key: cord-0687810-07m2r8j1 authors: Gonenc, I. I.; Elcioglu, N. H.; Martinez Grijalva, C.; Aras, S.; Grossmann, N.; Praulich, I.; Altmueller, J.; Kaulfuss, S.; Li, Y.; Nuernberg, P.; Burfeind, P.; Yigit, G.; Wollnik, B. title: Phenotypic distinctions of BLM- and RMI1-associated Bloom syndrome date: 2021-11-10 journal: nan DOI: 10.1101/2021.11.02.21265560 sha: 7785d3a10aad93df65fb9dd7da6b75cdb78b49f7 doc_id: 687810 cord_uid: 07m2r8j1 Bloom syndrome (BS) is an autosomal recessive disease with characteristic clinical features of primary microcephaly, growth deficiency, skin lesions, cancer predisposition, and immunodeficiency. Here, we report the clinical and molecular findings of eight patients from six families diagnosed with BS. We identified causative mutations in all families, three different homozygous mutations in BLM and one causative homozygous mutation in RMI1. The homozygous c.581_582delTT (p.Phe194*) and c.3164G>C (p.Cys1055Ser) mutations in BLM have already been reported in BS patients, while the c.572_573delGA (p.Arg191Lysfs*4) is novel. Interestingly, whole-exome sequencing revealed a homozygous loss-of-function mutation in RMI1 in two BS patients of a consanguineous Turkish family. All BS patients had primary microcephaly, intrauterine growth delay, and short stature, presenting the phenotypic hallmarks of BS. However, a narrow face, skin lesions, and upper airway infections were observed only in some of the patients. Overall, patients with homozygous BLM mutations had a more severe BS phenotype compared to patients carrying the homozygous RMI1 mutation, especially in terms of immunodeficiency and associated recurrent infections. Low-level immunoglobulins were observed in all BLM-mutated patients, emphasizing the immunodeficiency profile of the disease, which should be considered as an important phenotypic characteristic of BS, especially in the current Covid-19 pandemic era. Bloom syndrome (BS, MIM 210900) is a rare congenital disorder first described as "congenital telangiectatic erythema resembling lupus erythematosus" 1 by the dermatologist David Bloom in 1954 2 . Clinical characteristics of the syndrome are primary microcephaly, growth deficiency, short stature, photosensitivity, cancer predisposition, and immunodeficiency 3 . BS is inherited in an autosomal recessive fashion 4 and is mainly caused by biallelic loss-of-function (LoF) mutations in the BLM gene 5, 6 . BLM is located on chromosome 15q26.1 and encodes one of the five human RecQ helicases, the BLM helicase (RECQL3) 7, 8 . The BLM helicase has essential roles during DNA replication and repair processes, which makes it an important component of the cell for the maintenance of genomic stability 9 . Accordingly, cells from BS patients show high levels of genomic instability, e.g., chromosome aberrations and mitotic defects, which overall contribute to the pathogenesis of the disease 2, 10 . As an evidence of genomic instability, BS patient cells show elevated rates of sister chromatid exchange (SCE) 11 , which was used as a cytogenetics test in the diagnosis of BS when the molecular diagnostic tools were limited. The BLM helicase forms part of a four-subunit protein complex, known as the BTR complex or the BLM dissolvasome, which additionally includes the topoisomerase III alpha (TOP3A, MIM 601243) and the RecQ-mediated genome instability protein 1 (RMI1, MIM 610404) and 2 (RMI2, MIM 612426) [12] [13] [14] . The BTR complex dissolves DNA intermediates such as Gquadruplexes, D-loops, and Holliday junctions, which occur during different steps of the homologous recombination repair pathway 8, 15, 16 . Furthermore, the BTR complex is the only known protein complex that can dissolve Holliday junctions without the occurrence of crossovers between the homologous DNA strands 17, 18 . Biallelic LoF mutations, small and large indels, as well as mutations affecting correct splicing of BLM were described in BS patients 2, 19 . Moreover, LoF mutations in other members of the BTR complex, i.e., TOP3A, RMI1, and RMI2, have been recently linked to BS 6, 20, 21 . The phenotypic characteristics of patients with biallelic TOP3A mutations were similar to those of patients with recessive BLM mutations in . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint terms of primary microcephaly, growth retardation, and café-au-lait spots 4, 20 . However, dilated cardiomyopathy, which is not a typical characteristic of BS, was also reported in TOP3Amutated patients 20 . Another study reported a large homozygous deletion covering the whole RMI2 gene to be associated with a BS phenotype in two patients and the diagnosis was confirmed by increased SCE rates although the phenotypic features of the patients were milder than in the common BS phenotype 21 . Here, we report a patient cohort of eight individuals from six families presenting with main characteristics of BS phenotype. We identified one novel and two previously described homozygous, deleterious mutations in BLM in six individuals and defined the clinical phenotype of patients with a homozygous LoF variant in RMI1 that has recently been reported 20 . We describe the molecular and clinical findings of the patients and compare the phenotypic expression. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint All subjects or their legal representatives gave written informed consent to the molecular genetic analyses and the publication of the results. This study was performed according to the Declaration of Helsinki protocol and approved by the local institutional review board (University Medical Center Göttingen, Germany). Samples of genomic DNA from participating family members were extracted from peripheral blood lymphocytes in EDTA solution by standard extraction procedures. In the physical exam of the patients, the height, weight, and occipitofrontal head circumference (OFC) measurements were recorded and evaluated according to the percentile values of the corresponding age groups in Turkish children 22 . Genomic DNA samples of three patients were screened directly for mutations in BLM (NM_000057.4) using specific primers for each exon of the gene including the exon-intron boundaries. Following amplification, Sanger sequencing of the amplified regions was done by BigDye Terminator v3.1 (Applied Biosystems) method on an ABI 3500XL sequencer and the results were visualized using the 4Peaks software 23 . When a mutation was detected, the parents were also investigated for the corresponding region in the same way. An NGS-based in-house microcephaly panel (Agilent Technologies, Santa Clara, CA) covering 78 microcephaly-related genes including the intron-exon boundaries was used for the diagnosis of one patient (www.humangenetik-umg.de). DNA was extracted from blood and enriched with the SureSelect QXT method, PCR-amplified, and sequenced on the Illumina is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint All variants detected by NGS-based techniques were confirmed by the standard Sanger sequencing method as explained above. . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint In this study, we assessed the clinical phenotypes and analyzed the molecular basis of eight patients with Bloom syndrome from six families. The molecular analyses revealed three distinct homozygous mutations in BLM in six patients from five families (Table 1) . Additionally, a 5-bp deletion in RMI1 was detected in two cousins from a consanguineous family 20 . All families originated from same country and consanguinity between parents was reported in three families with homozygous BLM mutation patients. In one patient, F1/II-4, we identified by WES a novel homozygous mutation in BLM (c.572_573delGA; p.Arg191Lysfs*4) and confirmed this causative variant by Sanger sequencing. In this consanguineous family, the parents were heterozygous carriers (Fig. 1a) . The prediction of this variant on transcription level was the introduction of a premature stop codon in the third exon resulting in a truncated protein that would lack important domains of the helicase enzyme, e.g., ATP-binding domain and the helicase domain 30 (Fig. 1b and 1c ). This novel mutation was also identified in another patient, F2/II-2, in homozygous state by using an in-house built microcephaly multigene-panel test. The results were confirmed by Sanger sequencing and the parents were heterozygous for the mutation. A known homozygous nonsense mutation in BLM (c.581_582delTT;p.Phe194*) 31 was identified in patient F3/II-2 by WES analysis. In the ExAC database, the allele frequency of this variant was 8 x 10 -6 (rs367543026) and was predicted to be pathogenic. The same variant was detected in another patient (F4/II-2), in whom the BLM coding regions were directly sequenced as the clinical diagnosis of BS had already been given. Furthermore, molecular investigation of BLM revealed a homozygous missense variant (c.3164G>C;p.Cys1055Ser) 5 in two siblings (F5-II/2 and F5-II/3) whose consanguine parents were heterozygous carriers. This missense mutation was located in exon 16 and had an allele frequency of 17 x 10 -6 (rs367543029). The position of the altered cysteine was on the C-terminal part of the well- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint conserved RecQ domain, which is important for the ion binding and the helicase enzyme activity of the BLM protein 32 (Fig. 1c) . Additionally, by performing WES in a BLM mutation-negative family with BS, we detected a homozygous truncating variant in RMI1 in two siblings presenting a BS phenotype and included their phenotypic examination into the present study 20 . The parents of both siblings were related as step-cousins and were heterozygous carriers for the detected variant. No other variant was detected which could explain the disease phenotype. We reinvestigated the clinical features of our patients in order to determine the spectrum of BLM-and RMI1-associated phenotypes. All the index patients included in this study suffered from intrauterine growth deficiency and had a low fetal head circumference ( Table 2) . Some of the prominent features of BS such as microcephaly and growth deficiency could be already observed in the prenatal period. After birth, which mostly occurred at term, microcephaly and growth retardation continued in all patients resulting in a global developmental delay and short stature, which was in line with the current clinical synopsis of BS 6 ( Table 3 ). All patients were examined in either childhood or early adolescence and were mainly referred to our clinic due to growth retardation. The most severe case of growth deficiency and microcephaly was patient F4/II-2, presenting with -6 SD in height, -4.9 SD in weight, and -7.6 SD in head circumference. All other patients shared a similar pattern of postnatal growth failure for all three parameters (Table 3 ). In addition, patient F1/II-4 received growth hormone treatment for three years in early childhood, however, the growth values stayed low. A narrow face, sunlight sensitivity, and café-au-lait spots were observed on some but not all of the patients with homozygous BLM mutations. Café-au-lait spots in the form of hypo-and hyperpigmentation were common among the BLM-mutated patients except for one patient. On the other hand, sunlight-sensitive skin lesions, which are considered as a major phenotypic is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint characteristic of BS, were present only in two of our BLM-mutated patients (F2/II-2 and F3/II-2). Interestingly, neither of our two patients with RMI1 mutation showed any dermatological characteristics of BS. Immunodeficiency by means of low immunoglobulin levels (e.g., IgG, IgA, IgM, and/or IgG1) was detected in all patients carrying homozygous mutations in BLM (Table 3) In line with the clinical definition of BS, which includes primary microcephaly and growth delay, all of our eight patients presented pre-/postnatal microcephaly and short stature 2 . However, dermatological features, e.g., photosensitivity and café-au-lait spots, were heterogeneous characteristics among the BLM-mutated patients. Of note, neither of the patients with RMI1 mutation showed any skin phenotype of BS. Similarly, immunodeficiency was presented by the patients with homozygous BLM mutations but not by RMI1-mutated patients. Overall, we highlight that among our patient cohort, the individuals with homozygous mutations in BLM showed more severe BS phenotypic features than the individuals with the homozygous RMI1 mutation. . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint Evaluation of phenotypes and mutational spectra of rare diseases such as Bloom syndrome often faces the challenge of a small number of patients. Although the disease was described in 1954 1 , the most comprehensive BS patient cohort was reported in 2007 6 . The study reported 134 BS patients and extensively described the causative mutations in BLM 6 . Yet, 9 out of 134 BS patients lacked a disease-causing mutation in BLM, indicating a likely genetic heterogeneity 2,6 . In this study, we report a patient cohort of eight individuals diagnosed with BS. The molecular analyses revealed one novel and two previously described LoF mutations in BLM in homozygous state in six of the patients. Moreover, we included a previously described homozygous truncating variant in RMI1 into our study 20 . Our molecular diagnosis algorithm specific for BS comprises BLM gene screening, panel screening, and/or WES. The causative mutations that were detected via NGS-based technique were confirmed by Sanger sequencing in the patients and the parents. This methodology, which includes NGS-based approaches, could be considered as a new standard for the molecular diagnosis of Bloom syndrome since other genes than BLM are associated with BS phenotype, e.g., TOP3A, RMI1, and RMI2 20,21 . Therefore, further associations of the BS phenotype to genes involved in various pathways of maintenance of genomic stability might be expected and WES analysis would thus be of high interest to identify such novel links. As an example, we hypothesize that patients in the Bloom syndrome registry 6 with no detected BLM mutations likely have one or more causative mutations in other genes or have at least intronic deleterious variants in BLM, which could be detected by next-generation sequencing techniques. In the current study, one novel frameshift mutation in BLM (c.572_573delGA;p.Arg191Lysfs*4) was detected by WES analysis in one BS patient. The variant was identified as damaging by various in-silico prediction tools due to a premature stop codon in the N-terminal domain of the protein. Therefore, we report this homozygous variant as a disease-causing mutation in BLM detected in two individuals for the first time. Although the two patients were from different families, we hypothesize a founder effect for this variant, considering the high rates of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint consanguinity in the country of origin. Parents of Family 1 reported consanguinity from second degree. Parents of Family 2 claimed no relation although both were originating from the same village with less than 1,000 inhabitants. Therefore, we assume a common ancestor for this variant. Furthermore, a known mutation (BLM: c.581_582delTT;p.Phe194*) was detected in homozygous state in two BS patients coming from unrelated families. In an earlier study, this mutation was reported homozygously in one individual with BS phenotype 31 . The 2-bp deletion was deleterious due to the introduction of a premature stop codon, causing a truncated protein with missing enzymatic domains (Fig. 1c) . The third mutation detected in BLM was the extensively studied (c.3164G>C;p.Cys1055Ser) missense mutation 5 . This mutation affects one of the four highly-conserved cysteine domains which bind zinc ion 32, 33 . The ion binding to this domain is essential for the ATPase and helicase enzyme activity as well as for the stabilization of the BLM protein 34, 35 . Therefore, this amino acid change was detrimental to the enzyme activity and considered to be deleterious as a variant, resulting in the BS phenotype. Cancer predisposition is an important clinical feature in BS as it greatly affects patients' life span and quality. In our patient cohort, we have not detected any malignancy except one incident of Wilms tumor in one patient who underwent surgery in early childhood. The lack of cancer incidence in our patient cohort could be due to the young age of the patients: our oldest patient was at adolescent ages, while the mean age of onset of cancer in BS patients is 24 years 6 . In addition, patient F1/II-4 received growth hormone treatment for three years; however, the growth parameters did not change. The BS characteristics of this patient were heterogeneous as skin lesions and a narrow face were missing and, therefore, growth hormone treatment was started before the completion of molecular diagnosis. We want to note that great care must be taken while deciding on growth hormone treatment, bearing in mind the possibility of malignancy increase for BS patients with pronounced cancer predisposition 36 . We observed a phenotypic variety among the BS patients depending on the causative mutations. The strongest phenotypic variance among the BS patients was observed between . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint patients with homozygous LoF mutations in different genes, namely BLM and RMI1. The patients with homozygous BLM mutations showed a more severe BS phenotype in terms of photosensitivity and immunodeficiency in comparison to the RMI1-mutated patients. The gene products of the two genes; BLM helicase and RMI1 proteins, form part of a protein complex named the BTR complex, which dissolves DNA intermediates. Once the complex is near the chromatin, BLM helicase acts on the branches of entangled DNA by moving them closer to form a hemicatenated structure 17, 37 . Then, the topoisomerase III alpha protein separates the two strands by nicking to generate the final non-crossover products 38 . RMI1 stimulates the process by associating with both BLM and TOP3A 12, 39 . The major role within the complex may possibly be assigned to BLM and TOP3A proteins since they are the enzymatic parts acting on the DNA 40 . Nevertheless, lack of RMI1 protein has been shown to lead to defective cell proliferation due to deficient phosphorylation of BLM 13, 41 . In summary, we conclude that mutations in the different members of the BTR complex have a varying impact on the severity of the BS phenotype, especially when comparing BLM and RMI1-associated phenotypes. A distinct phenotypic characteristic of BS is the pronounced immunodeficiency profile 42 . Strikingly, all our patients with homozygous BLM mutations showed immunity problems resulting in recurrent infections mainly of the upper respiratory tract. However, patients with the homozygous RMI1 mutation did not show this clinical feature. Furthermore, we examined two siblings with BS in one family and observed the phenotypic variance based on a sibship. Infection rates were higher for the younger patient, although both children suffered from immunodeficiency and had similar BS characteristics. Based on our evaluations, we emphasize that the frequent infections in BS are as important as any other life-threatening element of BS like cancer predisposition. Due to the low levels of immunity, BS patients should be considered susceptible to any infection such as the Covid-19 pandemic that arose in 2020. In summary, in eight patients presented with BS from six families, we detected one novel homozygous frameshift mutation in BLM in two patients from two families, two already known . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint mutations in three families, and one homozygous truncating mutation in the RMI1 gene in a consanguineous family 20 . All variants were homozygous loss-of-function mutations associated with the BS phenotype. Moreover, patients with homozygous mutations in the BLM gene showed more severe BS phenotype characteristics in terms of photosensitivity, immunodeficiency, and infection rate than the patients carrying the homozygous RMI1 variant. The immunodeficiency profile of BS is an important characteristic of the disease. Thus, we highlight that extra care must be taken to protect BS patients, especially in the current Covid-19 pandemic. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint data are not publicly available due to privacy or ethical restrictions. Processed genetic data generated or analyzed within this study are available upon request. Written informed consent of all participants or their legal representatives was obtained prior to participation in the study. This study was performed according to the Declaration of Helsinki protocol and approved by the local institutional review board (University Medical Center Göttingen, Germany) under approval number 3/2/16. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; https://doi.org/10.1101/2021.11.02.21265560 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint . CC-BY-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 10, 2021. ; F6/1 § female 34 weeks n/a -1,7 n/a 6-10 -4 -3.8 -5 n/a n/a n/a n/a RMI1 F6/2 § female 38 weeks n/a -3,1 n/a 11-15 -2,9 -0,9 -5 n/a n/a n/a n/a The z-scores are calculated using the Turkish children's mean for age and gender. The following abbreviations are used: n/a, data not available; N, normal. † The individual received growth hormone treatment in early childhood. ‡ Wilms tumor was detected and operated in early childhood. § These individuals have been already reported 20 . Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs: Probably a Syndrome Entity Bloom's Syndrome: Clinical Spectrum, Molecular Pathogenesis, and Cancer Predisposition Bloom syndrome: a mendelian prototype of somatic mutational disease Bloom's syndrome. I. Genetical and clinical observations in the first twentyseven patients The Bloom's Syndrome Gene Product Is Homologous to RecQ Helicases Syndrome-causing mutations of the BLM gene in persons in the Bloom's Syndrome Registry Human RecQ Helicases in DNA Double-Strand Break Repair The Bloom's Syndrome Gene Product Is a 3'-5' DNA Helicase RecQ Helicases: Conserved Guardians of Genomic Integrity BT -DNA Helicases and DNA Motor Proteins RecQ helicases: multifunctional genome caretakers A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes A double holliday junction dissolvasome comprising BLM, topoisomerase IIIα, and BLAP75 BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome The Bloom's syndrome helicase unwinds G4 DNA Binding and melting of D-loops by the Bloom syndrome helicase The Bloom's syndrome helicase suppresses crossing over during homologous recombination A Structural Guide to the Bloom Syndrome Complex Bloom syndrome: an analysis of consanguineous families assigns the locus mutated to chromosome band 15q26 Mutations in TOP3A Cause a Bloom Syndrome-like Disorder Loss of RMI2 Increases Genome Instability and Causes a Bloom-Like Syndrome Reference values for weight, height, head circumference, and body mass index in Turkish children Groothuis T. 4Peaks SIFT web server: predicting effects of amino acid substitutions on proteins MutationTaster2: mutation prediction for the deep-sequencing age A method and server for predicting damaging missense mutations M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity Improved splice site detection in Genie A Novel Mutation in PIGA Associated with Multiple Congenital Anomalies-Hypotonia-Seizure Syndrome 2 (MCAHS2) in a Boy with a Combination of Severe Epilepsy and Gingival Hyperplasia Human RecQ Helicases in DNA Repair, Recombination, and Replication Three New BLM Gene Mutations Associated with Bloom Syndrome Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein Chapter 10 -Role of Zinc-Binding Domains of RecQ Helicases RecQ helicases: multiple structures for multiple functions? Bloom Syndrome in Short Children Born Small for Gestational Age: A Challenging Diagnosis Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration The dissolution of double Holliday junctions Functional role of BLAP75 in BLMtopoisomerase IIIalpha-dependent holliday junction processing The BLM dissolvasome in DNA replication and repair BLAP75, an essential component of Bloom's syndrome protein complexes that maintain genome integrity Immunodeficiency in Bloom's Syndrome We are grateful to all patients for participating in this study and Karin Boss for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Research Group FOR 2800 "Chromosome Instability:Cross-talk of DNA replication stress and mitotic dysfunction", SP5 and SPZ to B. Wollnik and Germany's Excellence Strategy, Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC; EXC 2067/1-390729940) to B. Wollnik. The authors declare no conflict of interest. Pedigrees and family histories are available upon request. The whole-exome sequencing raw