Cytogenetic Studies of Rwandan Pediatric Patients Presenting with Global Developmental Delay, Intellectual Disability And/Or Multiple

Home , Ring chromosome 15

Journal of Tropical Pediatrics, 2016, 62, 38–45 doi: 10.1093/tropej/fmv065 Advance Access Publication Date: 27 October 2015 Original Paper Cytogenetic Studies of Rwandan Pediatric Patients Presenting with Global Developmental Delay, Intellectual Disability and/or Multiple

Congenital Anomalies Downloaded from by Annette Uwineza,1,2 Janvier Hitayezu,1 Mauricette Jamar,2 Jean-Hubert Caberg,2 Seraphine Murorunkwere,1 1 2 1

Ndinkabandi Janvier, Vincent Bours, and Leon Mutesa http://tropej.oxfordjournals.org/

1Center for Medical Genetics, College of Medicine and Health Sciences, University of Rwanda, Huye, Rwanda 2Center for Human Genetics, Centre Hospitalier Universitaire Sart-Tilman, University of Liege, Liege, Belgium Correspondence: Leon Mutesa, Center for Medical Genetics, College of Medicine and Health Sciences, University of Rwanda, Huye, Rwanda. Tel: (þ250) 788451013. E-mail or

ABSTRACT

Global developmental delay (GDD) is defined as a significant delay in two or more developmental at McMaster University Library on April 8, 2016 domains: gross or fine motor, speech/language, cognitive, social/personal and activities of daily living. Many of these children will go on to be diagnosed with intellectual disability (ID), which is most commonly defined as having an IQ <75 in addition to impairment in adaptive functioning. Cytogenetic studies have been performed in 664 Rwandan pediatric patients presenting GDD/ID and/or multiple congenital abnormalities (MCA). Karyotype analysis was performed in all patients and revealed 260 chromosomal abnormalities. The most frequent chromosomal abnormality was Down syndrome and then Edward syndrome and Patau syndrome. Other identified chromosomal abnormalities included 47,XX,þdel(9)(q11), 46,XY,del(13)(q34) and 46,XX,der(22)t(10;22) (p10;p10)mat. In conclusion, our results highlight the high frequency of cytogenetically detectable abnormalities in this series, with implications for the burden on the healthcare. This study demon- strates the importance of cytogenetic analysis in patients with GDD/ID and MCA.

KEYWORDS: global developmental delay, intellectual disability, multiple congenital anomalies, karyotype, chromosomal abnormality, Rwandan pediatric patients

INTRODUCTION motor, speech/language, cognitive, social/personal Global developmental delay and intellectual disabil- and activities of daily living [2]. Many of these ity (GDD/ID) are common clinical conditions and children would later be diagnosed with ID, which affect 1–3% of children worldwide [1]. is most commonly defined as having an IQ <75 GDD is defined as significant delay in two in addition to impairment in adaptive functioning or more developmental domains: gross or fine [3].

38 Cytogenetic studies of Rwandan pediatric patients 39

To date, there is little or no data on the incidence hundred thirty-six (80%) were aged <5 years and of GDD/ID and cytogenetic studies in Sub-Saharian presented GDD while 128 children (20%) were aged Africa. The birth prevalence of congenital anomalies >5 years and presented ID. In 128 children diag- in the developing world is underestimated by defi- nosed with ID, the majority had moderate ID ciencies in diagnostic capabilities and lack of reliabil- (n ¼ 63), while 26 had mild ID, 32 severe ID and 7 ity of medical records and health statistics [4]. Birth profound ID (Table 1). defect registry allows more reliable estimates of the In addition, a blood sample was taken for karyo- prevalence at birth of congenital anomalies in a type analysis and for DNA extraction for further mo- population. However, cytogenetics analysis in indi- lecular cytogenetic testing. This study was approved viduals with various congenital abnormalities, dys-

morphic features, GDD and/or ID is generally Downloaded from considered as an essential component in the overall Table 1. Clinical characteristics of the 664 evaluation process [5]. Chromosomal disorders are Rwandan pediatric patients with ID/GDD and/ recognized as one of the major causes of childhood or MCA morbidity and mortality in developed countries [6]. Characteristics Number (percentage) However, these disorders have not received much at- http://tropej.oxfordjournals.org/ tention in developing countries, because the main Gender priority health problems responsible for childhood Total cohort 664 morbidity and mortality are caused by socioeco- Gender nomic and environmental factors rather than genetic Male 360 (54%) aetiologies [7]. The present study aimed at deter- Female 304 (46%) mining the prevalence and pattern of microscopically Mean age 2.6 years visible chromosomal abnormalities among children Global development 536 (80%)

referred to our clinical department for diagnosis and delay (child with at McMaster University Library on April 8, 2016 evaluation of GDD/ID associated or not with mul- <5 years) tiple congenital abnormalities (MCA). Intellectual disability 128 (20%) (child >5 years) Level of intellectual disability MATERIALS AND METHODS Mild 26 (4%) Moderate 63 (9.5%) Patients Severe 32 (5%) A total number of 664 Rwandan children with Profound 7 (1.5%) GDD/ID and/or MCA were transferred to the de- Facial dysmorphism partment of Medical Genetics at University Absent 173 (26%) Teaching Hospitals of Kigali and Butare (CHUK Present 491 (74%) and CHUB) and the Rwandan Military hospital Congenital heart defect (RMH) for genetic investigations from January 2010 Absent 457 (69%) to December 2014. During consultation, all patients Present 207 (31%) were seen by a geneticist who collected patient’s Hand and limb abnormalities demographic data (age, sex), a complete family his- Absent 629 (95%) tory and performed a physical examination. Patients Present 35 (5%) with GDD/ID but in whom the clinical history and Uro-genital malformations investigations revealed a non-genetic condition such Absent 641 (96.5%) as a history of birth asphyxia, and neonatal/ perinatal Present 23 (3.5%) infection were excluded. Epilepsy Among the patients, 360 were boys (54%) while Absent 607 (91%) 304 where girls (46%). The age of referral ranged be- Present 57 (9%) tween 1 day to 15 years (mean age 2.6). Five 40 Cytogenetic studies of Rwandan pediatric patients by the Rwandan National Ethics Committee In total, abnormal karyotypes were identified in (N394/RNEC/2013). Signed informed consent 260 patients (39%) with GDD/ID associated with or forms and permission for publication of this report without congenital malformation. Numerical abnor- and any accompanying images were obtained from malities were found in 246 (37%) patients whereas the parents or legal guardians of all patients. structural abnormalities were detected in 14 (2%) patients. Cytogenetic analysis Trisomy 21 was the most frequent abnormality A karyotype analysis was performed in each patient. and found in 203 patients (30%). Among these A sample of 5 ml of peripheral blood was obtained cases, 197 were free trisomy 21, five cases were due from each patient and was cultured and harvested ac- to a Robertsonian translocation where the chromo- cording to conventional protocol. Conventional some 21 was associated with another acrocentric Downloaded from chromosome analyses were performed with a reso- chromosome and one trisomy 21 was associated lution of 550 bands (Q-banding) on peripheral with an extra chromosome X (48,XXX,þ21) lymphocytes. At least 20 metaphases were counted (Table 2). Remaining chromosomal abnormalities for each patient and karyotyped. The karyotypes were characterized by free trisomy 18 in 23 patients; were analysed according to guidelines from the http://tropej.oxfordjournals.org/ International System for Human Cytogenetic Table 2. Chromosomal abnormalities in 664 Nomenclature (ISCN 2013). pediatric patients referred for investigation of congenital anomalies, dysmorphic features, DNA extraction GDD and/or ID DNA extraction was performed in order to perform Karyotype Number of further molecular analyses. Ten to five millilitres of children blood sample were collected into EDTA tube. A gen- (n ¼ 664) omic DNA of each patient was extracted from per- at McMaster University Library on April 8, 2016 ipheral lymphocytes using the phenol–chloroform Numerical abnormalities standard method. 47,XY,þ21 114 47,XX,þ21 83 Confirmatory analysis 48,XXX,þ21 1 Multiplex ligation-dependent probe amplification 47,XY,þ18 11 (MLPA) was performed to confirm the results of 47,XX,þ18 12 karyotype at the Center for Human Genetics at the 47,XY,þ13 6 University of Liege/Belgium. MLPA analysis was car- 47,XX,þ13 14 ried out according to the manufacturer’s instructions 45,X0 4 (MRC Holland, Amsterdam, Netherlands), using the 47,XXX 1 SALSA probe mix P036 and P070 Human Telomere Structural chromosomal abnormalities and SALSA probe mix P245 Microdeletion 46,XX,þ21,rob(21;21)(q10;q10) 2 Syndrome. Amplification products were analyzed 46,XY,þ21,rob(21;21)(q10;q10) 2 using capillary electrophoresis on ABI PRISM 3100 46,XX,þ21,rob(21;22)(q10;q10) 1 Genetic Analyzer. The obtained data were analyzed 47,XX,þdel(9)(q11) 1 using the Sequence Pilot software (JSI medical sys- 46,XX,der(22)t(10;22)(p10;p10)mat 1 tems, Kippenheim, Germany). (46,XX,dup (1)(p35p31) 1 46,XY,del(13)(q34) 1 47,XX,þmar (chr18) 1 RESULTS 47,XX,þmar (chr 22) 2 During the period of January 2010 to December 46, XX,r(15)(p10q26) 1 2014, 664 children were referred to the department 47,XX,þ18,t(5,6)(p10;p10) 1 of Medical Genetics at the University of Rwanda for Total 260 diagnostic investigation of GDD/ID (Table 1). Cytogenetic studies of Rwandan pediatric patients 41 Downloaded from http://tropej.oxfordjournals.org/ at McMaster University Library on April 8, 2016

Fig. 1. Karyotype results. one patient had trisomy 18 associated with a unbal- karyotypes, including 46,XX,r(15)(p10q26) and dupli- anced translocation 47,XX,þ18,t(5,6)(p10;p10). In cation (46,XX,dup (1) (p35p31), were confirmed by addition, 20 patients presented free trisomy 13, four array-CGH [8]. had Turner syndrome (45,X0) and one patient had 47,XXX. Other identified rare chromosomal abnormalities DISCUSSION were 47,XX,þdel(9)(q11), 46,XY,del(13)(q34) and Cytogenetic analysis performed in 664 patients re- 46,XX,der(22)t(10;22)(p10;p10)mat (Fig. 1). These vealed chromosomal abnormalities in 39% of pa- anomalies were confirmed by MPLA (Fig. 2). Three tients. Our findings showed high frequency of karyotypes with extrachromosomal marker have been chromosomal abnormalities compared to other stud- found and one karyotype was confirmed by array- ies ([Table 3]; 16.5% Mundhofir et al.[9]; 16.1% CGH revealing that the marker was derived from Balkan et al.[10]; 29.3% Duarte et al.[7]; 27% chromosome 18 (arr [hg19] 18p11.32p11.21 Aboussair et al.[11] and 17% Kim et al.[12]). (108,760-14,241,744) x3; 18p11.21q11.2 (15,345,079- Moreover, our frequency is in the lower range to 18,270,513) x3; see article Uwineza et al.[8]), while those found by Dayakar et al. (56%) [13] and other revealed markers came from the chromosome Thillainathan et al. (50.6%) [14]. 22, which have been confirmed by fluorescent in situ First of all, the high frequency compared to other hybridization (FISH). Moreover, other two abnormal studies may be due to the different inclusion criteria 42 Cytogenetic studies of Rwandan pediatric patients Downloaded from MLPA result showed a duplicaon 10p using the kit0245 http://tropej.oxfordjournals.org/

Duplicaon 9p kit 036 telomere at McMaster University Library on April 8, 2016

Duplicaon 9p kit 070 telomere

FIG.2.MLPA results. consisting of combination of individuals (children) sex abnormality (48,XXX,þ21). This patient presented presenting GDD/ ID associated or not with MCA in typical features of Down syndrome, and this combin- our cohort. The increased number of chromosomal ation has already been reported previously [15]. abnormalities can also be explained by the absence Edwards syndrome represented 3.6% in our cohort, of prenatal diagnostic technique in Rwanda. whichincluded23freetrisomy18andonecaseoftri- Down syndrome was the most frequent chromo- somy 18 caused by balanced translocation between the somal abnormality as reported in other previous cyto- chromosome 5 and 6. Interestingly, one patient aged 2 genetic studies [7, 9, 10, 13]. One patient with Down months presented clinical features suggesting trisomy syndrome combined an autosomal abnormality with a 18, which included facial dysmorphism, dolichocephaly, Cytogenetic studies of Rwandan pediatric patients 43

Table 3. Frequency of chromosomal abnormalities in current study compared with previous studies

Study (year) Overall frequency Country Inclusion criteria Current study 39% Rwanda Children with GDD/ID and MCA. Aged between 0–15 years. Balkan et al.[10] 16.1% India All patients referred for cytogenetic studies. Aged between 0–50 years. Mundhoﬁr et al.[9] 16.5% Indonesia Intellectually disabled individuals. Aged 6–25 years. Kim et al.[12]) 17.5% South Korea Patients with suspected chromosomal abnormalities.

Aboussair et al.[11] 27% Morocco Patients being referred for cytogenetic analysis. Downloaded from Aged between 0–50 years. Duarte et al.[7] 29.3% Brazil Patients being referred for cytogenetic analysis. Thillainathan et al.[14] 50.6% Sri Lanka Children with suspected chromosomal abnormalities. Aged between 0 and 12 years.

Dayakar et al.[13] 56% Turkey Children with MCA http://tropej.oxfordjournals.org/ Aged 0–15 years. microretrognatia, hypertelorism with low weight sloping forehead and flat occiput (Fig. 2F). He also (1.68 kg) and ventricular septal defect (Fig. 3). had hypospadias. He presented clinical features usu- Unfortunately, this child died before we could take his ally found in the group 3 of deletion 13q defined by DNA for further molecular analysis. Ballarati et al.[18]. Patau syndrome was confirmed in 3% of patients. A 3-year-old girl with a severe developmental In our study, patients with Edwards and Patau syn- delay presented a trisomy 10p with a monosomy 22q at McMaster University Library on April 8, 2016 dromes died before reaching one year of age. (46,XX,der(22)t(10;22)(p10;p10)mat). This abnor- Frequency of these abnormalities is higher than mality was the result of a maternal balanced recipro- those from other studies [7, 10] apart from Edward cal translocation (46,XX,t(10,22)(p10,p10)). Pure syndrome frequency which was quite similar to that trisomy 10p is rare chromosomal disorder, found by Thillainathan et al. (2.3%) [14]. characterized by some common findings such as glo- Ring chromosome 15 was found in one patient, bal developmental delay, intellectual disability, hypo- who presented typical clinical features of this syn- tonia, left lip or palate, congenital heart defects and drome characterized by developmental delay and fa- renal anomalies, clubbed foot, flexion anomalies and cial dysmorphism (Fig. 2E) [16]. hip dysplasia. Craniofacial anomalies include wide Three chromosomal markers were found in our sutures and fontanelles, microcephaly, dolichoceph- cohort, and further molecular analyses were per- aly, hypertelorism, ear anomalies and downturned formed to better characterize their origins. One was corners to the mouth [19]. Our patient share some related to chromosome 18 and the other two to similar clinical features with other patients like global chromosome 22. Patients with markers of chromo- psychomotor development delay, craniofacial dys- some 22 had typical features of Cat eye syndrome. morphic features (Fig. 3) characterized by dolicho- Clinical features of patients affected by Cat eye syn- cephaly with trigonocephaly, high arched eyebrows, drome (Fig. 2C) are characterized by the presence of upslanting eyelids, large philtrum, low set ears, prom- multiple malformations primarily involving the eyes, inent chin, severe muscle atrophia, abducted and ears, anorectal and urogenital systems [17]. flexed lower limbs. One patient had a deletion 13qter (46,XY,del In our cohort, we only had six cases of sex (13)(q34)). Our patient was referred for psycho- chromosomal abnormalities—four cases of Turner motor development delay, facial dysmorphism char- syndrome, one case of 47,XXX and one case of acterized by hypertelorism, strabismus, webbed neck, 48,XXX,þ21. This was a low frequency compared to 44 Cytogenetic studies of Rwandan pediatric patients Downloaded from http://tropej.oxfordjournals.org/ at McMaster University Library on April 8, 2016

Fig. 3. Facial dysmorphic features of patients with chromosomal abnormalities. other studies [7, 14], and similar to that found by ACKNOWLEDGEMENTS Mundhofir et al.[9]. We hypothesized that this low Our sincere thanks go to the WBI (Wallonie Bruxelles frequency can be explained by the fact that patient International) for their fellowship, the University of Rwanda with sex chromosome abnormalities have less severe and Center for Human Genetics at University of Liege. We phenotype than other chromosomal abnormalities are grateful for the support from the Coope´ration Universitaire au De´veloppement (CUD). We also thank all and consult at a later age when they are adolescents lab technicians from the Center for Human Genetics at the and not entering puberty normally, or adults with in- University of Liege and the University of Rwanda. This study fertility. This hypothesis was later verified in our co- could not have been completed without the cooperation of hort of patients with sex development disorders patients and their parents; we are very thankful. (data not shown). In conclusion, the high rate of chromosomal REFERENCES abnormalities (39%) found in our cohort shows the 1. Shevell M, Ashwal S, Donley D, et al. Practice parameter: evalu- importance of cytogenetic evaluation in patients with ation of the child with global developmental delay: report of GDD/ID associated or not with MCA. With the the Quality Standards Subcommittee of the American Academy of Neurology and The Practice Committee of the high rate of patients affected by Down syndrome Child Neurology Society. Neurology 2003;60:367–80. (30%), this demonstrated the necessity to implement 2. Moeschler JB. Medical genetics diagnostic evaluation of new cytogenetic techniques with higher resolution the child with global developmental delay or intellectual such as FISH, MLPA and Array-CGH disability. Curr Opin Neurol 2008;21:117–22. Cytogenetic studies of Rwandan pediatric patients 45

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