Identification of a Single Exon Deletion Using NGS in a Patient with Perlman Syndrome

reprinted from december 2019

pathology ◆ laboratory medicine ◆ laboratory management Identification of a single exon deletion using NGS in a patient with Perlman syndrome CAP TODAY and the Association for nephromegaly, hydronephrosis with by PCR specifically targeting DIS3L2 Molecular Pathology have teamed up megaureters, bilateral diffuse nephro- exon 9 using exon 6 as a positive PCR to bring molecular case reports to CAP blastomatosis, and renal dysplastic control (Fig. 2B, next page). Precise TODAY readers. AMP members write changes (Fig. 1). Also noted was or- breakpoints could not be determined the reports using clinical cases from their ganomegaly of the liver, spleen, and due to the nature of the targeted assay own practices that show molecular test- thymus. The lungs showed mild pul- used. Homozygous deletions of exon ing’s important role monary hypoplasia 9 in DIS3L2 have been reported in in diagnosis, prog- nosis, and treat- with diffuse hyaline multiple individuals from at least four 2-4 ment. The following membrane disease. families with Perlman syndrome report comes from A skin biopsy was and are listed in DECIPHER (#318416, Children’s Hospital case report sent to our labora- https://decipher.sanger.ac.uk/). The deletion of Philadelphia and tory for genetic test- of exon 9 is predicted to result in an Driscoll Children’s Hospital, Corpus ing with suspicion of Perlman syn- in-frame mRNA product, and it may Christi, Tex. If you would like to submit drome (mutation in DIS3L2 gene) or be mediated by nonallelic homolo- a case report, please send an email to the other potential causes for diseases gous recombination between LINE-1 AMP at [email protected]. For more informa- with elevated cancer risk in an over- repeats located in the introns.3 The tion about the AMP and all previously grown fetus, such as Simpson-Golabi- identification of theDIS3L2 exon 9 published case reports, visit www.amp.org. Behmel syndrome (GPC3). For these Jinhua Wu, PhD; Jeffrey Schubert, PhD reasons, a comprehensive hereditary A Xiaonan Zhao, PhD; Elizabeth Fanning, MS cancer panel of 130 genes (including Zhiqian Fan, MS; Lisa Sutton, MD DIS3L2) was performed. Marilyn M. Li, MD The comprehensive hereditary We report a case of a 34-week-gesta- cancer panel did not identify a patho- tion male infant with prenatal over- genic or likely pathogenic variant growth born to a 19-year-old G1P0 according to the American College mother. No significant family history of Medical Genetics and Genomics/ or consanguinity were reported. The Association for Molecular Pathology infant was delivered emergently by germline variant classification guide- B cesarean section due to pregnancy- lines,1 and no variants were observed induced hypertension and polyhy- in the DIS3L2 gene. However, ana- dramnios. At birth, the infant was lysis of regions with low sequence noted to have anasarca and ascites coverage on the panel discovered no with a massively distended abdomen sequence reads aligned to the genomic and enlarged kidneys. Despite resus- region of chr2:233028163-233028348, citative efforts, the infant expired at corresponding to exon 9 of DIS3L2 one day of life. A complete autopsy (NM_152383.4), indicating a homo- demonstrated clinical and histologic zygous deletion of DIS3L2 exon 9 Fig. 1. Renal histology from patient’s autopsy. features suggestive of Perlman syn- as visualized using Integrative Ge- A. Disorganized renal parenchyma and diffuse drome. There were significant urinary nomics Viewer (IGV) (Fig. 2A, next nephroblastomatosis (20 ×). B. Higher power view tract abnormalities, including massive page). The deletion was confirmed of a small foci of blastema (40 ×). 2 | CAP TODAY homozygous deletion is diagnostic genes as can NGS-based tests. Astuti, et al., established a molecu- of Perlman syndrome for the patient. Perlman syndrome, first described lar basis for Perlman syndrome in Next-generation sequencing tech- in the 1970s,5-7 is a rare, autosomal re- 20122 by identifying regions of homo- nology allows interrogation of mul- cessive overgrowth syndrome that can zygosity in consanguineous Perlman tiple genes in parallel to evaluate present prenatally with fetal macroso- syndrome families. With further ge- multiple variant types, including mia and polyhydramnios. Patients netic mapping, germline homozygous SNVs, small indels, exonic dele- also show characteristic dysmorphic deletions of DIS3L2 exons were found tions/duplications, and gross copy facial features, including deep-set in multiple consanguineous families, number changes, as well as fusions eyes, low-set ears, inverted V-shaped and additional biallelic DIS3L2 single and alternative splicing, through a upper lip, prominent forehead, and a nucleotide variants, including splice- single test, using DNA and RNA. broad, flat nasal bridge. There is a high site and missense mutations, were PCR was used to confirm the exon 9 rate of neonatal mortality in patients found to segregate with disease in homozygous deletion in this case with Perlman syndrome. Those who non-consanguineous patients. The since we knew what we were look- do survive commonly exhibit devel- authors further demonstrated through ing for; however, this method would opmental delay and renal abnormali- functional in vitro studies that recom- have had limited utility as a first-line ties and are at a high risk for develop- binant DIS3L2 protein with exon 9 test because it cannot detect the same ing nephroblastomatosis and Wilms homozygous deletion displayed sub- spectrum of variants in as many tumor at an early age.8,9 stantially reduced ribonuclease activ-

Fig. 2. Next-generation sequencing and PCR results showing homozygous deletion of DIS3L2 exon 9. A. Upper panel: screenshot from Integrative Genomics Viewer showing DIS3L2 exons 8, 9, and 10 of the patient’s sequencing data compared with a normal control. Sequence reads (red and blue boxes) are seen as expected in exons 8 and 10 of DIS3L2 in the patient, but no reads for exon 9 are present. Lower panel: zoom-in screenshot of exons 8, 9, and 10 of DIS3L2 in the patient compared with a normal control. B. PCR confirmation visualized by TapeStation: A1, ladder; B1, patient’s sample DIS3L2 exon 9 (no band); C1, control sample exon 9; D1, patient’s DIS3L2 exon 6 (patient intragenic control); E1, control sample exon 6; F1, blank. CAP TODAY | 3 ity compared with wild-type DIS3L2. hereditary cancer panel in a patient Stolte-Dijkstra I, van Essen AJ. Perlman syn- Additionally, loss of this gene in cell who died soon after birth. This result drome: four additional cases and review. Am lines led to increased rates of aneu- not only provides a molecular diagno- J Med Genet. 1999;86(5):439–446. ploidy, errors in mitosis, and abnormal sis of Perlman syndrome but also 5. Liban E, Kozenitzky IL. Metanephric ham- expression of mitotic proteins. carries important implications for the artomas and nephroblastomatosis in siblings. Cancer. 1970;25(4):885–888. In this case, a confirmed molecular family in its decision-making for fu- 6. Perlman M, Goldberg GM, Bar-Ziv J, Da- diagnosis of Perlman syndrome has ture pregnancies. Our experience novitch G. Renal hamartomas and nephro- two primary impacts on patient care. demonstrates that an NGS-based as- blastomatosis with fetal gigantism: a familial First, in a situation of a neonatal death, say can detect copy number variants syndrome. J Pediatr. 1973;83(3):414–418. a definitive diagnosis can help bring at the single exon level through use of 7. Perlman M, Levin M, Wittels B. Syndrome closure to the pregnancy for this fam- a carefully designed bioinformatics of fetal gigantism, renal hamartomas, and ily by providing a direct answer to pipeline and thorough sequence data nephroblastomatosis with Wilms’ tumor. Can- cer. 1975;35(4):1212–1217. what caused their loss. Second, and investigation. 8. Alessandri JL, Cuillier F, Ramful D, most significantly, this diagnosis has 1. Richards S, Aziz N, Bale S, et al. Standards et al. Perlman syndrome: report, prenatal important clinical implications for this and guidelines for the interpretation of se- findings and review. Am J Med Genet A. couple’s family planning and future quence variants: a joint consensus recom- 2008;146A(19):2532–2537. pregnancies. As part of the report is- mendation of the American College of Medi- 9. Perlman EJ. Pediatric renal tumors: practi- sued in this case, we recommended cal Genetics and Genomics and the Asso- cal updates for the pathologist. Pediatr Dev ciation for Molecular Pathology. Genet Med. Pathol. 2005;8(3):320–338. genetic testing for this family to aid in 2015;17(5):405–424. the assessment of recurrence risk. If 2. Astuti D, Morris MR, Cooper WN, et al. both parents are found to be asymp- Germline mutations in DIS3L2 cause the Dr. Wu and Dr. Schubert are clinical ge- tomatic carriers for heterozygous Perlman syndrome of overgrowth and nomic scientists, Dr. Zhao is genomic sec- pathogenic variants in DIS3L2, there Wilms tumor susceptibility. Nat Genet. 2012; tion lead scientist, Fanning is NGS analyst, is a 25 percent chance of their having 44(3):277–284. Fan is clinical laboratory section supervisor, another child with Perlman syndrome 3. Higashimoto K, Maeda T, Okada J, et al. and Dr. Li is vice chief—all in the Division of Genomic Diagnostics, Department of and a 50 percent chance of their hav- Homozygous deletion of DIS3L2 exon 9 due to non-allelic homologous recombina- Pathology and Laboratory Medicine, Chil- ing a child who is a carrier. tion between LINE-1s in a Japanese patient dren’s Hospital of Philadelphia. Dr. Sutton In sum, we identified a homozy- with Perlman syndrome. Eur J Hum Genet. is a pediatric pathologist, Department of Pa- gous deletion of a single exon of 2013;21(11):1316–1319. thology and Laboratory Medicine, Driscoll DIS3L2 gene using an NGS-based 4. Henneveld HT, van Lingen RA, Hamel BC, Children’s Hospital, Corpus Christi, Tex.

Test yourself Here are three questions taken from the case report. Answers are online now at www.amp.org/casereports and will be published next month in CAP TODAY. 1. What sequencing technology was used in this case report? What somal recessive manner that is associated with Wilms tumor susceptibility. types of variants can be detected by this method? DIS3L2. a. Next-generation sequencing. It detects single nucleotide variants, small dele- d. Perlman syndrome is a genetic disorder due to loss of function of specific tions and insertions, and copy number variation (DNA), and it can also detect genes. In newborns, symptoms include weak muscles, poor feeding, and slow alternative splicing and gene fusions (RNA). development. RET. b. Sanger sequencing. It detects single nucleotide variants, small deletions and 3. insertions, and copy number variations. What is the inheritance pattern of Perlman syndrome? What is c. Third-generation sequencing using Nanopore technology. It detects single the recurrence risk of having another child with Perlman syndrome nucleotide variants and small deletions and insertions. It does not detect copy in this family? a. It follows an autosomal recessive pattern of inheritance. Therefore, there is a number variations. 25 percent chance of the couple having another child with Perlman syndrome d. Sanger sequencing. It detects single nucleotide variants only. and a 50 percent chance of having a child who is a carrier. 2. What is Perlman syndrome? A mutation in what gene causes b. It follows an autosomal dominant pattern of inheritance. Since only the proband this disease? was tested in this family, parents should be tested for this variant as well to a. Perlman syndrome is a congenital overgrowth syndrome inherited in an auto- determine whether this variant is inherited or de novo. somal recessive manner that is associated with Wilms tumor susceptibility. c. It follows an autosomal recessive or autosomal dominant pattern of inheritance. DIS3L1 and DIS3L2. The risk for another child with Perlman syndrome is 100 percent for this b. Perlman syndrome is a genetic disorder due to loss of function of specific family. genes. In newborns, symptoms include weak muscles, poor feeding, and slow d. It follows an autosomal recessive pattern of inheritance. Since only the proband development. DIS3L2. was tested in this family, parents should be tested for this variant as well to c. Perlman syndrome is a congenital overgrowth syndrome inherited in an auto- determine if both parents are a carrier of this deletion.