J Autism Dev Disord DOI 10.1007/s10803-015-2394-9
ORIGINAL PAPER
Diagnostic Yield of Chromosomal Microarray Analysis in a Cohort of Patients with Autism Spectrum Disorders from a Highly Consanguineous Population Watfa Al-Mamari • Abeer Al-Saegh • Adila Al-Kindy • Zandre Bruwer • Fathiya Al-Murshedi • Khalid Al-Thihli
Ó Springer Science+Business Media New York 2015
Abstract Autism Spectrum Disorders are a complicated group of disorders characterized with heterogeneous genetic etiologies. The genetic investigations for this group of disorders have expanded considerably over the past decade. In our study we designed a tired approach and studied the diagnostic yield of chromosomal microarray analysis on patients referred to the Genetic and Developmental Medicine clinic in Sultan Qaboos University in Oman for autism spectrum disorders in a highly consanguineous population. Copy number variants were seen in 27 % of our studied cohort of patients and it was strongly associated with dysmorphic features and congenital anomalies. Keywords Autism Autism spectrum disorders Chromosomal microarray Array-CGH Diagnostic yield Consanguinity
Introduction Autism is a complex neurodevelopmental disorder defined as a persistent communication and social interaction deficits in multiple situations. The major manifestations of autism include impairment in social communication and behavioral problems such as fixated (restricted) interests and repetitive behaviors (DSM-5, American psychiatry association). Autism Spectrum Disorders (ASDs) are estimated to affect around 1:150 individuals (Newschaffer
W. Al-Mamari A. Al-Saegh (&) A. Al-Kindy Z. Bruwer F. Al-Murshedi K. Al-Thihli Genetics Department, College of Medicine, Genetic and Developmental Medicine Clinic, Sultan Qaboos University Hospital, PO Box 35, 123 Muscat, Sultanate of Oman e-mail: [email protected]
et al. 2007) and it occurs more frequently in males than females. Although twin studies have shown a strong genetic contribution to the etiology of ASDs, these disorders usually have complex multifactorial etiology (Fombonne 2005 and Freitag 2007). A genetic etiology corresponding to known chromosomal rearrangements or single gene disorders is currently recognized only in 10–20 % of the patients, partly reflecting the etiological heterogeneity of ASDs (Battaglia and Bonaglia 2006; Ravnan et al. 2006; Schaefer and Mendelsohn 2008). The recently published consensus statement of the International Standard Cytogenomic Array (ISCA) recommends the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with developmental delay/intellectual disability, ASD, or multiple congenital anomalies (Miller et al. 2010). ASDs may also be seen in association with a number of recessively inherited disorders like creatine deficiency syndromes (Schulze 2013), adenylosuccinate lyase deficiency (Perez-Duenas et al. 2012) and phenylketonuria (Baieli et al. 2003). There is also propensity for recessively inherited disorders to occur at relatively higher frequency in countries with high rates of consanguinity like Oman, a country where consanguinity rates could be as high as 50 % in some regions (Al-Thihli et al. 2014). These factors pose concerns about whether the diagnostic yield of CMA among patients with ASDs maybe lower in these countries than previously quoted in other studies, particularly that to date no study to our knowledge- has been conducted in a highly consanguineous population to address this issue. The developmental Pediatrician (WM) and clinical geneticists (AS, AK, FM & KT) at the Genetic and Developmental Medicine Clinic (GDMC) adapted a tiered approach for the diagnostic work up of patients with ASDs.
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J Autism Dev Disord
The approach is depicted in the flow chart shown in Fig. 1. The GDMC is the only diagnostic clinic for patients with ASD in Oman. This study describes the diagnostic yield of chromosomal microarray following 20 months of clinical application of this approach.
Methods The medical records and array-CGH results of all patients diagnosed with ASDs between January 2012 and September 2013 were reviewed. All patients were included when calculating the diagnostic yield provided they met the eligibility criteria for CMA adapted in the clinic at the time this test was carried out. Patients were considered to be eligible for CMA if they meet the following criteria: 1. 2.
An established clinical diagnosis of ASD and At least one of the following clinical determinants: a.
b. c.
Negative history for consanguinity or apparently sporadic occurrence (even if consanguinity is present) One or more major congenital malformation 3 or more minor dysmorphic features
Fig. 1 Diagnostic workup of patients with ASD
d.
Negative first tier testing if a,b, and c were absent.
DNA extracted from blood was sent for array-CGH at Wessex Regional Genetics Laboratory, the United Kingdom (www.wrgl.org.uk). The array-CGH provided uses Oxford Gene Technology’s (ISCA) 8 9 60 K oligo array platform, providing an average backbone resolution of 240 kb. The array-CGH chip was designed by ISCA and validated by the National Genetics Reference Laboratory (Wessex). CytoSure Interpret version 3.4.8 was the software used for array analysis. For further details of the array design and probes used see: http://www.ngrl.org.uk/Wes sex/array.htm.
Results A total of 230 patients were diagnosed with ASDs through the GDMC between January 2012 and September 2013. 223 patients (97 %) had associated intellectual disability (ID) or developmental delay (DD). A total of 100, 74 males and 26 females, met the adapted eligibility criteria for CMA. A total of 27 patients (27 %), 17 males and 10 females, had copy number variants (CNVs). Seven (7) patients had CNVs that overlap
Clinical Diagnosis of Ausm Spectrum Disorder
No clinical features sugges ve of a specific clinical diagnosis
Tier 1
Ammonia Lactate Plasma amino acids Urine organic acids Acylcarni ne profiling (dried blood spot) Urine crea ne & guanidinoacetate
Abnormal first er tes ng
Tier 2
Clinical features suggest a specific diagnosis
Referral to clinical biochemical gene cs service for further evalu on
Normal first
te
Chromosomal microarray (CMA)* Fragile X (if CMA is nega ve)
Specific molecular gene c tes ng or CMA
* CMA was done in all ents with ASD who have any of the following features: 1) n ve history for consanguinity, 2) apparently sporadic occurrence (even when consanguinity is present), 3) one or more major congenital malform ons, or 4) three or more minor dysmoprhic features.
123
Unknown
Unknown
Pathogenic
NK 300 kb deletion
470 kb duplication
arr[hg18] Xq21.31(87,839,749-88,139,807)X0
3 6
Dysmorphic features, cardiac anomaly, urogenital anomaly
4 5
Dysmorphic features
arr[hg18] Xp22.13(17,167,331-17,639,471)X2
NK
Diagnosed with clinical depression Maternally inherited 110 kb deletion within the intron of NRXN1 arr[hg19] 2p16.3(50,735,595-50,845,789)X1 4 4
Unusual behaviour
NK 318 kb duplication arr[hg18]10p15.3(268,741-586,626)X4 4 3
Dysmorphic features, microcephaly
51 kb deletion arr[hg18]7q35(146,020,240-146,071,372)X1 10 2
Dysmorphic features
2.1 Mb deletion arr22q11.21(17,041,699-19,125,890)X1 Dysmorphic features 4 1
Microarray finding Additional findings Age at diagnosis with ASD Number of patients
Table 1 Clinical and cytogenetic details of patients positive for copy number variants
Size of the deletions/ duplication
Discussion In consistence with the already established role of CMA in the diagnostic work up of patients with ASDs, this study emphasizes this role even in a country with high rates of consanguinity like Oman. Although the concern about missin g an underlying recessive etiology might be deterrent to ordering CMA in the initial diagnostic assessment of patients with ASD in consanguineous families, the diagnostic yield could still be high enough to argue for this consideration in appropriately selected group of patients. 27 % of patients diagnosed through our clinic had CNVs, and even when excluding the nine CNVs of uncertain clinical significance, the diagnostic yield of CMA becomes 18 %. Both figures fall within the range of diagnostic yields reported for CMA in different published studies (Miller et al. 2010; Vissers et al. 2003), and both are notably higher than that universally reported for conventional karyotype. Positive diagnostic yield was significantly higher in patients with associated congenital malformations or dysmorphic features. It is worth noting that almost all patients diagnosed with ASDs through our clinic had associated developmental delay (DD) or intellectual disability (ID). This is higher than the prevalence of 40 % reported by the Autism and Developmental Disabilities Monitoring Network Year 2008 (Gamsiz et al. 2013). Although referral and ascertainment bias cannot be reliably excluded, the relatively higher prevalence of DD/ID maybe hypothesized to be due to increased runs of homozygosity among our cohort compared to the previously reported cohort of patients. Gamsiz et al. showed that in in simplex ASD-affected families, probands with an IQ B 70 showed more ROH
Unknown
(Curran et al. 2013) Pathogenic this deletion is within intron of CNTNAP2 (member of neurexin superfamily Below average intelligence Maternally Inherited
Pathogenic 22q11.2 microdeletion syndrome – De novo
De novo Vs inherited
Phenotype of the affected parent
Significance
References
genomic imbalances defined in a CNV database for patients with ID, DD, ASD, or multiple congenital anomalies. Five (5) patients had CNVs that overlap genomic coordinates for previously published or well-recognized deletion or duplication syndromes. Six (6) patients had CNVs that contain OMIM morbid genes. The remaining nine (9) patients had CNVs of uncertain clinical significance. Table 1 shows details of the copy number variants identified in this cohort. Patients with dysmorphic features were more likely to have CNV compared with those considered non-dysmorphic (22/27 vs. 21/73, respectively, p = 0.0002). Congenital anomalies were also significantly associated with CNVs (16/27 compared to 12/73 without CNVs, p = 0.0005). The frequency of reported history of consanguinity was not significantly different between the CNV-positive and CNV-negative group of patients (p = 0.6639). Consanguinity was seen in 31 (31 %) of our studied families with ASD (31/100).
(Dabell et al. 2013)
J Autism Dev Disord
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123
6
5
3
3
5
3
9
5
4
7
8
4
5
8
9
10
11
12
13
14
15
16
17
18
19
Age at diagnosis with ASD
7
Number of patients
Table 1 continued
Dysmorphic features
Dsymorphic features and macrocephaly
Dysmorphic, polymicrogyria
Dysmorphic features
Dysmorphic features
Seizures
Dysmorphic features
Dysmorphic features, musculoskeletal anomalies, anal anomaly and cardiac anomalies
Dysmorphic features, anal anomaly, seizures
Dysmorphic features, hearing loss, musculoskeletal anomalies and urogenital anomalies
Dysmorphic features
Dysmorphic features
Dysmorphic features
Additional findings
12.3 Mb deletion contains 48 genes out of which 11 are OMIMMorbid enteries 2.5 Mb deletion
arr[hg19]10q25.1q26.11(107,352,476-119,663,083)X1
arr[hg19]22q11.21(18,894,820-21,440,515)X1
arr[hg18]7q11.23(72,404,24873,780278)X1.rsa7q11.23(p029)X1
arr16q12.2(56,659,661-56,692,859)X1
arr[hg18]1p22.3(68, 253,21486,565,053),3p24.3(19,194,234,19,629,776)X3dn.ish dup (3)(p24.3p24.3)
arr[hg18]14q32.2q32.31(99,908,703100,558,651)X3pat, 17p11.2(21,136,13121,147,826)X3mat,17p11.2(21,136,13121,147,826)X3pat
arr7p22.2p21.1(2,811,886-19,358,897)X3
arr[hg18]Xp11.3(46,258,090-46,382,273)X3
1.4 Mb deletion
33 kb deletion
2. 312 kb deletion within 1p22.3
De novo
Maternally inherited
History of speech delay
Pathogenic (duplication with 3p24.3)
Normal Maternally inherited deletion 1. 436 duplication kb within 3p24.3
Pathogenic (William’s syndrome)
Pathogenic
Benign CNV (312 kb deletion within 1p22.3)
Pathogenic Below average intelligence. Diagnsoed with depression, and renal stones
Paternally inherited 650 kb duplication within the 14q32.2to14q32.31. 12 kb duplication within 17p11.2
De novo Duplication
Pathogenic
125 kb duplication contains part of the gene ZNF
De Novo
Pathogenic
Pathogenic22q11.2 microdeletion syndrome
Pathogenic (similar patient reported, Rooney et al. 1989)
Pathogenic member of neurexin superfamily
Unknown
Unknown
Unknown
Significance
16.5 Mb duplication involves 87 genes out of which 6 are OMIMMorbid enteries
Normal
Normal
Phenotype of the affected parent
Pathogenic
Paternally inherited
De Novo
De Novo
Paternally inherited
NK
NK
NK
De novo Vs inherited
De Novo
60 kb duplication
56 kb duplication within CNTN4 gene
arr[hg18]3p26.3(2,351,629-2,407,891)X3
arr[hg19]2q22.2(143,352,620143,931,358)X3,3p26.3(2,376,629-2,432,891)X3
500 kb duplication
680 kb duplication
5 kb deletion
Size of the deletions/ duplication
arr[hg19]5p12p11(45,615,361-46,115,173)X3
arr[hg19]7p21.1(17,254,111-17,932,752)X3
arr[hg19]5q22.2(112,132,377-112,137,084)X1
Microarray finding
(Lugtenberg et al. 2006)
(Curran et al. 2013)
(Curran et al. 2013)
References
J Autism Dev Disord
4
2
8
7
6
7
4
21
22
23
24
25
26
27
Dysmorphic features
Dysmorphic features
Dysmorphic features
microcephaly
Dysmorphic features
Dsymorphic features, seizures
Behavioural abnormalities
Dysmorphic features, musculoskeletal anomaly
Cleft palate
Musculoskeletal anomalies
Dysmorphic features
Additional findings
arr[hg19] 3p26.3(2,376,629-2,432,891)X3
arr[hg19] Xp22.11(22,156,80422,438,749)X2,2p16.3(51,067,05551,218,716)X1,7q32.1q32.3(128,972,820131,984,276)X1
arr[hg19]6q27(168,790,739-171,005,522)X1
arr[hg19] 14q32.33(105,017,254-107,287,708)X1
arr[hg19]2q22.2(143,352,620-143,931358)X3, 3p26.3(2,376,629-2,432,891)X3
Arr[hg19]20p13(83,092-956,262)X3
Arr[hg18]3p26.3(2,693,622-2,994,093)X3
10.886 Mb duplication
Arr3[hg18]q27.3q29(186,918,265197,803,820)X3,9p24.3p23(215.366-12,907,826)X1
57 kb duplication
3. Duplication involving Xp22.11 with minimum size of 280 kb
2. Interstitial deletion involving 7q32.1 to 7q32.3 with a minimum deletion of 3 Mb
NK
NK
3 findings: 1. Interstitial deletion within 2p16.3 of about 150 kb within NRXN1 gene
NK
NK
NK
NK
NK
De novo
De novo Vs inherited
2.3 Mb terminal deletion
2.3 Mb deletion
60 kb duplication within CNTN4
870 kb duplication
300 kb duplication. Duplication disrupts CNTN4
12.693 Mb deletion
Size of the deletions/ duplication
Microarray finding
Patients with autistic spectrum disorder (ASD) found to have copy number variants (CNVs)
4
Age at diagnosis with ASD
20
Number of patients
Table 1 continued Phenotype of the affected parent
The duplication is within CNTN4 gene –Pathogenic
3. Unknown-novel
2. Unknown- novel
1. Pathogenic (Dabell et al. 2013)
(Striano et al. 2006)
Terminal deletions of 6q-Pathogenic
14q terminal deletion syndromePathogenic
Pathogenic
NK-novel
Pathogenic
Pathogenic
Significance
(Roohi et al. 2009)
References
J Autism Dev Disord
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J Autism Dev Disord
than their unaffected siblings, whereas probands with an IQ [ 70 do not show this excess (Gamsiz et al. 2013). This calls for this observation to be studied in a highly consanguineous population like ours to explore whether it is consistent to merit etiological exploration. This study emphasizes the importance of array-CGH in the initial diagnostic evaluation of patients with ASD even in countries with high consanguinity rate. Conflict of interest disclose.
The authors do not have conflict of interest to
References Al-Thihli, K., Al-Murshedi, F., Al-Hashmi, N., Al-Mamari, W., Islam, M. M., & Al-Yahyaee, S. A. (2014). Consanguinity, endogamy and inborn errors of metabolism in Oman: A crosssectional study. Human Heredity, 77(1–4), 183–188. doi:10. 1159/000362686. Baieli, S., Pavone, L., Meli, C., Fiumara, A., & Coleman, M. (2003). Autism and phenylketonuria. Journal of Autism and Developmental Disorders, 33(2), 201–204. Battaglia, A., & Bonaglia, M. C. (2006). The yield of subtelomeric FISH analysis in the evaluation of autistic spectrum disorders. American Journal of Medical Genetics Part C Seminars in Medical Genetics, 142C(1), 8–12. doi:10.1002/ajmg.c.30077. Curran, S., Ahn, J. W., Grayton, H., Collier, D. A., & Ogilvie, C. M. (2013). NRXN1 deletions identified by array comparative genome hybridisation in a clinical case series—further understanding of the relevance of NRXN1 to neurodevelopmental disorders. Journal of Molecular Psychiatry, 1(1), 4. doi:10.1186/ 2049-9256-1-4 Dabell, M. P., Rosenfeld, J. A., Bader, P., Escobar, L. F., El-Khechen, D., Vallee, S. E., et al. (2013). Investigation of NRXN1 deletions: Clinical and molecular characterization. American Journal of Medical Genetics Part A, 161(4), 717–731. doi:10. 1002/ajmg.a.35780 Fombonne, E. (2005). Epidemiology of autistic disorder and other pervasive developmental disorders. Journal of Clinical Psychiatry, 66(Suppl 10), 3–8. Freitag, C. M. (2007). The genetics of autistic disorders and its clinical relevance: A review of the literature. Mol Psychiatry, 12(1), 2–22. doi:10.1038/sj.mp.4001896. Gamsiz, E. D., Viscidi, E. W., Frederick, A. M., Nagpal, S., Sanders, S. J., Murtha, M. T., et al. (2013). Intellectual disability is associated with increased runs of homozygosity in simplex autism. The American Journal of Human Genetics, 93(1), 103–109. doi:10.1016/j.ajhg.2013.06.004.
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Lugtenberg, D., Yntema, H. G., Banning, M. J., Oudakker, A. R., Firth, H. V., Willatt, L., et al. (2006). ZNF674: A new kruppelassociated box-containing zinc-finger gene involved in nonsyndromic X-linked mental retardation. The American Journal of Human Genetics, 78(2), 265–278. doi:10.1086/500306 Miller, D. T., Adam, M. P., Aradhya, S., Biesecker, L. G., Brothman, A. R., Carter, N. P., et al. (2010). Consensus statement: Chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. The American Journal of Human Genetics, 86(5), 749–764. doi:10.1016/j.ajhg.2010.04.006. Newschaffer, C. J., Croen, L. A., Daniels, J., Giarelli, E., Grether, J. K., Levy, S. E., et al. (2007). The epidemiology of autism spectrum disorders. Annual Review of Public Health, 28, 235–258. doi:10.1146/annurev.publhealth.28.021406.144007. Perez-Duenas, B., Sempere, A., Campistol, J., Alonso-Colmenero, I., Diez, M., Gonzalez, V., et al. (2012). Novel features in the evolution of adenylosuccinate lyase deficiency. European Journal of Paediatric Neurology, 16(4), 343–348. doi:10.1016/j.ejpn. 2011.08.008. Ravnan, J. B., Tepperberg, J. H., Papenhausen, P., Lamb, A. N., Hedrick, J., Eash, D., et al. (2006). Subtelomere FISH analysis of 11 688 cases: An evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities. Journal of Medical Genetics, 43(6), 478–489. doi:10.1136/jmg.2005.036350. Roohi, J., Montagna, C., Tegay, D. H., Palmer, L. E., DeVincent, C., Pomeroy, J. C., et al. (2009). Disruption of contactin 4 in three subjects with autism spectrum disorder. Journal of Medical Genetics, 46(3), 176–182. doi:10.1136/jmg.2008.057505 Rooney, D. E., Williams, K., Coleman, D. V., & Habelt, A. (1989) A case of interstitial deletion of 10q25.2—q26.1. Journal of Medical Genetics, 26(1), 58–60. doi:10.1136/jmg.26.1.58. Schaefer, G. B., & Mendelsohn, N. J. (2008). Genetics evaluation for the etiologic diagnosis of autism spectrum disorders. Genetics in Medicine, 10(1), 4–12. doi:10.1097/GIM.0b013e31815efdd7. Schulze, A. (2013). Creatine deficiency syndromes. [Review]. Handbook of Clinical Neurology, 113, 1837–1843. doi:10. 1016/B978-0-444-59565-2.00053-8. Striano, P., Malacarne, M., Cavani, S., Pierluigi, M., Rinaldi, R., Cavaliere, M. L., et al. (2006). Clinical phenotype and molecular characterization of 6q terminal deletion syndrome: Five new cases. American Journal of Medical Genetics Part A, 140(18), 1944–1949. Vissers, L. E., de Vries, B. B., Osoegawa, K., Janssen, I. M., Feuth, T., Choy, C. O., et al. (2003). Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. The American Journal of Human Genetics, 73(6), 1261–1270. doi:10.1086/379977.
ORIGINAL PAPER
Diagnostic Yield of Chromosomal Microarray Analysis in a Cohort of Patients with Autism Spectrum Disorders from a Highly Consanguineous Population Watfa Al-Mamari • Abeer Al-Saegh • Adila Al-Kindy • Zandre Bruwer • Fathiya Al-Murshedi • Khalid Al-Thihli
Ó Springer Science+Business Media New York 2015
Abstract Autism Spectrum Disorders are a complicated group of disorders characterized with heterogeneous genetic etiologies. The genetic investigations for this group of disorders have expanded considerably over the past decade. In our study we designed a tired approach and studied the diagnostic yield of chromosomal microarray analysis on patients referred to the Genetic and Developmental Medicine clinic in Sultan Qaboos University in Oman for autism spectrum disorders in a highly consanguineous population. Copy number variants were seen in 27 % of our studied cohort of patients and it was strongly associated with dysmorphic features and congenital anomalies. Keywords Autism Autism spectrum disorders Chromosomal microarray Array-CGH Diagnostic yield Consanguinity
Introduction Autism is a complex neurodevelopmental disorder defined as a persistent communication and social interaction deficits in multiple situations. The major manifestations of autism include impairment in social communication and behavioral problems such as fixated (restricted) interests and repetitive behaviors (DSM-5, American psychiatry association). Autism Spectrum Disorders (ASDs) are estimated to affect around 1:150 individuals (Newschaffer
W. Al-Mamari A. Al-Saegh (&) A. Al-Kindy Z. Bruwer F. Al-Murshedi K. Al-Thihli Genetics Department, College of Medicine, Genetic and Developmental Medicine Clinic, Sultan Qaboos University Hospital, PO Box 35, 123 Muscat, Sultanate of Oman e-mail: [email protected]
et al. 2007) and it occurs more frequently in males than females. Although twin studies have shown a strong genetic contribution to the etiology of ASDs, these disorders usually have complex multifactorial etiology (Fombonne 2005 and Freitag 2007). A genetic etiology corresponding to known chromosomal rearrangements or single gene disorders is currently recognized only in 10–20 % of the patients, partly reflecting the etiological heterogeneity of ASDs (Battaglia and Bonaglia 2006; Ravnan et al. 2006; Schaefer and Mendelsohn 2008). The recently published consensus statement of the International Standard Cytogenomic Array (ISCA) recommends the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with developmental delay/intellectual disability, ASD, or multiple congenital anomalies (Miller et al. 2010). ASDs may also be seen in association with a number of recessively inherited disorders like creatine deficiency syndromes (Schulze 2013), adenylosuccinate lyase deficiency (Perez-Duenas et al. 2012) and phenylketonuria (Baieli et al. 2003). There is also propensity for recessively inherited disorders to occur at relatively higher frequency in countries with high rates of consanguinity like Oman, a country where consanguinity rates could be as high as 50 % in some regions (Al-Thihli et al. 2014). These factors pose concerns about whether the diagnostic yield of CMA among patients with ASDs maybe lower in these countries than previously quoted in other studies, particularly that to date no study to our knowledge- has been conducted in a highly consanguineous population to address this issue. The developmental Pediatrician (WM) and clinical geneticists (AS, AK, FM & KT) at the Genetic and Developmental Medicine Clinic (GDMC) adapted a tiered approach for the diagnostic work up of patients with ASDs.
123
J Autism Dev Disord
The approach is depicted in the flow chart shown in Fig. 1. The GDMC is the only diagnostic clinic for patients with ASD in Oman. This study describes the diagnostic yield of chromosomal microarray following 20 months of clinical application of this approach.
Methods The medical records and array-CGH results of all patients diagnosed with ASDs between January 2012 and September 2013 were reviewed. All patients were included when calculating the diagnostic yield provided they met the eligibility criteria for CMA adapted in the clinic at the time this test was carried out. Patients were considered to be eligible for CMA if they meet the following criteria: 1. 2.
An established clinical diagnosis of ASD and At least one of the following clinical determinants: a.
b. c.
Negative history for consanguinity or apparently sporadic occurrence (even if consanguinity is present) One or more major congenital malformation 3 or more minor dysmorphic features
Fig. 1 Diagnostic workup of patients with ASD
d.
Negative first tier testing if a,b, and c were absent.
DNA extracted from blood was sent for array-CGH at Wessex Regional Genetics Laboratory, the United Kingdom (www.wrgl.org.uk). The array-CGH provided uses Oxford Gene Technology’s (ISCA) 8 9 60 K oligo array platform, providing an average backbone resolution of 240 kb. The array-CGH chip was designed by ISCA and validated by the National Genetics Reference Laboratory (Wessex). CytoSure Interpret version 3.4.8 was the software used for array analysis. For further details of the array design and probes used see: http://www.ngrl.org.uk/Wes sex/array.htm.
Results A total of 230 patients were diagnosed with ASDs through the GDMC between January 2012 and September 2013. 223 patients (97 %) had associated intellectual disability (ID) or developmental delay (DD). A total of 100, 74 males and 26 females, met the adapted eligibility criteria for CMA. A total of 27 patients (27 %), 17 males and 10 females, had copy number variants (CNVs). Seven (7) patients had CNVs that overlap
Clinical Diagnosis of Ausm Spectrum Disorder
No clinical features sugges ve of a specific clinical diagnosis
Tier 1
Ammonia Lactate Plasma amino acids Urine organic acids Acylcarni ne profiling (dried blood spot) Urine crea ne & guanidinoacetate
Abnormal first er tes ng
Tier 2
Clinical features suggest a specific diagnosis
Referral to clinical biochemical gene cs service for further evalu on
Normal first
te
Chromosomal microarray (CMA)* Fragile X (if CMA is nega ve)
Specific molecular gene c tes ng or CMA
* CMA was done in all ents with ASD who have any of the following features: 1) n ve history for consanguinity, 2) apparently sporadic occurrence (even when consanguinity is present), 3) one or more major congenital malform ons, or 4) three or more minor dysmoprhic features.
123
Unknown
Unknown
Pathogenic
NK 300 kb deletion
470 kb duplication
arr[hg18] Xq21.31(87,839,749-88,139,807)X0
3 6
Dysmorphic features, cardiac anomaly, urogenital anomaly
4 5
Dysmorphic features
arr[hg18] Xp22.13(17,167,331-17,639,471)X2
NK
Diagnosed with clinical depression Maternally inherited 110 kb deletion within the intron of NRXN1 arr[hg19] 2p16.3(50,735,595-50,845,789)X1 4 4
Unusual behaviour
NK 318 kb duplication arr[hg18]10p15.3(268,741-586,626)X4 4 3
Dysmorphic features, microcephaly
51 kb deletion arr[hg18]7q35(146,020,240-146,071,372)X1 10 2
Dysmorphic features
2.1 Mb deletion arr22q11.21(17,041,699-19,125,890)X1 Dysmorphic features 4 1
Microarray finding Additional findings Age at diagnosis with ASD Number of patients
Table 1 Clinical and cytogenetic details of patients positive for copy number variants
Size of the deletions/ duplication
Discussion In consistence with the already established role of CMA in the diagnostic work up of patients with ASDs, this study emphasizes this role even in a country with high rates of consanguinity like Oman. Although the concern about missin g an underlying recessive etiology might be deterrent to ordering CMA in the initial diagnostic assessment of patients with ASD in consanguineous families, the diagnostic yield could still be high enough to argue for this consideration in appropriately selected group of patients. 27 % of patients diagnosed through our clinic had CNVs, and even when excluding the nine CNVs of uncertain clinical significance, the diagnostic yield of CMA becomes 18 %. Both figures fall within the range of diagnostic yields reported for CMA in different published studies (Miller et al. 2010; Vissers et al. 2003), and both are notably higher than that universally reported for conventional karyotype. Positive diagnostic yield was significantly higher in patients with associated congenital malformations or dysmorphic features. It is worth noting that almost all patients diagnosed with ASDs through our clinic had associated developmental delay (DD) or intellectual disability (ID). This is higher than the prevalence of 40 % reported by the Autism and Developmental Disabilities Monitoring Network Year 2008 (Gamsiz et al. 2013). Although referral and ascertainment bias cannot be reliably excluded, the relatively higher prevalence of DD/ID maybe hypothesized to be due to increased runs of homozygosity among our cohort compared to the previously reported cohort of patients. Gamsiz et al. showed that in in simplex ASD-affected families, probands with an IQ B 70 showed more ROH
Unknown
(Curran et al. 2013) Pathogenic this deletion is within intron of CNTNAP2 (member of neurexin superfamily Below average intelligence Maternally Inherited
Pathogenic 22q11.2 microdeletion syndrome – De novo
De novo Vs inherited
Phenotype of the affected parent
Significance
References
genomic imbalances defined in a CNV database for patients with ID, DD, ASD, or multiple congenital anomalies. Five (5) patients had CNVs that overlap genomic coordinates for previously published or well-recognized deletion or duplication syndromes. Six (6) patients had CNVs that contain OMIM morbid genes. The remaining nine (9) patients had CNVs of uncertain clinical significance. Table 1 shows details of the copy number variants identified in this cohort. Patients with dysmorphic features were more likely to have CNV compared with those considered non-dysmorphic (22/27 vs. 21/73, respectively, p = 0.0002). Congenital anomalies were also significantly associated with CNVs (16/27 compared to 12/73 without CNVs, p = 0.0005). The frequency of reported history of consanguinity was not significantly different between the CNV-positive and CNV-negative group of patients (p = 0.6639). Consanguinity was seen in 31 (31 %) of our studied families with ASD (31/100).
(Dabell et al. 2013)
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6
5
3
3
5
3
9
5
4
7
8
4
5
8
9
10
11
12
13
14
15
16
17
18
19
Age at diagnosis with ASD
7
Number of patients
Table 1 continued
Dysmorphic features
Dsymorphic features and macrocephaly
Dysmorphic, polymicrogyria
Dysmorphic features
Dysmorphic features
Seizures
Dysmorphic features
Dysmorphic features, musculoskeletal anomalies, anal anomaly and cardiac anomalies
Dysmorphic features, anal anomaly, seizures
Dysmorphic features, hearing loss, musculoskeletal anomalies and urogenital anomalies
Dysmorphic features
Dysmorphic features
Dysmorphic features
Additional findings
12.3 Mb deletion contains 48 genes out of which 11 are OMIMMorbid enteries 2.5 Mb deletion
arr[hg19]10q25.1q26.11(107,352,476-119,663,083)X1
arr[hg19]22q11.21(18,894,820-21,440,515)X1
arr[hg18]7q11.23(72,404,24873,780278)X1.rsa7q11.23(p029)X1
arr16q12.2(56,659,661-56,692,859)X1
arr[hg18]1p22.3(68, 253,21486,565,053),3p24.3(19,194,234,19,629,776)X3dn.ish dup (3)(p24.3p24.3)
arr[hg18]14q32.2q32.31(99,908,703100,558,651)X3pat, 17p11.2(21,136,13121,147,826)X3mat,17p11.2(21,136,13121,147,826)X3pat
arr7p22.2p21.1(2,811,886-19,358,897)X3
arr[hg18]Xp11.3(46,258,090-46,382,273)X3
1.4 Mb deletion
33 kb deletion
2. 312 kb deletion within 1p22.3
De novo
Maternally inherited
History of speech delay
Pathogenic (duplication with 3p24.3)
Normal Maternally inherited deletion 1. 436 duplication kb within 3p24.3
Pathogenic (William’s syndrome)
Pathogenic
Benign CNV (312 kb deletion within 1p22.3)
Pathogenic Below average intelligence. Diagnsoed with depression, and renal stones
Paternally inherited 650 kb duplication within the 14q32.2to14q32.31. 12 kb duplication within 17p11.2
De novo Duplication
Pathogenic
125 kb duplication contains part of the gene ZNF
De Novo
Pathogenic
Pathogenic22q11.2 microdeletion syndrome
Pathogenic (similar patient reported, Rooney et al. 1989)
Pathogenic member of neurexin superfamily
Unknown
Unknown
Unknown
Significance
16.5 Mb duplication involves 87 genes out of which 6 are OMIMMorbid enteries
Normal
Normal
Phenotype of the affected parent
Pathogenic
Paternally inherited
De Novo
De Novo
Paternally inherited
NK
NK
NK
De novo Vs inherited
De Novo
60 kb duplication
56 kb duplication within CNTN4 gene
arr[hg18]3p26.3(2,351,629-2,407,891)X3
arr[hg19]2q22.2(143,352,620143,931,358)X3,3p26.3(2,376,629-2,432,891)X3
500 kb duplication
680 kb duplication
5 kb deletion
Size of the deletions/ duplication
arr[hg19]5p12p11(45,615,361-46,115,173)X3
arr[hg19]7p21.1(17,254,111-17,932,752)X3
arr[hg19]5q22.2(112,132,377-112,137,084)X1
Microarray finding
(Lugtenberg et al. 2006)
(Curran et al. 2013)
(Curran et al. 2013)
References
J Autism Dev Disord
4
2
8
7
6
7
4
21
22
23
24
25
26
27
Dysmorphic features
Dysmorphic features
Dysmorphic features
microcephaly
Dysmorphic features
Dsymorphic features, seizures
Behavioural abnormalities
Dysmorphic features, musculoskeletal anomaly
Cleft palate
Musculoskeletal anomalies
Dysmorphic features
Additional findings
arr[hg19] 3p26.3(2,376,629-2,432,891)X3
arr[hg19] Xp22.11(22,156,80422,438,749)X2,2p16.3(51,067,05551,218,716)X1,7q32.1q32.3(128,972,820131,984,276)X1
arr[hg19]6q27(168,790,739-171,005,522)X1
arr[hg19] 14q32.33(105,017,254-107,287,708)X1
arr[hg19]2q22.2(143,352,620-143,931358)X3, 3p26.3(2,376,629-2,432,891)X3
Arr[hg19]20p13(83,092-956,262)X3
Arr[hg18]3p26.3(2,693,622-2,994,093)X3
10.886 Mb duplication
Arr3[hg18]q27.3q29(186,918,265197,803,820)X3,9p24.3p23(215.366-12,907,826)X1
57 kb duplication
3. Duplication involving Xp22.11 with minimum size of 280 kb
2. Interstitial deletion involving 7q32.1 to 7q32.3 with a minimum deletion of 3 Mb
NK
NK
3 findings: 1. Interstitial deletion within 2p16.3 of about 150 kb within NRXN1 gene
NK
NK
NK
NK
NK
De novo
De novo Vs inherited
2.3 Mb terminal deletion
2.3 Mb deletion
60 kb duplication within CNTN4
870 kb duplication
300 kb duplication. Duplication disrupts CNTN4
12.693 Mb deletion
Size of the deletions/ duplication
Microarray finding
Patients with autistic spectrum disorder (ASD) found to have copy number variants (CNVs)
4
Age at diagnosis with ASD
20
Number of patients
Table 1 continued Phenotype of the affected parent
The duplication is within CNTN4 gene –Pathogenic
3. Unknown-novel
2. Unknown- novel
1. Pathogenic (Dabell et al. 2013)
(Striano et al. 2006)
Terminal deletions of 6q-Pathogenic
14q terminal deletion syndromePathogenic
Pathogenic
NK-novel
Pathogenic
Pathogenic
Significance
(Roohi et al. 2009)
References
J Autism Dev Disord
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J Autism Dev Disord
than their unaffected siblings, whereas probands with an IQ [ 70 do not show this excess (Gamsiz et al. 2013). This calls for this observation to be studied in a highly consanguineous population like ours to explore whether it is consistent to merit etiological exploration. This study emphasizes the importance of array-CGH in the initial diagnostic evaluation of patients with ASD even in countries with high consanguinity rate. Conflict of interest disclose.
The authors do not have conflict of interest to
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