Clin Genet 2014 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12459

Short Report

Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population Fedick AM, Shi L, Jalas C, Treff NR, Ekstein J, Kornreich R, Edelmann L, Mehta L, Savage SA. Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population. Clin Genet 2014. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014

A.M. Fedicka,b , L. Shic , C. Jalasd , N.R. Treffa,b , J. Eksteine , R. Kornreichc , L. Edelmannc , L. Mehtac and S.A. Savagef

Hoyeraal–Hreidarsson syndrome (HH) is a clinically severe variant of dyskeratosis congenita (DC), characterized by cerebellar hypoplasia, microcephaly, intrauterine growth retardation, and severe immunodeficiency in addition to features of DC. Germline mutations in the RTEL1 gene have recently been identified as causative of HH. In this study, the carrier frequency for five RTEL1 mutations that occurred in individuals of Ashkenazi Jewish descent was investigated in order to advise on including them in existing clinical mutation panels for this population. Our screening showed that the carrier frequency for c.3791G>A (p.R1264H) was higher than expected, 1% in the Ashkenazi Orthodox and 0.45% in the general Ashkenazi Jewish population. Haplotype analyses suggested the presence of a common founder. We recommend that the c.3791G>A RTEL1 mutation be considered for inclusion in carrier screening panels in the Ashkenazi population.

a Department of Microbiology and Molecular Genetics, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, USA, b Reproductive Medicine Associates of New Jersey, Basking Ridge, NJ, USA, c Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, d Bonei Olam, Center for Rare Jewish Genetic Disorders, Brooklyn, NY, USA, e Dor Yeshorim, The Committee for Prevention of Jewish Diseases, Brooklyn, NY, USA, and f Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD, USA

Conflict of interest

Key words: Ashkenazi Jewish – carrier screening – dyskeratosis congenita – Hoyeraal–Hreidarsson syndrome – RTEL1

The authors declare no conflicts of interest.

Corresponding author: Sharon A. Savage, MD, FAAP, Chief, Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 9609 Medical Center Dr., Rm. 6E456, MSC 9772, Rockville, MD 20850, USA. Tel.: +1 240 2767241; fax: +1 240 2767836; e-mail: [email protected] Received 23 May 2014, revised and accepted for publication 14 July 2014

Hoyeraal–Hreidarsson syndrome (HH) is a severe variant of dyskeratosis congenita (DC, MIM#615190), a cancer-prone inherited bone marrow failure syndrome caused by aberrant telomere biology (1). The triad of nail dysplasia, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC but variable in onset. Patients with DC may have other medical problems, including pulmonary fibrosis, liver abnormalities, and stenosis

of the esophagus, lacrimal ducts, and/or urethra. In addition to DC-associated phenotypes, individuals with HH have microcephaly, cerebellar hypoplasia, severe immunodeficiency and intrauterine growth retardation (IUGR) (1). Several different mutations occurring in both homozygous and compound heterozygous states in RTEL1 (regulator of telomere elongation helicase 1, MIM

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Fedick et al. #608833) have been identified in HH and DC (2–6). RTEL1 is an evolutionarily conserved DNA helicase that contributes to telomere maintenance, DNA replication, double stranded break repair, and genomic stability (7–10). The dysfunction of RTEL1 due to germline mutations is associated with shortened telomeres and DC/HH phenotype. Several of the reported RTEL1-associated HH cases were of Ashkenazi Jewish (AJ) descent. Two were homozygous for c.3791G>A mutation (splice variant uc021wge.1, p.R1264H) (6), and one was compound heterozygous for c.1548G>T (p.M516I) and c.2992C>T (p.R998*) (2, 3). The common ancestry shared among these cases suggests that certain RTEL1 mutations may be more prevalent in this population. Therefore, we performed population screening of these mutations in two different healthy AJ populations.

cycles at 95∘ C for 15 s and 60∘ C for 1 min. Allelic discrimination was performed on the 7900s and data were analyzed using TaqMan Genotyper v1.2 software, with samples used in the initial validation included as controls. If samples did not amplify, they were not included in the carrier frequency calculation. The Wilson score interval was used to calculate confidence intervals (CI) for carrier frequencies. Sanger sequencing was performed on samples with the mutant alleles to confirm the genotypes (GENEWIZ, South Plainfield, NJ). Owing to the high number of heterozygous carriers found for the c.3791G>A mutation, DNA fingerprinting using 40 highly polymorphic TaqMan SNP assays was performed to ensure that none of the anonymous samples were from related individuals or duplicated. Sequenom MassARRAY genotyping

Materials and methods Study participants

The samples from Orthodox AJ adults used in this study were obtained with written consent from self-identified AJs enrolled in the carrier testing Dor Yeshorim program for use in research (11). Consent form information included that patient material would be used for clinical testing and that excess material would be de-identified and used for research purposes to characterize single gene disorders in the AJ population. The samples from self-identified AJ individuals used in the second carrier frequency experiment were obtained from the Mount Sinai Genetic Testing Laboratory. These samples were obtained in conjunction with clinical testing from patients who consented to having their samples anonymized and used for research studies [45 CFR part 46.101(b)(4)]. TaqMan genotyping

The initial carrier frequency study was performed on 1032–1048 samples from the Dor Yeshorim program for the c.3791G>A, c.1548G>T, c.2992C>T, and c.2288G>T mutations (Table 1). Alternative nomenclature based on different RTEL1 splice variants for these mutations are listed in the footnotes of Table 1. TaqMan genotyping assays were designed for these mutations using File Builder software [Life Technologies (LTI), Carlsbad, CA], with VIC and FAM labeled probes for the detection of the normal and mutant alleles, respectively (Table S1, Supporting Information). A synthetic control was made for the c.2288G>T mutation by creating a gBlock (Integrated DNA Technologies, Coralville, IA), which was added at a 1:1 ratio with DNA from an individual known to possess the major allele for the mutation site to create a heterozygous control as DNA was unavailable. The DNA samples were not normalized prior to plating, but quantities fell within the suggested range of 1–20 ng. Samples were run on the GeneAmp® PCR System 9700 (LTI) at the following setting: holds at 50∘ C for 2 min and 95 ∘ C for 10 min, and then 40

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Targeted genotyping for the c.1548G>T, c.2288G>T, c.2941C>T, c.2992C>T, and c.3791G>A mutations was performed on a Sequenom MassARRAY system [Sequenom Inc. (SI), San Diego, CA] by using Sequenom iPLEX Pro chemistry and Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. Assay sequences were designed by MassARRAY Typer Assay Editor v4.0 (SI) (Table S2). DNA samples from 2240 adult individuals of self-reported AJ ancestry were normalized to 5–20 ng/μl and multiplex polymerase chain reaction (PCR) was carried out for each sample on a GeneAmp PCR System 9700 (LTI) at the following settings: a hold at 95∘ C for 2 min, and then 45 cycles at 95∘ C for 30 s, 56∘ C for 30 s, and 72∘ C for 1 min. After PCR, amplicons were treated with shrimp alkaline phosphatase by incubating at 37∘ C for 30 min. A single base extension reaction was performed for each sample at the following settings: 95∘ C for 30 s, then 40 cycles consisting of a hold at 95∘ C for 5 s followed by five cycles at 52∘ C for 5 s and 80∘ C for 5 s, and then a hold at 72∘ C for 3 min. Allelic discrimination was performed by Sequenom Typer Viewer v4.0 (SI). Samples genotyped as heterozygous were confirmed through Sanger sequencing. The Wilson score interval was used to calculate CI for carrier frequencies (12). Haplotyping

Fourteen single nucleotide polymorphisms (SNPs) previously used to characterize the c.3791G>A mutation were genotyped to perform haplotype analyses (6). Both commercially available and custom designed TaqMan genotyping assays were used. Genotyping was performed for the 20 heterozygous carrier samples identified in both sample groups, as well as 1 homozygous affected control and 100 random anonymous AJ samples that had not been previously genotyped. Quantitative PCR (qPCR) and allelic discrimination were performed following the same TaqMan settings described above. Data were analyzed using TaqMan Genotyper v1.2 software. Genotyping results for each SNP site were manually compared

p.M516I p.R998* p.G763V p.R981W p.R1264H

Protein

Chr20: 62319118 Chr20: 62324564 Chr20: 62321514 Chr20: 62324513 Chr20: 62326972

1037 1045 1048 N/A 1032

2 0 0 N/A 10

No. of No. of heterozygous wild type carriers 0.192% (0.05–0.70%) 0.00 0.00 N/A 0.960% (0.52–1.76%)

Carrier frequency (95% CI) 2239 2237 2229 2237 2227

1 0 0 0 10

No. of No. of heterozygous wild type carriers

0.045% (0.01–0.24%) 0.00 0.00 0.00 0.447% (0.25–0.82%)

Carrier frequency (95% CI)

Mount Sinai Genetic Testing Laboratory samples, general AJ population

9629 9630 9630 9630 9629

1d 0 0 0 1g

0.01% 0 0 0 0.01%

No. of No. of heterozygous Carrier wild type carriers frequency

Public databasesa

*, stop codon; AJ, Ashkenazi Jewish; CI, confidence interval; No., number; N/A, not assessed. a Data combined from genotypes present in dbSNP, 1000 Genomes (2500 individuals, http://www.1000genomes.org/home) and ESP (6468 individuals, http://evs.gs.washington. edu/EVS/), and ClinSeq population (http://genome.gov/20519355). b Reference sequence for nomenclature is NM_032957. c c.1548G>T (p.M516I) has been reported as c.G1476T (p.M492I) based on alternative isoforms and NM_016434. d One heterozygous carrier of c.1548G>T is reported in the ESP dataset of 4286 individuals of European American ancestry. e c.2992C>T (p.R998*) has been reported as c.C2920T (p.R974*) based on alterative isoforms and NM_016434. f c.3791G>A is rs201540674 in the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) and based on NM_016434, isoform uc021wge.1. g One heterozygous carrier of c.3791G>A is reported in the ClinSeq dataset of 662 participants of European ancestry.

c.2992C>T c.2288G>T c.2941C>T c.3791G>Af

e

c.1548G>Tc

cDNAb

Genomic coordinate (GRCh37)

Dor Yeshorim samples, Orthodox AJ population

Table 1. RTEL1 variants genotyped in Ashkenazi Jewish populations compared with public databases

Carrier screening of RTEL1 mutations

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Fedick et al.

Fig. 1. Allelic discrimination plots for RTEL1 genotyping. (a) TaqMan allelic discrimination results. For all plots, the VIC probe is represented by the x-axis, and the FAM probe is represented by the y-axis. Water was used as the no template control (NTC). (b) Sequenom allelic discrimination results. For all plots, the low mass allele is represented by the x-axis, and the high mass allele is represented by the y-axis.

with the homozygous affected control sample to determine the at-risk haplotype.

Table 2. Shared haplotype for the c.3791G>A mutation in the Ashkenazi Jewish population

Results

Location

The heterozygous carrier frequency for the c.3791G>A mutation in the Dor Yeshorim AJ samples was 0.960% (95% CI 0.52–1.76%), based on the presence of 10 heterozygous carriers and 1032 wild-type samples (Table 1, Fig. 1a). The genotypes for all heterozygous carrier samples were 100% concurrent between the TaqMan and Sanger sequencing methods. Fingerprinting results indicated that all 10 heterozygous c.3791G>A mutations came from unrelated individuals (data not shown). The carrier frequency for the c.1548G>T mutation was 0.192% (95% CI 0.05–0.70%), based on the presence of two heterozygous carriers and 1037 wild-type samples. No carriers were detected for the c.2288G>T mutation (1048 samples tested) or the c.2992C>T mutation (1045 samples tested). A second carrier frequency experiment using the Sequenom MassARRAY was then conducted utilizing an additional 2240 samples from the general AJ population (Table 1, Fig. 1b). The carrier frequency for the c.3791G>A mutation in this population was 0.447% (95% CI 0.25–0.82%), based on the presence of 10 heterozygous and 2227 wild-type samples. The carrier frequency for the c.1548G>T mutation was 0.045% (95% CI 0.01–0.24%), based on the presence of 2239 wild-type samples and 1 heterozygous carrier. No carriers were detected for the c.2992C>T mutation (2237 samples tested), the c.2288G>T mutation (2229 samples tested), or the c.2941C>T mutation (2237 samples tested). We then investigated the haplotype of the c.3791G>A mutation to further evaluate the previously reported founder effect of this mutation. Our data suggests that all of the heterozygous samples shared a common haplotype

chr20:62293118 chr20:62294015 chr20:62305274 chr20:62309554 chr20:62319464 chr20:62319561 chr20:62319604 chr20:62320968 chr20:62321655 chr20:62322288 chr20:62324289 chr20:62326110 chr20:62326579 chr20:62327199

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rs number

SNP alleles

AJ haplotype allele

rs41308088 rs2297434 rs2297437 rs41309367 rs2738785 rs2777940 rs2777941 rs6062302 rs2236506 rs3208007 rs3848672 rs3208008 rs41309931 rs1056990

C/T C/T G/A C/T C/T C/G A/C C/T A/G C/T C/T C/A G/T A/G

C T G T C G C C A C C C G A

across 14 different SNP sites (Table 2), consistent with both the prior study (6) and the haplotype observed in the homozygous affected control. Subsequent genotype and haplotype determination of 100 additional c.3791G wild-type Ashkenazi samples indicated that this at-risk haplotype was present in 23% of this population. Discussion

We found a nearly 1% carrier frequency for the c.3791G>A RTEL1 (R1264H) mutation in the Orthodox AJ population and 0.45% in the general AJ population. Based on this, we estimate a 1 in 40,000 and a 1 in 197,530 incidence of homozygosity in these populations, respectively. In contrast, a carrier frequency of approximately 1 in 9600 is reported in publically available databases (dbSNP37, 1000 Genomes, and the NHLBI

Carrier screening of RTEL1 mutations Exome Sequencing Project), which suggests an approximately 1 in 100 million incidence of homozygosity in the general, non-AJ population (Table 1) (6). The higher than expected carrier frequency in AJ individuals raises the question of why there are not an equivalent number of reported cases of HH among the Ashkenazim. The reported cases with HH due to c.3791G>A homozygous RTEL1 mutations had significant unclassified immunodeficiencies and enteropathies that were not completely understood (6). The severity of the IUGR, cerebellar hypoplasia, immunodeficiency, enteropathy, and/or developmental delays suggests that either other patients with homozygous or compound heterozygous mutations may have died before the diagnosis of HH was made or that affected fetuses did not survive pregnancy. The haplotyping data suggested a common haplotype for this mutation in the AJ population, even though the haplotype was also seen in 23% of c.3791G wild-type samples from AJ individuals. It is possible that this mutation is on a common AJ ancestry haplotype or that the alleles are not in perfect linkage disequilibrium with the mutation. Trios were not available in our study to set phase, so the heterozygous alleles cannot be guaranteed to belong to a specific haploblock in both the carrier and wild-type samples. We also sought to understand four other RTEL1 mutations that may have occurred in the Ashkenazi population based on history and communication with the authors of the manuscripts (2, 3). The c.2288G>T mutation may or may not be an Ashkenazi mutation because it was identified to be compound heterozygous with the AJ founder mutation in a patient of unknown ethnicity (2). The c.2992C>T mutation was reported to occur in an individual of self-identified Ashkenazi ancestry. The c.2941C>T mutation was reported in compound heterozygosity in several individuals of unknown ethnicity. The fact that no carriers were found in over 1000 Ashkenazi individuals suggests that the c.2288G>T, c.2992C>T, and c.2941C>T mutations may not be common in AJ individuals or may be private mutations occurring only in reported families. Additionally, the individual with a compound heterozygous mutation of c.1548G>T was a self-identified Ashkenazi Jew and the carrier frequency of this mutation was very rare in this population (A mutation be included in mutation screening panels specific for the Ashkenazi population. This mutation meets the criteria for inclusion set by the American College of Medical Genetics (ACMG), which includes a 1% frequency, a well-understood disease history, and a significant morbidity rate in affected patients (13). Even though the c.1548G>T mutation has a frequency of only 0.192% in the AJ populations studied here, we believe that this mutation, and possibly other RTEL1 mutations, should also be considered for carrier screening since there is a chance of compound heterozygosity in this population. The advances made in high-throughput screening

programs (14) have made screening for additional and/or rare mutations possible and affordable, with many commercial labs already offering ethnic screening panels that extend beyond the scope of mutations specifically recommended by the ACMG and other such organizations (15). As the ACMG, along with the American College of Obstetricians and Gynecologists, has acknowledged that individuals may want to be screened for additional disorders, we suggest that information for all of the RTEL1 mutations be made available when receiving genetic counseling to aid patients in making informed decisions (16, 17). Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.

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