The CNVs in the 17q25.3 region were found to be rare events, with a prevalence of 0.0008% (15/16,664) observed in our cohort. Further, the CNVs spanned the entire 17q25.3 region with variable breakpoints and no smallest region of overlap. The subjects were observed to have a wide range of clinical features, with neurodevelopmental disorders (ASD, ID, DD) as the most common feature in 80%, expressive language disorder in 33%, followed by cardiovascular malformations in 26% cases. Our results are largely in agreement with the study by Prost et al., with one major variation regarding penetrance, as Prost et al. reported a higher penetrance of ~60% for cardiac malformation [5]. The difference in penetrance of traits can be attributed to the variable CNV regions between the cases in both studies, the genes involved, and the inheritance pattern of these CNVs. Herein, we have compiled the cases reported in literature along with the cases in our cohort, and proposed genotype-phenotype correlations for a subset of gene(s) impacted in these CNVs. The detailed phenotype for each case is listed in supplementary file 3. Since this is a retrospective study where medical records were retrieved for phenotyping, the rates of clinical features should considered to be lower bound estimates.
Three cases with isolated 17q25.3 CNVs
Case 1: The subject was diagnosed with Sanfillipo syndrome type A, and his father had the same diagnosis. The sequencing analysis of the SGSH gene identified a homozygous deletion of 11 nucleotides beginning at position 1272 (c.1272_1282del11), which is expected to cause a reading frame shift, resulting in an aberrant protein. The CMA detected, arr[hg19] 17q25.3(78,183,648-78,190,994)x1, approximately 7 kb in size, deleting exons 2-8 in the SGSH gene. The CNV analysis clarified the results obtained with sequencing analysis, where the homozygous call for 11 nucleotide deletion resulted from 11 nucleotide deletion on one allele, and a deletion of exons 2-8 on the other allele. Further, heparan N sulfatase (Heparan sulfaminidase) was found to be deficient which is consistent with Sanfilippo syndrome type A. Activity of N acetyglucosamine 6 sulfatase (type D) was normal ruling against multiple sulfatase. The father was found to be carrier with a heterozygous 11 nucleotide deletion, but maternal testing could not be performed to determine the origin of the CNV, and to determine if mother was also a carrier.
Overall, the results were consistent with the clinical diagnosis of Sanfillipo syndrome type A in this individual, as the SGSH gene is associated with Mucopolysaccharidosis type IIIA (Sanfilippo A), AR (MIM #252900). Interestingly, only SNVs in the SGSH gene have been associated with Sanfilippo A, and to our knowledge, this is the first case with a deletion in the SGSH gene (no case in reported in ClinVar, or published literature), that appears to be causal for Sanfillipo syndrome type A syndrome.
Case 2: The subject was diagnosed with ASD, developmental delay, low muscle tone, posterior cerebellar artery syndrome and intellectual disability. At the time of exam, his physical features included a receding hairline, a narrow nasal bridge, a deep philtrum, and flat feet arches. He has family history of a maternal first cousin with speech delay, and his maternal half uncle and his daughter diagnosed with ASD. The subject had a normal 46,XY constitution by karyotyping and showed no evidence of trinucleotide repeat amplification within the FMR1 gene.
The CMA detected, arr[hg19] 17q25.3(80,190,108-80,552,040)x3 pat, approximately 362 kb in size. The duplication included SLC16A3, CSNK1D, CD7, SECTM1, TEX19, UTS2R, OGFOD3, HEXD, CYBC1, NARF, FOXK2 genes, of which FOXK2 was partially duplicated. Importantly, two cases in literature were found with FOXK2 gene duplications with similar clinical features. First, a subject with ID was reported with a 494 kb duplication (DICEPHER database), which also partially duplicated the FOXK2 gene, as observed in our subject. Second, a subject was reported with partial tetrasomy of 17q25.3, with a segment translocated to 10q disrupting the FOXK2 gene [7]. Notably, depletion of Foxk transcription factors have been observed in association with genome-wide transcriptional mis-regulation and developmental arrest in zebrafish embryos [8].
Case 3: The subject was diagnosed with ADHD, developmental disorder of scholastic skills, impulsive, poor social cues, difficulty processing emotions, delayed physical development, speech delay, and possible exposure to alcohol during pregnancy. Notable facial features include dysmorphic facial features, and congenital deformities of the skull/face/jaw, and Café au lait spot on inner buttock. The family history was unknown for this subject. She had a normal 46,XX constitution by karyotyping and showed no evidence of trinucleotide repeat amplification within FMR1 gene.
The CMA detected, arr[hg19] 17q25.3(77190712-77234523)x1, approximately 44 kb in size, resulting in a single exon deletion of the RBFOX3 gene. The gene has been predicted to be intolerant to dosage change (pLI score of 1), while functional studies show an important role in brain development, with knockout of the gene showing reduced brain mass, abnormal hippocampal dentate gyrus, but normal total body mass, and increased susceptibility to seizures [9,10].
Neurodevelopmental disorder and cardiac findings in two cases with RNF213 dosage change.
Case 4: The subject was diagnosed with mixed receptive-expressive language disorder, selective mutism, anxiety disorder, coordination impairment, and a heart murmur. An evaluation for ASD was performed, but did not result in a diagnosis of ASD. He has had growth issues from a young age, including short stature and poor weight gain. His physical appearance includes mild over folding of the left upper helix, a slightly high arched anterior palate, shortening of fifth fingers, minimal 3-2 toe syndactyly bilaterally, slightly dark and dysplastic of the fifth toenails, and several small café au lait spots. He was also noted to have additional points on the back of his teeth, a trait that is shared with his father. The subject had a normal karyotype of 46,XY, and showed no evidence of trinucleotide repeat amplification within the FMR1 gene.
The CMA detected, arr[hg19] 17q25.3(80,190,108-80,552,040)x3 pat, approximately 99 kb in size. The duplication included and partially duplicated SLC26A11 and RNF213 genes, of which RNF213 (MIM #613768) is associated with autosomal dominant and recessive susceptibility to Moyamoya disease-2. The clinical symptoms of Moyamoya disease vary widely and include, but are not limited to, headaches, recurrent transient ischemic attacks, epileptic seizures, or disturbances of speech and cognition, developmental delays or disability, and cardiovascular malformations. The literature has demonstrated SNVs in RNF213 to be strongly associated with Moyamoya disease, but no report of a CNV in this region has been associated with the disease. Receptive-expressive language disorder, coordination impairment, development delays observed in this patient overlap with the clinical features of Moyamoya disease. Importantly, heterozygotes have been reported to have a milder phenotype compared to homozygotes, with the inheritance pattern being both AR and AD [11-14]. Furthermore, knockout of SLC26A11 in mice have demonstrated impaired motor performance [15]. The presence of a cytogenetic aberration in an unaffected parent may be considered evidence that the change is not pathogenic; however, many genetic conditions exhibit variable expressivity, where the same abnormality may cause disease in some individuals and not in others.
Case 5: The subject was diagnosed with a mild left pulmonic stenosis, a 2/6 systolic murmur, bruxism, and central apnea. He has a medical history of feeding through a J-tube, gastroesophageal reflux, allergic rhinitis, periodic fevers, and newborn jaundice. He has behavioral concerns of screaming fits and sensitivity to loud noises. Notable physical features include a full nasal tip, slightly uplifted earlobes, macrocephaly, syndactyly of the third and fourth fingers on the left hand, a large anterior fontanelle, and primary upper extremity hypertonia. The subject’s sample was sequenced for the first coding exon of the HRAS gene and the entire GCHD gene and showed a normal result. Further, several biochemical markers that included, oligosaccharides, mucopolysaccharides, lysosomal activity, and organic acids were found to be normal with slightly elevated urine glutarylcarnitine levels.
The CMA detected arr[hg19] 17q25.2q25.3(72,852,763-78,232,958)x3~4 dn, 17q25.3(78,239,107-78,643,088)x1 dn, approximately 5380 kb and 404 kb in size, respectively. The triplication encompassed several genes including SEPTN9 and EXOC7, while the deletion harbored RNF213, ENDOV, NPTX1, RPTOR, and MIR4730 genes (Figure 2). The SEPTN9 gene is associated with autosomal dominant hereditary neuralgic amyotrophy (MIM #604061), and RNF213 (MIM #613768) is associated with Moyamoya disease 2. Further, the EXOC7 gene has been associated with neurodevelopmental disorder with autosomal recessive seizures and brain atrophy, while RPTOR gene has been reported to be intolerant to dosage change (pLI score of 1), with functional studies demonstrating that the loss of the RPTOR gene in mice neural progenitor cells affects normal development in young age and may contribute to alleviate KA seizure‐induced behavioral abnormalities, suggesting that raptor protein plays an important role in seizure comorbidities [16]. As an apparently de novo CNV, there is an increased likelihood that the 17q25.2q25.3 duplication and the 17q25.3 deletion contribute at least in part to the phenotypes observed in the patient.
Three cases with SEPTN9 gene duplication
In addition to case 5, two other cases harbored duplication overlapping the SEPTN9 gene
Cases 8 and 6: The subject (case 8) was diagnosed with expressive language disorder, learning disability, dyslexia, dolichocephalic, mild scoliosis, pes planus (flat foot), and reactive airway disease. He has a medical history of attention issues, speech delay, umbilical hernia, undescended testicle, and newborn jaundice. Notable physical features include ear anomalies (low set, posteriorly rotated, folded helix), high arched and narrow palate, long face, and down slated palpebral fissures, and overall low physical measurements. Family history includes learning disability for maternal uncle and low overall physical measurements for paternal great-uncle. The diagnostic testing for this subject included, karyotyping that showed 47,XY,+mar[16]/46,XY[14], FISH that confirmed the marker as chromosome 17 material (Figure 3 a and b).
The CMA detected, arr[hg19] 2q11.2(98,448,884-98,811,103)x3 mat, 15q11.2(22,770,421-23,620,154)x3 pat, 17q25.1q25.3(72,832,054-76,221,428)x3 dn, approximately 362 kb, 850 kb, and 3389 kb in size, respectively, defining the region chromosome 17 that constitutes the marker chromosome (Figure 3c). The large duplication on 17q harbored the SEPTN9 gene, that might be associated with the physical features and most likely the speech delay, expressive language disorder (focal paresis), while the EXOC7 gene deletion might be associated with learning disability. Given, the parents were unaffected, there is an increased likelihood that the 17q25.1q25.3 duplication contributes at least in part to the phenotypes observed in the patient.
Another patient (with suspected stickler syndrome was found to have arr[hg19] 17q25.3(75,346,425-75,403,493)x3, approximately 57 kb in size, duplicating a single exon of SEPTN9 gene.
Two cases with B3GNTL1 gene duplication
Case 9: The subject was diagnosed with ASD, intellectual disability, and expressive language disorder. She has a medical history of delayed development, speech delay, and was born premature at 27 weeks. Notable physical features include a Café au lait spot. She has family history of a maternal and paternal cousin with ASD. She had a normal 46,XX constitution on evaluation with karyotyping, and normal whole exome sequencing (ES) results. The CMA detected, arr[hg19] 17q25.3(809,76,346-81,041,938)x3 mat, 19q13.41(52,940,574-53,158,834)x3 mat, approximately, 66 kbp and 218 kbp in size. The duplication on 17q harbored the B3GNTL1 and METRNL genes.
Another, subject (case 10) was diagnosed with cerebral palsy, hypertonia, and spasticity. He has a medical history of delayed development, delayed milestones, and muscle spasms. He also has behavioral concerns of OCD-like behaviors. Notable physical features include an upturned nasal tip, Café au lait spot on lower anterior neck, the fifth fingers are shortened bilaterally, and progressively dystonic features. The subject’s previous testing was found to be normal for organic acids, total and free creatinine, amino acids, and acylcarnitine profile. The ES analysis detected a heterozygous missense variant (ADARc.3019G>A) that was found to be associated with Dyschromatosis symmetrica hereditaria, AD, but did not explain the observed phenotype. The CMA detected, arr[hg19] 6q24.1(141,957,503-142,116,632)x1mat, 17q25.3(80,864,260-81,007,175)x3mat, approximately 159 kbp and 143 kbp in size. The duplication of 17q region harbored TBCD and B3GNTL1 genes.
It is interesting to note that Prost el al. reported two cases of focal dosage changes including TBCD and B3GNTL1 genes (one case each with loss and gain), sparring the distal METRNL gene. Both reported cases had developmental delays and no cardiac malformation, as observed in our cases. Further, all three cases with duplication of TBCD and B3GNTL1 genes show facial dysmorphology. Thus, dosage changes involving these two genes appears to be associated with developmental delay, facial dymorphology and no cardiac malformation. The two cases reported in literature were de novo, while both our cases harbored CNVs that were maternally inherited. The mother was unaffected, supporting incomplete penetrance and variable expressivity for this change.
Limitations of the study
Though the study presents a set of cases with rare CNVs of the same cytogenetic band (17q25.3), there was no smallest region of overlap, which makes it difficult to ascertain definitive genotype-phenotype association. However, since the incidence of CNVs of 17q25.3 region are rare, even in the disease population, and given that this is a gene-rich region of the genome, documentation of these cases is extremely important and may lead to definitive genotype-phenotype association in the future. Importantly, the cases 1 to 7 are the most informative, as these do not harbor any other CNV (and in some cases no sequencing alteration) suggesting a causal role of these CNVs/genes. Further, though the remaining cases (8 to 15) have additional CNVs, which complicates association, all are of uncertain significance, and encourage further analysis of CNVs in the 17q25.3 region, given the rarity and a broader phenotype association of genes in this region. Finally, evaluating the mechanism was beyond the scope of this work, but these gene associations warrant further investigation of these genes in the future.