metabolized by TPMT enzyme (4).
TPMT enzyme deficiency is inherited in an autosomal recessive manner (4). Previous studies in
Caucasians and African-Americans have shown that most of them possess high activity ~90%,
10% shows intermediate activity and 0.3% low activity. Those who show high activity have the
TPMT*1/*1 (wild-type) genotype. Those who exhibit intermediate activity have the
heterozygous TPMT genotype and possess one TPMT variant allele. They may experience
moderate to severe myelosuppression. Finally the ones who show low activity of the enzyme
have the homozygous TPMT variant genotype and they have the highest risk of developing severe
3
myelosuppression. Such patients should not be treated with a thiopurine drug or the drug dose
should be reduced (5, 6).
TPMT displays genetic polymorphisms including 10 different allelic variants to date, of which
the most commonly studied are TPMT *2, *3A, *3B, *3C, and *4 (3).The TPMT*3A allele
comprises two transitions, 460G>A and 719A>G, which in turn produce the amino acid
substitutions Ala154Thr and Tyr240Cys, respectively, where as the TPMT*3B or TPMT*3C
comprises only the 460G>A or 719A>G transition (7).
According to the genotype analysis done in previous studies, the TPMT *3A mutation is known
to be the TPMT ancestral mutant allele as it is found in the African, Caucasian and Asian
populations (8). Further studies have shown that TPMT*3C is known to be the predominant
mutant allele reported in Asian and African populations whereas TPMT*3A is the predominant
mutant allele found in Caucasians (9). In the present study we describe the implementation and
development of a Tetra-primer Amplification refractory mutation system (ARMS) assay for the
detection of TPMT*3C (719A>G) rs1142345 and TPMT*3B (460G>A) rs1800460 single
nucleotide polymorphisms (SNPs) in order to study the predominant polymorphisms of TPMT
gene in the Sri Lankan population.
Method
Genomic DNA was extracted from 30 peripheral blood samples from a general Sri Lankan
population (10 samples each from three ethnic groups – Sinhalese, Moor and Tamil) using
QIAamp DNA Mini Kit and both the TPMT polymorphisms were genotyped in each sample.
PCR amplification was performed using primer sets for each SNP (pair of common outer primers
and a pair of inner/allele specific primers) (Table 1). Primers were designed using Primer 1
(primer design for tetra-primer ARMS-PCR) tool. Allele specificity was maximized by
introducing a 3` mismatch in each of the allele specific primers. Both SNPs were amplified
4
separately. Annealing temperatures for each allele specific primer were optimized and then
combined. Reactions consisted of a total volume of 25 µl containing , 12.4 µL of double distilled
water, 5 µl of 5X buffer, 0.5 µl of 10 mM dNTPS, 0.5 µl of each primer, 3 µL of 25 mM MgCl2,
0.1 µl of 5U/µL Taq DNA polymerase (Promega Corp,USA) and 2 µl of genomic DNA. An
initial denaturation step at 94°C for 5 min was followed by 30 cycles consisting of denaturation
step at 94°C for 45s, annealing for 45s at 59°C, and extension at 72°C for 45s. The final
extension was subsequently performed at 72°C for 10 min. The DNA fragments were
subsequently analyzed using a 3% agarose gel electrophoresis and stained with ethidium bromide
(0.03g/ml). The identity of positive control was confirmed by automated DNA sequencing on an
ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, CA, USA), using Big Dye
Terminator chemistry version 3.1 (Applied Biosystems).
Results
Tetra-primer ARMS-PCR, which employs two primer pairs to amplify the two different alleles
of a SNP in a single PCR reaction was designed and implemented in order to determine the
predominant polymorphisms of TPMT gene in the Sri Lankan population. Agarose gel
electrophoresis images showing the genotyping results are given in Figure 1. Out of the 30
samples used, none were found to be heterozygous or homozygous for both mutations. All were
of the wild type genotype (TPMT*3C – AA and TPMT*3B – GG).
5
Table 1- Oligonucleotide primers used in this assay
TMPT*3C TPMT*3B
Figure1. Images of PCR amplicons containing SNP loci of TPMT*3C (NM_0003672:c.719A>G) andTPMT*3B (NM_000367.2:c.460G>A) after agarose gel electrophoresis. Sizes of the amplicons areindicated by arrows. Lane 1 shows the 500bp DNA ladder, Lane 2-3: samples showing wild typegenotype of TPMT*3C (Homozygous wild - AA) consisting of 475bp and 191bp amplicons. Lane 6-7:samples showing wild type genotype of TPMT*3B (Homozygous wild - GG) consisting of 506bp and263bp amplicons. Lanes 4 and 8 : Control sample.
# Primer name Sequence1 TPMT*3C-Forward (Inner Primer -A allele)
rs1142345 5’ GAATTGACTGTCTTTTTGAAAAGTTCTA 3’
2 TPMT*3C-Reverse (Inner primer -G allele)rs1142345 5’TGTCTCATTTACTTTTCTGTAAGTATAC 3’
Reference : Roberts RL, Barclay ML, Gearry RB, Kennedy MA. A multiplexed allele-specificpolymerase chain reaction assay for the detection of common thiopurine S-methyltransferase (TPMT) mutations. Clinica chimica acta; international journal ofclinical chemistry. 2004;341(1-2):49-53.
v
Thiopurine S-methyltransferase, encodedby the TPMT gene, is the major enzymein hematopoietic cells responsible for theinactivation of thiopurines. Thiopurinedrugs are used in transplantation and thetreatment of several disorders includingchronic inflammatory diseases andhematological malignancies. Thiopurinedrugs have a narrow therapeutic indexand can cause life-threatening toxicity,which has been associated with anaccumulation of TPMT’s substrates, 6-thioguanine.Individuals with intermediateenzyme activity are heterozygous for onevariant in TPMT while those with low orno activity are homozygous or compoundheterozygous for variants in TPMT.
Reasons for Referral:
Individuals with conditions requiringtreatment with thiopurine drugs
Limitations:
Not all variants with known impact on enzymeexpression and activity are tested in this assay.Rare genetic alterations at primer binding sitesmay result in diagnostic errors.
Testing Methodology:
DNA amplification by Tetra-primer allelespecific PCR and analyzing PCR products byagrose gel electrophoresis.
Requisition must accompany specimen. Prior toany genetic testing we request that the subjectsign our consent form and submit it with thesample. To receive our forms, additionalinformation, please contact our unit.
Turnout time: 2 weeks
References
Seki T, Tanaka T, Nakamura Y. Genomic structureand multiple single-nucleotide polymorphisms(SNPs) of the thiopurine S-methyltransferase(TPMT) gene. Journal of human genetics.2000;45(5):299-302.
Srimartpirom S, Tassaneeyakul W, KukongviriyapanV, Tassaneeyakul W. Thiopurine S-methyltransferasegenetic polymorphism in the Thai population. Britishjournal of clinical pharmacology. 2004;58(1):66-70.
Performing Laboratory:Human Genetics UnitFaculty of MedicineUniversity of ColomboKynsey Road, Colombo 8, Sri LankaPhone (94-011) 2695 300, 2689 545 (Direct)Fax (94-011) [email protected]://www.hgucolombo.or
Thiopurine-S-Methyltransferase (TPMT) Genotyping
Human Genetics Unit, Faculty of Medicine, University Colombo
vi
Confidential Molecular Genetic Laboratory Test Report Date:
Human Genetics UnitFaculty of Medicine
University of ColomboKynsey Road, Colombo 8, Sri Lanka
Because of their complexity and their potential implications for other family members, all genetic tests should beaccompanied by genetic counseling.
Prof. Rohan W. Jayasekara MBBS (Ceylon), PhD (Newcastle), C.Biol., MSB (London) – Medical Geneticist and DirectorProf. Vajira H. W. Dissanayake MBBS (Colombo), PhD (Nottingham) – Medical Geneticist
Remarks: Based on this individual’s Genetic Result:
If heterozygous for one mutation – indicates an intermediate metabolizer ofthiopurine drugs. Hence this patient is at a risk of developing side effects orhematologic toxicity, and may require a lower dosage.
If homozygous/compound heterozygous - indicates a poor metabolizer ofthiopurine drugs and is at high risk for life-threatening hematologic toxicity ifgiven full doses of thiopurine drugs. Alternative therapy or greatly reduced dosageshould be considered for this patient.
If no mutations are detected –Since the above two mutations were not detected, this patient is having the wildtype genotype which is consistent with normal TPMT enzyme activity. Standarddoses of thiopurine drugs are less likely to be toxic in individuals with thisgenotype
Prof. Vajira H. W. Dissanayake MBBS, PhD, FNASSLMedicalGeneticist
………………………..
Analysis Performed by: ………………………..
MOLECULAR CYTOGENETICS REPORT
A MATERNALLY INHERITED PARTIAL
TRISOMY 1q (q44qter) AND PARTIAL
TRISOMY 15 (pterq22) IN A CHILD WITH
SILVER RUSSELL & PARTIAL TRISOMY
15q SYNDROME
1
ABSTRACT
A male infant with partial trisomy 1q syndrome and partial trisomy 15q syndrome
47,XY,+der(15)t(1;15)(q44;q22) is described. The baby presented with feeding difficulty,
developmental delay and dysmorphic features including macrocephaly, triangular face, high
nasal bridge, low set ears with a simple and malformed left ear, long philtrum, bilateral single
The inheritance of a derivative unbalanced chromosome creates a partial trisomy or partial
monosomy creating an unbalanced genotype that results in abnormal phenotypic features. Partial
trisomy 1q syndrome is a rare chromosomal abnormality, arising in most cases with de novo
translocation, duplication or insertion [1]. Two major partial trisomy 1q syndromes with regard
to the breakpoint localization have been described, one being a proximal partial trisomy ( 1q32-
qter) and the other as distal partial trisomy (1q42-qter) [2, 3]. Distal partial trisomy 1q syndrome
is often accompanied by other chromosome aberrations, which makes the definition of a
phenotype difficult [4].
A number of individuals have been reported with duplications of the proximal portion of 15q22
having 47 chromosomes, with the extra chromosome being a de novo bisatellited chromosome
15 [5] . Partial trisomy of the proximal part of the long arm of chromosome 15 can arise from a
balanced parental translocation, as a result of parental mosaicism or de novo which in turn leads
to either partial monosomy, or partial trisomy for different autosomes [6].
In this study, we report the results of standard cytogenetics and Fluorescence in situ
Hybridization (FISH). The phenotype of the child is also described and compared with other
previous prenatal cases reported in the literature.
Case report
We report a male infant who was examined at the age of 1 ½ months and died at the age of 8
months. He was the only child of non consanguineous parents with a history of a previous first
trimester miscarriage. He was born at 36 weeks of gestation by cesarean section due to lack of
progression and fetal distress with a birth weight of 2.1Kg (<10th percentile) and length of
34cm.( At birth the only dysmorphic feature which was noted was bilateral syndactyly of the 2nd
3
and 3rd toes. After 4 days the baby was admitted to the hospital due to poor sucking and
diagnosed with pyloric stenosis, jaundice due to lactation failure and weight loss (weight –
1.9kg), though there was no sign of fever. Ramstead Pyloromyotomy was performed and he was
managed in the PBU (Premature Babies Unit) and referred for genetic analysis.
The baby presented with feeding difficulty, developmental delay and dysmorphic features;
macrocephaly, triangular face, high nasal bridge, low set ears with a simple and malformed left
ear, long philtrum, bilateral single palmer creases and bilateral syndactyly. Echocardiographydetected Atrial Septal Defect and ultrasound scanning of abdomen and brain showed no
abnormalities. At the age of 4 months his weight was 3.65Kg. Figure 1 shows some of the
clinical features of the patient.
4
A B
C
Figure 1: Some clinical features of the Patient: A- Large head, Triangular face, Long Philtrum, low set
ears, B-B/L (Bi-lateral) Syndactyly of toes, C- Single palmer crease
Methods
The study was approved by the Ethics Review Committee, Faculty of Medicine, University of
Colombo and written informed consent was obtained from both parents.
Karyotyping
Chromosome culture and karyotyping was performed on peripheral blood lymphocytes of the
baby and both the parents according to the standard procedures. Metaphase chromosome spreads
4
A B
C
Figure 1: Some clinical features of the Patient: A- Large head, Triangular face, Long Philtrum, low set
ears, B-B/L (Bi-lateral) Syndactyly of toes, C- Single palmer crease
Methods
The study was approved by the Ethics Review Committee, Faculty of Medicine, University of
Colombo and written informed consent was obtained from both parents.
Karyotyping
Chromosome culture and karyotyping was performed on peripheral blood lymphocytes of the
baby and both the parents according to the standard procedures. Metaphase chromosome spreads
4
A B
C
Figure 1: Some clinical features of the Patient: A- Large head, Triangular face, Long Philtrum, low set
ears, B-B/L (Bi-lateral) Syndactyly of toes, C- Single palmer crease
Methods
The study was approved by the Ethics Review Committee, Faculty of Medicine, University of
Colombo and written informed consent was obtained from both parents.
Karyotyping
Chromosome culture and karyotyping was performed on peripheral blood lymphocytes of the
baby and both the parents according to the standard procedures. Metaphase chromosome spreads
5
were digested and stained using GTG banding technique. Chromosome spreads were observed
under light microscope (BX 61) and analyzed using Cytovision 3.1 soft ware. Out of 26
metaphase spreads, 14 were analyzed and 8 were karyotyped under a banding resolution of 450.
FISH (Fluorescence in situ Hybridization)
A preliminary FISH experiment was performed on the harvested whole blood lymphocyte cell
suspension of the baby using commercial fluorescent labeled probes of chromosomes 1 (BAC
clone: RP11-624F6, gene- HNRNPU, 1q44 (chr1:245,003,602-245,037,844), color- Green) and
97,325,282), color- Red) according to the protocols and procedures described by Empire
Genomics LLC, NY, US (2014). According to the GTG banding technique we concluded that the
translocated regions of both chromosome 1 & 15 were present in the derivative chromosome 15
as 1q44-qter and 15pter-q22 respectively. FISH probes were chosen by selecting a gene (Figure
2) in each of these regions which correlated with the clinical phenotype of the patient. Though
RP11-1059N24 is not located within the 15pter-15q22 region, this probe was used to detect the
presence of derivative chromosome 15.
Initially, the cell culture was harvested using standard cytogenetics protocol. The fixative
(Carnoy’s 3:1 methanol: acetic acid) in cell suspension tube was changed until supernatant was
colourless and then re-fixed in fresh fixative prior to slide preparation. The slides were cleaned
by placing in a coplin jar with 70% alcohol for 5 minutes and then placing in a coplin jar with
fresh 3:1 acetic acid:methanol fixative. Three drops of cell suspension were added on to one slide
in a vertical angle. Then the slide was gently rotated, tipping slightly after ~15 seconds to drain
excess suspension. The slides were kept horizontally until a grainy appearance was observed and
the edges of the slide were dried. 10μl of each probe mixture was added on to two slides (2ul
6
probe + 8ul hybridization buffer) separately. A clean 22 x 22 cover slip was applied on to each
slide and the edges of the cover slip were sealed using rubber cement. The probes were
hybridized with metaphase chromosomes in a StatSpin®ThermoBrite® Hybridizer (Abbot
molecular); denaturation at 73◦C for 2 minutes/Hybridize at 37◦C for 16 hours. After
hybridization the slides were taken and the cover slips were removed. A pre-warmed WS1
(0.4xSSC/0.3% NP-40) at 73◦C was used to wash the slides for 2 min and transferred to WS2
(2xSSC/0.1% NP-40) at room temp/1min. The slides were dried in a dark room. Chromosomes
were counterstained with 10μl of 4’, 6-diamidino-2-phenylindole (DAPI) and covered with 22 x
22 cover slips. After 15-30 minutes the slides were visualized and the images were captured
using Olympus BX61 epifluorescence microscope (Olympus, Tokyo, Japan), ×100/1.3
magnification objective with CCD camera model ER-3339 (Applied Imaging, Newcastle, UK)
and analyzed using GenASIs software (Applied Spectral Imaging, USA).
A
B
Figure 2: A- Chromosomal region of HNRNPU gene (1q44); B- Chromosomal region of IGF1R
gene (15q26.3) (Source: UCSC genome browser)
Results
Chromosomal culture and karyotyping showed a marker chromosome resulting in an unbalanced
structural abnormality (Figure 2). Further analysis by parental screening showed a balanced
translocation between chromosome 1 and 15 in the mother; 46, XX,t(1;15)(q44;q22) and the
father having a karyotype with no structural and numerical abnormalities (46,XY). Therefore the
7
marker chromosome that was present in the child was the derivative chromosome 15 inherited
from the mother.
The origin of the additional material on chromosome 15 from the long arm of chromosome 1,
suggested by GTG banding pattern, was confirmed by FISH results (Figure 3). The FISH probe
on chromosome 1 (RP11-624F6) was localized in 1q44 and the signals were observed on both
normal chromosomes 1 and on the derivative chromosome 15. The FISH probe on chromosome
15 (RP11-1059N24) was localized in 15q26 and both the normal chromosomes 15 showed two
signals but it was absent on the derivative chromosome 15.
A B
C D
Figure 2. A-Karyogram of the proband bearing a derivative chromosome 15 inherited from mother, B-Ideogram of trisomy 15 with the derivative chromosome 15 illustrating the maternal translocationbetween chromosome 1 and 15, C - Karyogram of the mother bearing a balanced translocation betweenchromosome 1 and 15 : 46,XX,t(1;15)(q44;q22), D- Ideogram of maternal t((1;15)(q44;q22).
marker chromosome that was present in the child was the derivative chromosome 15 inherited
from the mother.
The origin of the additional material on chromosome 15 from the long arm of chromosome 1,
suggested by GTG banding pattern, was confirmed by FISH results (Figure 3). The FISH probe
on chromosome 1 (RP11-624F6) was localized in 1q44 and the signals were observed on both
normal chromosomes 1 and on the derivative chromosome 15. The FISH probe on chromosome
15 (RP11-1059N24) was localized in 15q26 and both the normal chromosomes 15 showed two
signals but it was absent on the derivative chromosome 15.
A B
C D
Figure 2. A-Karyogram of the proband bearing a derivative chromosome 15 inherited from mother, B-Ideogram of trisomy 15 with the derivative chromosome 15 illustrating the maternal translocationbetween chromosome 1 and 15, C - Karyogram of the mother bearing a balanced translocation betweenchromosome 1 and 15 : 46,XX,t(1;15)(q44;q22), D- Ideogram of maternal t((1;15)(q44;q22).
marker chromosome that was present in the child was the derivative chromosome 15 inherited
from the mother.
The origin of the additional material on chromosome 15 from the long arm of chromosome 1,
suggested by GTG banding pattern, was confirmed by FISH results (Figure 3). The FISH probe
on chromosome 1 (RP11-624F6) was localized in 1q44 and the signals were observed on both
normal chromosomes 1 and on the derivative chromosome 15. The FISH probe on chromosome
15 (RP11-1059N24) was localized in 15q26 and both the normal chromosomes 15 showed two
signals but it was absent on the derivative chromosome 15.
A B
C D
Figure 2. A-Karyogram of the proband bearing a derivative chromosome 15 inherited from mother, B-Ideogram of trisomy 15 with the derivative chromosome 15 illustrating the maternal translocationbetween chromosome 1 and 15, C - Karyogram of the mother bearing a balanced translocation betweenchromosome 1 and 15 : 46,XX,t(1;15)(q44;q22), D- Ideogram of maternal t((1;15)(q44;q22).
Figure 3 : A-FISH image of the BAC clone RP11-1059N24 (15q26.3) showing a double signal on bothnormal chromosomes 15, B- FISH image of the BAC clone RP11-624F6 (1q44-ter) showing a triplesignal on both normal chromosomes 1 and the derivative chromosome 15.
Discussion
The clinical phenotypes of partial trisomy 1q syndrome vary widely, due to the different
breakpoints on chromosome 1q and the extent of aberrations involved in other autosomes. Partial
trisomy 1q syndrome can be classified according to breakpoint position as 1q32-qter or 1q42-
qter [2, 3]. Duplication of 1q42-qter with no other involved chromosomal abnormality usually
presents as a mild phenotype, which may include macrocephaly with wide fontanelles, flat nasal