Genome-wide identification of disease-causing copy number variations in 450 individuals with anorectal malformations

CNV analysis

After the application of quality filter step I (gender mismatch), two patients were excluded. Through step II (call rate <98%), 40 patients were discarded and after step III (exceeded double of standard deviation), 12 more patients were excluded. In the remaining 396 individuals with ARM, a total of 6316 CNVs, were called. Within the remaining 4066 controls, a total of 43,323 CNVs were called. Regarding the quality filter for each CNV, filter steps (IV) and (V) led to the exclusion of 5026 CNVs among ARM individuals and the exclusion of 32,075 CNVs among healthy control individuals. Comparison of frequencies in cases and controls (VI) excluded additional 887 CNVs. In this filter step, we identified three individuals with trisomy 21 (not further mentioned). Application of filter steps VII and VIII filtered out 117 microdeletions and 35 microduplications. Filter step IX yielded a final count of 82 microdeletions and 49 microduplications. According to the annotation of the respective CNVs in gnomAD (gnomad.broadinstitute.org), OMIM (www.omim.org), UCSC (genome.ucsc.edu), MGI (www.informatics.jax.org), and PubMed (pubmed.ncbi.nlm.nih.gov), we ultimately prioritized and qPCR confirmed four microscopic chromosomal anomalies and nine submicroscopic CNVs comprising seven microdeletions (del2p13.2, del4p16.2, del7q31.33, del9p24.1, del16q12.1, del18q32, del22q11.21) and two microduplications (dup2p13.2, dup17q12).

Unbalanced chromosomal translocations

Two affected individuals carried unbalanced terminal translocations including 46,XX,der(11)t(11;16)(q24.2;q22.2) and 46,XX,der(22)t(22;9)(q11.21;pter) (Table 1).

Table 1 Unbalanced chromosomal translocations and microscopic chromosomal de novo duplications.

Individual 1 presented with an ARM in form of a perineal fistula, congenital intraventricular hemorrhage with hydrocephalus, bilateral clubfoot, dysplastic pelvic kidney (left), bilateral atresia of submandibular gland, left sided aplasia of the thyroid gland, choanal atresia, hearing disability, and macular delocalization. Her chromosomal anomaly comprised an unbalanced translocation 46,XX,der(11)t(11;16)(q24.2;q22.2) with terminal deletion 11q24.2-qter. The first deleted SNP is located at chromosomal position chr11:127.078.525 and the last deleted SNP at chr11:134.923.456. Deletion of 11q23.3-qter has been associated with Jacobsen syndrome (JBS, MIM #147791). JBS is a contiguous gene deletion syndrome involving terminal chromosome 11q. Key phenotypic features are pre- and postnatal growth retardation, intellectual disability (ID), characteristic facial dysmorphism, thrombo- or pancytopenia. Further clinical features are congenital heart defects (CHD), congenital anomalies of the kidneys and urinary tract (CAKUT), gastrointestinal tract, genitalia, central nervous system (CNS) anomalies and/or skeleton. Previously, Mattina et al. reported gastrointestinal tract malformations in 18–25% of individuals with JBS including pyloric stenosis, ARM, and less frequently, duodenal atresia, annular pancreas, or gut malrotation [15]. Besides terminal deletion 11q24.2-qter Individual 1 carried also an 18 Mb duplication of chromosomal region 16q22.2-qter. The first duplicated SNP is located at chromosomal position chr16:71.956.505, the last duplicated SNP at chromosomal position chr16:90.161.959. Key features described for partial trisomy 16qter (ORPHA:96106) include low birth weight, failure to thrive, hypotonia, ID, CHD, limb anomalies and joint contractures, facial dysmorphism with a high prominent forehead, down slanting and small palpebral fissures, hypertelorism, periorbital edema, low-set and abnormal ears, prominent nose, micrognathia, CAKUT, genital anomalies, and ARM. Interestingly, several reports in the literature associated dup16pterqter with ARM [16].

Individual 2 presented with ARM in form of a perineal fistula, kidney hypoplasia (left), bilateral hydronephrosis, persistent urogenital sinus, and small for gestational age. Her chromosomal anomaly comprised an unbalanced translocation with a 2.5 Mb deletion of chromosomal region 22q11.21. The first deleted SNP was located at chromosomal position chr22:18.875.445 and the last deleted SNP was located at chromosomal position chr22:21.461.607. The 22q11.2 deletion syndrome is the most common microdeletion syndrome. It is not surprising that several cases of ARM have been described among patients with this deletion [16] (see below Individual 14). Individual 2 also carried a 38.8 Mb duplication of 9p. The first duplicated SNP was located at chromosomal position chr9:209.325, the last duplicated SNP was located at chromosomal position chr9:39.021.035. Common features of a 9p trisomy include ID, craniofacial dysmorphisms, skeletal alterations, CNS anomalies, CHD, and less common CAKUT. While ARM has not been associated with trisomy 9p, several reports in the literature mention ARM in association with mosaic or non-mosaic complete trisomy 9 [16].

Microscopic chromosomal de novo duplications

Two affected individuals carried each a terminal duplication including 46,XXdup(3)(q26.31-q29) and 46,XXdup(17)(q25.3-qter) (Table 1).

Individual 3 presented with an isolated ARM. It is uncertain if he presented with additional minor stigmata. His chromosomal anomaly comprised a 25.1 Mb duplication of chromosome 3q26.31-q29 (Fig. 1a). The first duplicated SNP was located at chromosomal position chr3:171.615.261, the last at chromosomal position chr3:196.805.528. Common features of 3q duplication syndrome are a hirsutism, synophrys, broad nasal root, anteverted nares, downturned corners of the mouth, malformed ears, CHD, CAKUT and genital anomalies, ID, and growth retardation. To the best of our knowledge, this is the first report of duplication 3q26.31-q29 in an individual with ARM.

Fig. 1
figure 1

CNVs harboring putative ARM candidate genes.

Individual 4 presented with an isolated ARM in form of a perineal fistula. He did not present with any other congenital anomaly or stigmata. His chromosomal anomaly comprised an 0.48 Mb duplication of 17q25.3-qter (Fig. 1b). The first duplicated SNP was located at chromosomal position chr17:80.545.076, the last at chromosomal position chr17:81.033.874. To the best of our knowledge, this is the first report of duplication of 17q25.3-qter in an individual with ARM.

Rare inherited CNVs and CNVs of unknown inheritance

Six affected individuals carried very rare inherited submicroscopic CNVs (Table 2). All of these CNVs were absent in 4066 healthy inhouse controls (CNV frequency <0.0003) and DGV.

Table 2 Rare inherited CNVs and CNVs of unknown inheritance.

Individual 5 presented with an isolated ARM in form of a recto-vestibular fistula. Besides her ARM, she had no co-occurring anomalies. She carried a maternally inherited 316.074 bp deletion of chromosomal region 2p13.2. The first deleted SNP was located on chromosomal position Chr2:72.623.204, the last at chromosomal position Chr2:72.939.279. Within this deletion resides the developmental gene EXOC6B. EXOC6B has been associated with autosomal-recessive inherited spondyloepimetaphyseal dysplasia with joint laxity, type 3 (MIM #618395).

Individuals 6 and 7 are brothers who both presented with ARM in form of a perineal fistula. In addition, Individual 6 presented with a testicular appendage and right-sided deafness. His brother presented with additional epispadias. Both carried a maternally inherited 132,652 bp duplication of chromosomal region 2p13.2. The first duplicated SNP was located at chromosomal position Chr2:72.882.934, the last at chromosomal position Chr2:73.015.587. Within this duplication resides the above-mentioned developmental gene EXOC6B. While the above EXOC6B-associated spondyloepimetaphyseal dysplasia with joint laxity, type 3 does not present with any visceral organ affection, the findings in individual 5 and in the two brothers 6 and 7 suggests a possible involvement of EXOC6B or the respective genomic region in ARM formation.

Individual 8 presented with ARM in form of a rectoprostatic fistula, aganglionosis of the Meissner’s plexus (submucosa, M. Hirschsprung) and craniofacial Goldenhar syndrome. He carried a paternally inherited 265.097 bp deletion of chromosomal region 4p16.2. The first deleted SNP was located at chromosomal position Chr4:5.162.593, the last SNP at chromosomal position Chr4:5.427.691. Within this deletion resides the developmental gene STK32B, which has been associated with Ellis-van Creveld syndrome, an autosomal-recessive skeletal dysplasia with co-occurring genital anomalies, e.g., hypospadias.

Individual 9 presented with an unclassified ARM as part of her VATER/VACTERL association, with further phenotypic data missing. She carried a paternally inherited 47,083 bp deletion of chromosomal region 7q31.33. The first deleted SNP was located at chromosomal position Chr7:126.392.369, the last at chromosomal position Chr7:126.439.453. Within this deletion resides the developmental gene GRM8, so far associated with any human disease.

Individual 10 presented with ARM in form of a perineal fistula. He carried a 181.925 bp maternally inherited duplication of chromosomal region 17q1. The first duplicated SNP was located at chromosomal position Chr17:33.786.176, the last duplication at chromosomal position Chr17:33.968.102. Within this duplication reside five coding genes comprising AP2B, PEX12, SLFN12L, SLFN14, and SNORD7 and two long non-coding RNAs LINC02001 and LOC107985033. Of the coding genes, PEX12 has been associated with the autosomal-recessive Peroxisome biogenesis disorder 3A (Zellweger, MIM #614859). SLFN14 has been associated with the autosomal-dominant “Bleeding disorder, platelet-type, 20 (MIM #616913)”.

Individual 11 presented with ARM in form of a recto-vesical fistula as part of his VATER/VACTERL association. He presented with additional CHD, right-sided kidney agenesis, and cryptorchidism. His chromosomal anomaly comprised a 27.823 bp deletion of chromosomal region 9q24.1 (Table 2). The first deleted SNP was located on chromosomal position Chr9:49.73.080, the last at chromosomal position Chr9:5.000.904. Here, we had no parental samples available for testing and can therefore not conclude, if the CNV has occurred de novo or has been transmitted from a healthy parent. Within the deletion resides the developmental gene JAK2, a gene that has been associated with broad spectrum of human hematologic malignancies (MIM *147796) but not with congenital visceral anomalies.

Rare submicroscopic de novo CNVs

In total we detected and confirmed three submicroscopic de novo CNVs (Table 3).

Table 3 Patients with de novo CNVs.

Individual 12 presented with an ARM in form of a perineal fistula and right-sided auricular hypoplasia. She carried a 1.38 Mb de novo deletion of chromosomal region 16q12.1. The first deleted SNP was located at chromosomal position chr16:50.135.837, the last at chromosomal position chr16:51.522.044. Within the deleted region reside nine coding genes including ADCY7, HEATR3, PAPD5, BRD7, CYLD, NKD1, NOD2, SNX20, and SALL1. Furthermore, the deletion comprised two microRNAs (MIR6771 and MIR3181) and four long non-coding RNAs (LINC02127, LINC02168, LINC02178, LOC101927272).

Individual 13 presented with ARM in form of a rectoprostatic fistula, duplicated kidney with megaureter, and right-sided maldescensus testis. He carried a 0.81 Mb de novo deletion of chromosomal region 18q32 (Fig. 1c). The first deleted SNP was located at chromosomal position chr18:76.501.085, the last at chromosomal position 18:77.373.296. Within the deletion reside three coding genes comprising SALL3, ATP9B, and NFATC1.

Individual 14 presented with an ARM in form of a perineal fistula. In addition, she presented with patent ductus arteriosus, ventricular septal defect and pulmonary valve stenosis. Furthermore, she had luxation of the arytenoid cartilages and an abdominal wall hernia. Her chromosomal anomaly comprised an isolated 2.58 Mb deletion of 22q11.21 (see above Individual 2). The first deleted SNP was located on chromosomal position Chr22:18.875.445, the last at chromosomal position Chr22:21.461.607. DECIPHER database (DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources) [17] lists 1267 CNVs of chromosomal region 22q11.2. These comprise 667 heterozygous deletions. Within these 667 deletions are 14 deletions that have been described in association with ARM. The smallest region of overlap of all these deletions comprises 1.39 Mb. Here, further genetic studies are warranted to prioritize a possible ARM associated human disease-gene.

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