Outcomes from chromosomal microarray
From the 101 probands in whom CMA was performed, pathogenic CNVs were detected in 3 cases (2.9%) including two deletions and one duplication (Table 2). Proband ASD-076 had an 8Mb deletion at the 15q11.2 locus which encompassed 20 OMIM genes and is known to cause 15q11.2 deletion syndrome (OMIM#615656) or Angelman syndrome (OMIM#105830). Compared to the individuals with class II deletions (BP2-BP3; ISCA-37478), individuals with large class I deletions (BP1-BP3; ISCA-37404) at the 15q11.2 region are observed to have a high likelihood of language impairment and autistic traits, similar to that seen in the proband in our study (45). Patient ASD-103 was detected with a deletion of 0.19Mb size at the 9q34.3 locus which encompassed 6 OMIM genes and is associated with Kleefstra syndrome I (OMIM#610253). Individuals with > 1Mb deletion of the 9q34 locus have a severe phenotype such as congenital anomalies including heart defects, limb anomalies, seizures and respiratory distress. In contrast individuals having < 1Mb deletion are observed with a milder phenotype, which in part could explain the phenotype in the proband in the current study such as bruxism, drooling, subtle facial dysmorphism and recurrent episodes of vomiting (46, 47). Lastly, proband ASD-001 was detected with a 0.52Mb duplication on the 1q22 locus which consists of 8 OMIM genes. This is a rare CNV which has previously only been reported in a boy with intellectual disability and psychiatric disturbances (48). Multiple individuals in this family were affected and the duplication variant segregated with the neurological features in all family members with this variant. All CNVs in our cohort were de novo in origin and were observed exclusively in male probands.
Table 2
List of cases observed with pathogenic or likely pathogenic copy number variation using CMA and/or WES.
Sr No | Case ID | Chr No | Chromosomal microarray | Whole exome sequencing | CNV Type | Zygosity | Inheritance pattern | Inherited from | Variant classification | OMIM Disease (OMIM ID) |
Genomic coordinate | Size (Kbp) | Genomic coordinate | Variant |
1 | ASD-021 | 15 | - | - | ?_20279345_21145729_? | chr15:g.(?_20279345)_(21145729_?)del | loss | Het | AD | De novo | Variant of uncertain significance | Chromosome 15q11.2 deletion syndrome (#615656) |
2 | ASD-050 | 1 | arr[GRCh37] 1q22(155446632_155971760)x3 | 525 | ?_155611966_156081998_? | chr1:g.(?_155611966)_(156081998_?)dup | gain | Het | AD | De novo | Likely pathogenic | Chromosome 1q22 microduplication syndrome |
3 | ASD-076 | 15 | arr[GRCh37]15q11.2q13.2(22770421_30913574)x1 | 8,143 | ?_24955049_28272386_? | chr15:(?_24955049)_(28272386_?)del | loss | Het | AD | De novo | Pathogenic | Chromosome 15q11.2 deletion syndrome (#615656) |
4 | ASD-103 | 9 | arr[GRCh37]9q34.3(140338142_140533378)x1 | 195 | ?_137426248_137629023_? | chr9:g.(?_137426248)_(137629023_?)del | loss | Het | AD | De novo | Pathogenic | Kleefstra syndrome-1 (#610253) |
Chr = chromosome; No = number; Kbp = kilo basepair; CNV = copy number variation; Het = heterozygous; AD = autosomal dominant |
Outcomes from whole exome sequencing
WES was carried out in 99 of 101 cases, as the cohort contained two monozygotic twin pairs and only one proband from each twin pair was processed for WES. The 99 cases also included the three cases that yielded a result by CMA to assess the sensitivity of WES to detect CNVs. On an average, approximately 3 candidate gene(s) or variant(s) were identified per proband (Supplementary Table 3).
From the 101 patients, pathogenic and/ or likely pathogenic variants were identified in 30 cases (29.7%), of which, SNVs were detected in 27 cases (90%) and CNVs in 3 cases (10%) (Table 3). Interestingly, 3 CNVs detected by CMA were also identified by WES, however, a 0.8Mb de novo deletion encompassing the BP1 region of the 15q11.2 locus was detected by WES alone (Table 2). On further analysis, the lack of detection of the aforementioned CNV by CMA was due to the lack of probes covering this region on CytoScan™ Optima array.
Table 3
List of cases observed with pathogenic or likely pathogenic single nucleotide variation using WES
Sr No | Case ID | Chr No | Genomic coordinate | Ref allele | Alt allele | Gene (OMIM ID) | Transcript ID | Exon | Variant | Protein change | Zygosity | Mode of inheritance | Inherited from | Variant classification | OMIM Disease (OMIM ID) |
1 | ASD-001 | 20 | 62071010 | C | A | KCNQ2 (*602235) | ENST00000359125.2 NM_172107.4 | 6 | c.868G > T | p.Gly290Cys | Het | AD | De novo | Pathogenic | Developmental and epileptic encephalopathy 7 (#613720); |
2 | ASD-003 | 7 | 114299416 | G | A | FOXP2 (*605317) | ENST00000408937.3 NM_148898.4 | 13 | c.1549G > A | p.Ala517Thr | Het | AD | not Maternal a | Likely pathogenic | Speech-language disorder-1 (#602081) |
3 | ASD-010 | 2 | 166172149 | G | T | SCN2A (*182390) | ENST00000375437.2 NM_001040142.2 | 11 | c.1552G > T | p.Glu518Ter | Het | AD | De novo | Pathogenic | Developmental and epileptic encephalopathy 11 (#613721) |
4 | ASD-011 | 1 | 19212049 | - | GGTCTGCA | ALDH4A1 (*606811) | ENST00000375341.3 NM_003748.4 | 5 | c.363_370dup | p.Arg124LeufsTer9 | Comp het | AR | Maternal | Pathogenic | Hyperprolinemia, Type II (#239510) |
19209638 | C | G | 7 | c.658G > C | p.Ala220Pro | Paternal | Likely pathogenic |
5 | ASD-013 | 17 | 28530262 | T | TC | SLC6A4 (*182138) | ENST00000261707.3 NM_001045.6 | 14 | c.1745dup | p.Thr583AsnfsTer23 | Het | AD; - | Maternal | Likely pathogenic | {Obsessive-compulsive disorder} (#164230); {Anxiety-related personality traits} (#607834) |
6 | ASD-016 | X | 153363096 | CGGCG | - | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 1 | c.23_27del | p.Ala8GlufsTer32 | Het | XLD | De novo | Pathogenic | Rett syndrome (#312750) |
7 | ASD-017 | 3 | 184072060 | G | A | TBL1XR1 (*608628) | ENST00000457928.2 NM_024665.7 | 7 | c.679G > A | p.Asp227Asn | Het | AD | De novo | Likely pathogenic | Intellectual developmental disorder, autosomal dominant 41 (#616944); Pierpont syndrome (#602342) |
8 | ASD-020 | X | 153296473 | - | C | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 3 | c.842dup | p.Arg282ProfsTer61 | Het | XLD | De novo | Pathogenic | Rett syndrome (#312750) |
9 | ASD-022 | 3 | 184072060 | G | A | CLCN2 (*600570) | ENST00000265593.4 NM_004366.6 | 15 | c.1550C > T | p.Thr517Met | Het | AD | Paternal | Likely pathogenic | {Epilepsy, idiopathic generalized, susceptibility to, 11} (#607628) |
10 | ASD-034 | 5 | 134871150 | - | | NEUROG1 (*601726) | ENST00000314744.4 NM_006161.3 | 1 | c.228_231dup | p.Thr78Profs*122 | Homo | UNK | Maternal and Paternal | Likely pathogenic | - |
11 | ASD-045 | 19 | 39805801 | A | G | LRFN1 (*612807) | ENST00000248668.4 NM_020862.2 | 1 | c.176T > C | p.Val59Ala | Het | UNK | De novo | Likely pathogenic | - |
12 | ASD-046 | 7 | 147092836 | C | T | CNTNAP2 (*604569) | ENST00000361727.3 NM_014141.6 | 10 | c.1634C > T | p.Ala545Val | Het | AD | De novo | Likely pathogenic | {Autism susceptibility 15} (#612100) |
13 | ASD-047 | 18 | 52896081 | G | A | TCF4 (*602272) | ENST00000398339.1 NM_001243226.3 | 19 | c.2182C > T | p.Arg278Ter | Het | AD | De novo | Pathogenic | Pitt-Hopkins syndrome (#610954) |
14 | ASD-049 | 2 | 166245627 | T | A | SCN2A (*182390) | ENST00000375437.2 NM_001040142.2 | 27 | c.5311T > A | p.Tyr1771Asn | Het | AD | De novo | Likely pathogenic | Developmental and epileptic encephalopathy-11; (#613721); Benign familial neonatal-infantile seizures-3; (#607745); Episodic ataxia type 9 (618924) |
15 | ASD-053 | 5 | 149629805 | G | T | CAMK2A (*114078) | ENST00000348628.6 NM_015981.4 | 11 | c.884C > A | p.Ala295Asp | Het | AD | De novo | Likely pathogenic | Intellectual developmental disorder, autosomal dominant 53 (#617798) |
16 | ASD-054 | X | 44949169 | C | T | KDM6A (*300128) | ENST00000377967.4 NM_021140.4 | 25 | c.3730C > T | p.Leu1244Phe | Hemi | XL | Maternal | Likely pathogenic | Kabuki syndrome 2 (#300867) |
17 | ASD-057 | X | 153296824 | G | C | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 3 | c.491C > G | p.Pro164Arg | Het | XLD | De novo | Pathogenic | Rett syndrome (#312750) |
18 | ASD-058 | 17 | 60042409 | G | T | MED13 (*603808) | ENST00000397786.2 NM_005121.3 | 20 | c.4802C > A | p.Ser1601Ter | Het | AD | Maternal | Pathogenic | Intellectual developmental disorder, autosomal dominant 61 (#618009) |
19 | ASD-064 | X | 153296882 | G | A | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 3 | c.433C > T | p.Arg145Cys | Het | XLD | De novo | Pathogenic | Rett syndrome (#312750) |
20 | ASD-067 | X | 153296777 | G | A | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 3 | c.538C > T | p.Arg180Ter | Mosaic, somatic variant | XLD | De novo; post-zygotic event | Pathogenic | Rett syndrome (#312750) |
21 | ASD-072 | X | 70389622 | T | G | NLGN3 (*300336) | ENST00000358741.3 NM_181303.1 | 8 | c.2222T > G | p.Leu741Arg | Hemi | XL | De novo | Likely pathogenic | {Autism susceptibility, X-linked 1} (#300425) |
22 | ASD-074 | 9 | 77249655 | C | T | RORB (*601972) | ENST00000376896.3 NM_006914.4 | 3 | c.202C > T | p.Gln68Ter | Het | AD | Paternal (affected father) | Pathogenic | {Epilepsy, idiopathic generalized, susceptibility to, 15} (#618357) |
23 | ASD-080 | 17 | 29553492 | C | T | NF1 (*613113) | ENST00000358273.4 NM_001042492.3 | 18 | c.2041C > T | p.Arg681Ter | Het | AD | De novo | Pathogenic | Neurofibromatosis, type 1; (#162200) Neurofibromatosis-Noonan syndrome (#601321) |
24 | ASD-082 | 7 | 70231162 | G | A | AUTS2 (*607270) | ENST00000342771.4 NM_015570.4 | 9 | c.1531G > A | p.Gly511Arg | Het | AD | De novo | Likely pathogenic | Intellectual developmental disorder, autosomal dominant 26 (#615834) |
25 | ASD-084 | 2 | 97377437 | C | T | LMAN2L (*609552) | ENST00000377079.4 NM_001142292.2 | 7 | c.773G > A | p.Arg258His | Hom | AR | Maternal & Paternal | Likely pathogenic | ? Intellectual developmental disorder, autosomal recessive 52 (#616887) |
26 | ASD-085 | 5 | 125887798 | G | A | ALDH7A1 (*107323) | ENST00000409134.3 NM_001182.5 | 14 | c.1232C > T | p.Pro411Leu | Hom | AR | Maternal & Paternal | Likely pathogenic | Epilepsy, pyridoxine-dependent (#266100) |
27 | ASD-105 | X | 153296363 | G | A | MECP2 (*300005) | ENST00000453960.2 NM_001110792.2 | 3 | c.952C > T | p.Arg318Cys | Het | XL | De novo | Pathogenic | Autism susceptibility, X-linked 3 (#300496) |
Het: heterozygous; Hom: homozygous; Hemi: hemizygous; Comp het: Compound heterozygous; AD: autosomal dominant; AR: autosomal recessive; XL: X-linked; UNK: unknown |
a Paternal DNA sample was unavailable and the variant was not detected in maternal sample. |
Segregation analysis revealed that approximately 66.6% (n = 3 for CNVs and n = 17 for SNVs) of the cases were caused due to a de novo variant. De novo SNVs were found primarily in previously known ASD genes- MECP2, SCN2A, KCNQ2, TBL1XR1, CNTNAP2, TCF4, CAMK2A, NF1, AUTS2, FOXP2 and NLGN3. Of 17 de novo variants, 6 were predicted to be loss of function (pLOF) variants (35.2%) whereas the remaining were missense variants. Remarkably, 6 of the 17 patients had a de novo SNV in the MECP2 gene, which is associated with Rett syndrome (OMIM#312750). Of these, 5 were female and 1 was a male proband. Interestingly, in a rare case of the male proband aged 2.5 years with Rett syndrome, we observed that the variant c.538C > T (p.Arg180Ter) in the MECP2 gene originated through a post-zygotic de novo event which led to somatic mosaicism in the proband (Table 3) (49).
In our cohort of patients with pathogenic/ likely pathogenic variants, 5 probands (n = 5/30; 16.6%) were observed with biallelic or hemizygous variants in genes associated with NDD or metabolic disorders with a recessive mode of inheritance (Table 3). Specifically, biallelic variants were detected in (i) ALDH4A1 gene which is associated with hyperprolinemia type II (OMIM#239510), (ii) NEUROG1 gene which is associated with congenital cranial dysinnervation disorder and autism spectrum disorder (50), (iii) KDM6A gene which is associated with Kabuki syndrome 2 (OMIM#300867), (iv) LMAN2L gene which is associated with mental retardation 52 (OMIM#616887) and, (v) ALDH7A1 gene which is associated with pyridoxine dependent epilepsy (OMIM#266100).
In addition, 4 probands were identified with pathogenic/ likely pathogenic heterozygous variants, which were inherited from one of their parents. In 2 cases, the variants were inherited from unaffected mother and in 1 case the variant was inherited from an unaffected father. In the 4th case, pLOF variant c.202C > T (p.Gln68Ter) in the RORB gene was inherited from father who also had a clinical history of seizures (Supplementary Table 2; Supplementary Information 1). Of note, in one case (ASD-003), paternal sample was un-available, hence the mode of inheritance couldn’t be deduced.
Lastly, WES identified 22 VUS variants in 21 patients (n = 21/101; 20.8%; Supplementary Table 4). The variants were identified in genes that have previously been associated with or implicated in ASD etiology as per the Simons Foundation Autism Research Initiative (SFARI) Gene Database and Autism Database (AutDB). Of these, majority of the probands were detected with heterozygous variants (66.6%) which were inherited from either of the unaffected parents with equal distribution. Of note, 3 of the 21 patients following segregation analysis were detected with missense variants in the KMT2C gene (Kleefstra syndrome 2; OMIM#617768) which were inherited from a healthy parent. Whilst the majority of the cases have been reported with a de novo variant in the KMT2C gene, 4 reports observed variants being inherited from a healthy parent suggesting a potential oligogenic mode of inheritance (51–54).