Humu-2014-0041 Research Article

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families Periklis Makrythanasis1,3, Mari Nelis1,2, Federico A. Santoni1, Michel Guipponi3, Anne Vannier1, FrédériqueBéna3, Stefania Gimelli3, Elisavet Stathaki3, Samia Temtamy4, André Mégarbané5,6, Amira Masri4, Mona S. Aglan4, Maha S. Zaki4, Armand Bottani3, SivFokstuen3, Lorraine Gwanmesia3, Konstantinos Aliferis3, Mariana E. Bustamante1, Georgios Stamoulis1, Stavroula Psoni8, Sofia Kitsiou-Tzeli8, Helen Fryssira8, Emmanouil Kanavakis8, Nasir AlAllawi9, Abdelaziz Sefiani10,11, Sana’ Al Hait12, Siham C. Elalaoui10, Nadine Jalkh5, Lihadh AlGazali13,14, Fatma Al-Jasmi13,14, Habiba Chaabouni Bouhamed15, EbtesamAbdalla16, David N. Cooper17, Hanan Hamamy1, Stylianos E. Antonarakis1,3,18* 1. Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland 2. Current affiliation: Estonian Genome Centre, University of Tartu, Tartu, Estonia 3. Service of Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland 4. Department of Clinical Genetics, National Research Centre, Cairo, Egypt 5. Medical Genetics Unit, Saint Joseph University, Beirut, Lebanon 6. InstitutJerôme Lejeune, Paris, France 7. Pediatric Department, The University of Jordan, Amman, Jordan This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/humu.22617.

This article is protected by copyright. All rights reserved.

1

8. Department of Medical Genetics, University of Athens, Athens, Greece 9. Department of Pathology, College of Medicine, University of Dohuk, Dohuk, Iraq 10. Department of medical genetics, Institut National d'Hygiène, Rabat, Morocco 11. Centre de Génomique Humaine, Faculté de Médecine et de Pharmacie, Université Mohamed V Souissi, Rabat, Morocco 12. Genetics & IVF Department, The Farah Hospital, Amman, Jordan 13. Department of Paediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates 14. Department of Pediatrics, Tawam Hospital, United Arab Emirates University, Al-Ain, United Arab Emirates 15. Department of Human Genetics, University Tunis El Manar, Faculty of Medicine, Tunis, Tunisia 16. Department of Human Genetics , Medical Research Institute, Alexandria University, Alexandria, Egypt 17. Institute of Medical Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK 18. iGE3 Institute of Genetics and Genomics of Geneva, Geneva, Switzerland

This article is protected by copyright. All rights reserved.

2

*Correspondence should be addressed to: Stylianos E Antonarakis MD, DSc Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel-Servet 1211 Geneva, Switzerland tel +41-22-379-5708 fax +41-22-379-5706 email: [email protected]

Funding: This work was supported by grants from the Gebert Ruf Stiftung foundation, the European Union ERC (FP7-IDEAS-ERC; 249968), and the Swiss SNF to SEA and by a grant from the von Meissner foundation to PM. During this work PM was supported by a scholarship by the Bodossaki foundation. MN was supported by the Scientific Exchange Program between Switzerland and the New Member States of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This article is protected by copyright. All rights reserved.

3

ABSTRACT Rare, atypical and undiagnosed autosomal recessive (AR) disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively AR disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of AR inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s) and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH were then performed on one affected individual per family. High-confidence pathogenic variants were found in homozygosity in known disease-causing genes in 18 families (36%) (one by aCGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes. Keywords: Consanguinity, exome sequencing, homozygosity mapping, high throughput sequencing

This article is protected by copyright. All rights reserved.

4

INTRODUCTION The recent focus on the molecular basis of apparently Mendelian disorders has been successful in identifying thousands of pathogenic variants in protein-coding genes causing these phenotypes. Since the discovery of the first human gene mutations responsible for αand β- thalassemia in the late 1970s (Chang and Kan, 1979; Orkin, et al., 1978), a total of >3,100 protein-coding genes have been causatively linked to 3,684 inherited disorders in OMIM (Online Mendelian Inheritance in Man; http://www.omim.org), and a total of more than 150,000 different pathogenic variants have been identified (Cooper, et al., 2010). The recent rapid evolution of sequencing technologies and our increasing knowledge of the structure and function of the human genome have together served to accelerate these discoveries, thereby providing accurate diagnoses as well as a detailed understanding of the pathophysiology of a considerable number of inherited disorders. This notwithstanding, the majority of patients with rare autosomal recessive disorders remain undiagnosed even after performing available imaging, biochemical, pathological and genetic testing; thus, the current management of undiagnosed cases is suboptimal and the genetic counseling rather uninformative. High Throughput Sequencing (HTS) has made possible the identification of almost all variants in the coding regions of protein-coding genes. This provides the opportunity to explore the genotype-disease relationship in small nuclear families as it was shown in a proof of concept study by Ng et al. ( 2010).

This article is protected by copyright. All rights reserved.

5

Consanguineous marriage is practiced in a large number of human populations; rates attain 20-50% in several countries of the Mediterranean basin and the Middle East as well as in immigrant populations in western European countries (Al-Gazali, et al., 2006; Hamamy, et al., 2011). Reports in the literature (Stoll, et al., 1999; Stoltenberg, et al., 1999) and a recent study in the UK have indicated that children born to consanguineous couples have an increased risk of presenting with congenital anomalies (Sheridan, et al., 2013). Identifying the causative pathogenic gene lesion in consanguineous families seeking genetic counseling is not always successful despite the veritable array of currently available clinical and laboratory investigations. In such instances, prevention of the recurrence of the disease in future offspring is usually not possible. Moreover, the costs of imaging, biochemical and genetic testing for unresolved cases could easily exceed that of whole exome sequencing and may ultimately still not provide a precise diagnosis. In this study, we employed whole exome sequencing and genotype analysis to screen members of consanguineous families with likely recessive disorders. Our hypothesis was that because of the homozygosity of the causative defect, our diagnostic strategy would be successful in identifying the molecular basis of the disorder in at least a proportion of the participating patients. The minimum inclusion criteria were consanguinity among parents of affected, whilst the phenotypic spectrum was unrestricted. Together with a recently published study of non-consanguineous patients (Yang, et al., 2013), we anticipate that the results of this study will have important implications for molecular diagnostics and genetic counseling, and could lead to the broad establishment of exome sequencing as a key diagnostic tool for consanguineous families with undiagnosed genetic disorders outside specialized centers. This article is protected by copyright. All rights reserved.

6

MATERIALS AND METHODS Patients and nuclear families A total of 50 families participated in this study. The main criteria for inclusion were consanguinity among parents irrespective of the patient’s clinical phenotype. Families with at least two affected individuals were preferentially selected. Of these families, 45 were of Arab origin, either living in Arab countries or having immigrated to Greece or Switzerland. Three families were of Greek and two of Kurdish origin. Intellectual disability (ID, defined as an IQ0.3 were included in the analysis. The ROH were further defined as genomic regions demarcated by the first encountered heterozygous SNPs flanking each established homozygous region. Exome sequencing The exome was captured using the SureSelect Human All Exons v3(20 patients), v4(11 patients) and v5(19 patients) reagents (Agilent Inc®). Sequencing was performed in an IlluminaHiSeq 2000 instrument. Each exome library was indexed, separated into two equal halves and sequenced in two different lanes. The raw results were analyzed using a customized pipeline which utilizes published algorithms in a sequential manner (BWA(Li and Durbin, 2009) for mapping the reads, SAMtools (Li, et al., 2009) for detection of variants, Pindel (Wang, et al., 2010) for the detection of indels, ANNOVAR (Ye, et al., 2009) for the annotation). The entire coding sequence corresponding to the human RefSeq (Pruitt, et al., 2007) coding genes was used as the reference for the calculation of coverage and reads on This article is protected by copyright. All rights reserved.

9

target. All experiments were performed using the manufacturer’s recommended protocols without modifications. Bioinformatic analysis The ROH coordinates, the genotypes and the exome results were processed using an inhouse algorithm (CATCH v1.1, unpublished). CATCH additionally takes into account the family information and accepts different types of exclusion and inclusion filters. The final result consists of a list of those variants that respect the provided filters and assigns them to a different class according to how well they respect the segregation of the ROH. The filters used were: homozygous exonic and splicing variants (±6bp from the intron-exon junction) with a minimum allele frequency less than 0.02 in public [dbSNP version 137, 1000 Genomes (April 2012 release; http://www.ncbi.nlm.nih.gov/SNP/) ,(Genomes Project, 2010) Exome Variant Server (v.0.018; http://evs.gs.washington.edu/EVS/) ] and the local database (which includes 50 individuals with the same ethnic origin). Synonymous variants not affecting the splice site or variants that were found within segmental duplications were excluded. Only variants respecting the mentioned filters and the ROH segregation were considered for further analysis. The final list of variants was further evaluated based on the predicted pathogenicity scores provided by SIFT (http://sift.jcvi.org/, cutoff=0.447) (Adzhubei, et al., 2010) and Mutation Taster (http://www.mutationtaster.org/, cutoff:qualitative prediction as pathogenic)(Schwarz, et al., 2010). Two out of three were required in order to declare a variant to be possibly pathogenic or possibly benign. Variants were also evaluated based on This article is protected by copyright. All rights reserved.

10

their presence in the Professional (Schwarz, et al., 2010) version of HGMD (Stenson, et al., 2013)(version: 2013.1) and a literature search focusing on functional data. Evolutionary conservation scores were provided by PhyloP (comparison of 46 species) (Cooper, et al., 2005) and GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/) (Davydov, et al., 2010). Initial diagnoses were re-evaluated when variants (either found in HGMD or predicted to be pathogenic) were identified in causative genes known to be responsible for phenotypes corresponding to the clinical presentations of the patients. In the families in which no variants in known pathogenic genes were identified as being responsible for the clinical phenotype, a complementary approach was used. Each patient was assigned to a broad phenotypic category as they are defined in the Clinical Genomic Database (http://research.nhgri.nih.gov/CGD/, March 2014 version) (Supp. Table S2). The corresponding list of genes was downloaded and all the genes that typically displayed AR or X-linked inheritance (in families where all the affected individuals were male) were interrogated, irrespective of ROH and family segregation. Since none of the parents has been exome sequenced, a search for de novo variants was not possible. All variants identified by exome sequencing and discussed in this paper, were verified by Sanger sequencing in all family members. Only the variants that were respecting the segregation were retained. The variant nomenclature was controlled by Mutalyzer (https://mutalyzer.nl/, version 2.0.beta-29). The variants mentioned in this study have been submitted to LOVD (http://databases.lovd.nl/whole_genome/genes).

This article is protected by copyright. All rights reserved.

11

Array Comparative Genomic Hybridization (aCGH) Unless already performed by the referring clinical group, aCGH was carried out In order to identify homozygous deletions or duplications and to exclude the presence of a chromosomal aberration. Briefly a DNA sample from one patient per family was tested using the SurePrint G3 Human CGH Microarray Kit, 2x400K (Agilent Technologies, Santa Clara, CA) with 7.2 kb overall median probe spacing. Labeling and hybridization were performed according to the manufacturer's protocol. Data analysis was performed using Agilent Genomic Workbench Lite Edition 6.5.0.58. Probe positions are by reference to NCBI37 hg19. RESULTS aCGH results Nineteen patients for whom no aCGH data existed were tested during this study. On average, every patient had 15.7 (range: 9-26) Copy Number Variants (CNVs), 5.2 (range: 113) of which were duplications, whereas 10.5 (range: 5-20) were deletions. The CNVs varied in size from 1 to 2309 kb and on average every patient had 0.9 (range 0-5) novel CNVs (defined as CNVs not reported in the Database of Genomic Variants) and 1.6 (range 0-4) homozygous CNVs (Supp. Table S3). During the course of this project, we identified by aCGH a homozygous deletion of 32kb encompassing the first exon of VLDLR (MIM# 192977), known to be responsible for cerebellar hypoplasia and mental retardation with or without quadrupedal locomotion (also known as Disequilibrium syndrome (DES))(Boycott, et al., 2005)(MIM# 224050). The patients presented with ID and severe ataxia (they have never walked), compatible with the This article is protected by copyright. All rights reserved.

12

described phenotype of DES; the deletion segregated with the clinical phenotype through the family pedigree (family 4). ROH and Exome sequencing Samples from 50 affected individuals, one from each family, were sequenced after exome capture. Supp. Table S4 provides the number of reads per sample. Supp. Table S5 provides the ROH for the families where a probably causative variant was identified. On average, among the 21,335 exonic and 1,370 splicing variants identified per individual, 50.3% were synonymous (98.6% of which have already been catalogued in dbSNP137; Supp. Table S6). Using our algorithm, the putatively pathogenic homozygous variant was found in known disease-causing genes in 17 families (Table 2, Supp. Table S7). In one of these families, the patients harbored two high-confidence pathogenic variants whereas in two further families, high-confidence pathogenic variants explaining part of the phenotype were identified. The follow-up analysis identified one additional homozygous variant in family 12 (Table 2, Supp. Table S7). This variant was missed by the original approach because we made the incorrect assumption that the two affected cousins had the same condition. After identification of the variant, further clinical examination confirmed the genotypic-phenotypic correlation with the exome sequenced patient and especially the fact that despite similar clinical picture the affected cousin did not have the brain MRI anomalies. It must be noted that using the same criteria, no compound heterozygous variants or variants in X-linked genes were identified.

This article is protected by copyright. All rights reserved.

13

Families diagnosed with known recessive genes by exome sequencing In 15 families, we were able to identify variants in genes already known to be involved in recessive disorders and which were compatible with the clinical phenotypes (Table 2). In families 12, 26, 29, 30, 32, 37, 43, 44, 46, 48 and 49, pathogenic variants were identified in known genes responsible for disorders with a clinical description that correlated with the referred phenotypic description. These highly heterogeneous phenotypes could have been caused by mutations in a number of different genes; exome sequencing allowed the reliable detection of the causative variant and an accurate diagnosis of the syndrome in each patient. In family 1, the two affected females were homozygous for a known variant (NM_004407:c.1A>G:p.(Met1Val))(Feng, et al., 2006; Lorenz-Depiereux, et al., 2006) inDMP1 (MIM# 600980) reported to cause autosomal recessive hypophosphatemic rickets (MIM# 241520). The patients’ phenotype, previously described in detail (Chouery, et al., 2010), included diffuse hyperostosis and serum phosphorus only slightly below the normal limits (0.79mmol/L, lower normal limit: 0.81 mmol/L) at the age of the diagnosis. Similarly, one of the reported families (Lorenz-Depiereux, et al., 2006) with older patients also report osteosclerosis in the affected individuals and some of them have had serum phosphorus values just under the normal limits. In family 13, a novel variant was identified in FKTN (MIM# 607440) responsible for many muscular disorders as shown in Table 2. The family was ascertained with the preliminary diagnosis of familial microcephaly and developmental delay. The three patients have displayed hypotonia since an early age, and brain imaging revealed prominent frontal This article is protected by copyright. All rights reserved.

14

gyration, kinked corpus callosum, defective myelination along with a retrocerebellar cyst and mild vermian hypoplasia; all of these findings were compatible with the clinical spectrum described in Fukuyama congenital muscular dystrophy (FCMD)(Kobayashi, et al., 1998). However, all three patients had microcephaly (between -3.5 to -4.5 SD) and absence of pseudohypertophy while two of them were able to walk unaided. Plasma CPK levels measured more than once were within the normal range, in contrast to the markedly increased CPK levels typical of clinical presentations of this disease. The differences in the phenotypic spectrum could be attributed to the nature of the variant and/or unknown modifiers. In family 31, a novel variant was identified in SYNE1 (MIM# 608441). SYNE1 is a large gene with 146 exons and two different phenotypes attributed to it, with different modes of inheritance and different types of pathogenic mutation: the autosomal dominant EmeryDreifuss muscular dystrophy 4 (MIM# 612998) and the autosomal recessive spinocerebellar ataxia 8 (MIM# 610743). The SYNE1 variant identified in family 31 was found within the coding region that usually harbors mutations causing Emery-Dreifuss syndrome (Zhang, et al., 2007); however, the mutation created a stop codon in contrast to the missense variants usually identified. Interestingly, the patient’s phenotype is that of severe hypotonia since birth which does not correspond to Emery-Dreifuss dystrophy or spinocerebellar ataxia. A literature search revealed one case report of a consanguineous Palestinian family with a nonsense SYNE1 mutation and a similar clinical picture (Attali, et al., 2009). The description of this family increases further the phenotypic spectrum related to SYNE1 variants. In family 36, variants in two different genes known to cause monogenic disorders were identified. Both identified variants are found in genes of recessive intellectual disability This article is protected by copyright. All rights reserved.

15

[MTFMT (MIM# 611766) and MAN1B1 (MIM# 604346)] and the phenotype appears to be a combination of both disorders. Concerning MTFMT, lactic acidosis is not always detected (Tucker, et al., 2011) and sampling of cerebrospinal fluid had not been possible. Brain MRI failed to show any brain lesions but there have been reported cases with no detected lesions; on the other hand, the patients present strabismus and hypotonia both of which are regularly seen in patients with MTFMT mutations (Haack, et al., 2014). With respect to MAN1B1I, there are no published photographs of patients (Rafiq, et al., 2011) but the facial description correlates well with that of the patients reported here: wide, arched, sparse eyebrows, prominent nose, flat philtrum and thin upper lip. Based on this evidence we consider that the majority of the phenotype is explained by the MAN1B1 variant while it the same time it seems that the MTFMT variant contributes to a probably lower but uknown extent. In two families with patients with ID, variants explaining at least part of the phenotype have been identified. However, the molecular cause of ID remains unknown. In family 38, a variant in PYGM (MIM# 608455) could potentially account for the marked hypotonia of the patients. In the second, family 39, a homozygous frameshift variant was identified in PRX (MIM# 605725) known to cause Charcot-Marie-Tooth type 4F (CMT4F). The patients in family 39 do not show any of the classic CMT4F symptoms (early onset demyelinating sensory neuropathy) apart from a marked muscular weakness. It is interesting to note that there is a report of a Japanese patient with CMT4F (Tokunaga, et al., 2012), harboring a nonsense mutation in an adjacent codon (p.R1070*),with late onset presentation (in his late 20s). DISCUSSION This article is protected by copyright. All rights reserved.

16

Consanguinity is a known risk factor for the incidence of autosomal recessive disorders, and the risk to offspring increases with the level of consanguinity between the parents (Hamamy, 2012). In this study, we examined 50 consanguineous families with unresolved molecular diagnoses with the aim of diagnosing known disorders causing the clinical phenotype in the offspring. Using exome sequencing, we have identified 17 patients out of 50 with variants in known genes that can explain either the observed clinical phenotype or at least some aspects of it with variable degrees of confidence. Several notable conclusions can be drawn. 1. In 11 families, high-confidence pathogenic variants were identified by exome sequencing in known genes that correlated with the corresponding phenotypes. 2. In three families where causative variants were identified in DMP1, FKTN and SYNE1 respectively, significant new aspects pertaining to the natural history of the diseases/clinical phenotypes have been noted. In family 1 (with the DMP1 mutation), the clinical picture seen in childhood was different from that observed in adulthood. In family 13, the differential diagnosis of the clinical presentation did not include FKTN which would have been unlikely to have been included in the differential genetic workup. In family 31, our results expanded the phenotypic spectrum linked to SYNE1. 3. In family 36, homozygous variants were identified in two genes known to cause intellectual disability and the clinical phenotype may be considered to be due to a combination of the two variants.

This article is protected by copyright. All rights reserved.

17

4. In two families (38 and 39), variants that can partially account for the clinical phenotype were identified. In family 38, the variant in PYGM may account for the patients’ severe hypotonia but not the intellectual disability. In family 39, the variant in PRX may be associated with the patients’ muscle weakness and provides the opportunity to better anticipate the sensory neuropathy that could occur later in life. However, the molecular cause of their intellectual disability remains unknown. 5. In one family the pathogenic variant (a homozygous 32kb deletion) was detected by aCGH. The sample size offered by this study provides the possibility to perform a preliminary prediction regarding the total number of autosomal recessive disorders in human. We identified a causative mutation in 36% (18/50) of the families studied; since there are 1,828 molecularly-characterized recessive disorders currently listed in OMIM, we predict that at least 5,000 recessive human clinical phenotypes may exist. This crude estimate does not take into account the fact that some patients may have two or more genetic defects nor the fact that many genes may be responsible for several clinical phenotypes but it does concur with a recent estimate of a total of 12,000 to 15,000 monogenic disorders in human (Cooper, et al., 2010). In 32 families, no variants in known pathogenic genes were identified. With the exception of variants that were missed because no gene has been attributed to the specific phenotype, additional reasons include: A. Method related false negatives: i) protein-coding genes in whole or in part are not captured by the reagents currently employed, ii) insufficient coverage, iii) trinucleotide repeat expansions,; B. Analysis false negatives: iv) functional This article is protected by copyright. All rights reserved.

18

genomic elements other than protein-coding genes were not interrogated, v) false negative variant calling, vi) unsuspected problems in the analysis pipeline; C. Hypothesis-driven false negatives: vii) incorrect hypothesis for the mode of inheritance, viii) affected individuals in the same family may have genetically different disorders, ix) pathogenic variants with reduced penetrance may have resulted in false negative conclusions, x) pathogenic variants occurring in genomic regions identical by state between affected and non-affected family members. In a recently published study of diagnostic exome sequencing in patients suspected of having genetic disorders, a diagnostic rate of 25% was achieved (62/250 patients)(Yang, et al., 2013). Our slightly higher diagnostic rate (36%) is either due to the requirement for consanguinity, either to the more stringent inclusion criterion of at least 2 affected offspring or a combination of both. The methods employed here confirm the feasibility of using HTS as a diagnostic tool (Makrythanasis and Antonarakis, 2012) and could be adapted for researching any autosomal recessive disorder, whether in consanguineous or non-consanguineous families, in order to increase substantially the likelihood of an accurate molecular diagnosis and consequently increasing the standard of care. The HTS of exomes is also potentially useful for the detection of carrier status in consanguineous couples and the estimation of the risk for affected offspring, for family planning and reproductive decision making purposes (Bell, et al., 2011). Although the detection space would be limited to known disease genes, the prospective identification of

This article is protected by copyright. All rights reserved.

19

risk for an autosomal recessive disorder in the offspring would be substantially increased (Kingsmore, 2012). Conclusions A specific diagnosis of the patients’ main symptoms was successfully reached using exome sequencing and aCGH in 16 out of 50 patients participating in this study while in another 2 patients, a partial explanation of the clinical phenotype was obtained. This establishes HTS as an excellent first-tier diagnostic procedure. Precise diagnosis of the genetic disorder segregating in the family offers a wide range of future reproductive options including testing for carrier status with premarital and preconception counseling. Consanguineous marriages are culturally favored in a substantial number of human populations. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and establish more detailed causative links between mutant genotypes and clinical phenotypes.(El-Shanti, et al., 1999; Harville, et al., 2010; Pasutto, et al., 2007; Yoshida, et al., 2001)

This article is protected by copyright. All rights reserved.

20

ACKNOWLEDGMENTS We are grateful to the members of all families enrolled in this study. Conflicts of interest The authors declare no conflict of interest Authors contributions PM, HH and SEA wrote the manuscript, PM and MN performed the ROH and exome analyses PM, HH and SEA coordinated the study, FAS conceived the algorithms and wrote the bioinformatic pipelines, MG and AV performed the HTS and Sanger sequencing, FB, SG and ES performed and analyzed the aCGH, ST, AMe, AMa, MSA, MSZ, SF, LG, AB, KA SP, SKT, HF, EK, NA, AS, SA, SCE, NJ, LA, FA, HCB and EA examined the patients, described the phenotypic characteristics and contributed the DNA samples, MEB and GS analyzed exome data, DNC contributed HGMD mutation data and edited the paper, HH designed the study and coordinated the patient collection, SEA designed and conceived the general overview of the study. All authors contributed to the manuscript and approved the final version.

This article is protected by copyright. All rights reserved.

21

REFERENCES Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. 2010. A method and server for predicting damaging missense mutations. Nat Methods 7(4):248-9. Al-Gazali L, Hamamy H, Al-Arrayad S. 2006. Genetic disorders in the Arab world. BMJ 333(7573):8314. Attali R, Warwar N, Israel A, Gurt I, McNally E, Puckelwartz M, Glick B, Nevo Y, Ben-Neriah Z, Melki J. 2009. Mutation of SYNE-1, encoding an essential component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis. Hum Mol Genet 18(18):3462-9. Bell CJ, Dinwiddie DL, Miller NA, Hateley SL, Ganusova EE, Mudge J, Langley RJ, Zhang L, Lee CC, Schilkey FD and others. 2011. Carrier testing for severe childhood recessive diseases by nextgeneration sequencing. Sci Transl Med 3(65):65ra4. Boycott KM, Flavelle S, Bureau A, Glass HC, Fujiwara TM, Wirrell E, Davey K, Chudley AE, Scott JN, McLeod DR and others. 2005. Homozygous deletion of the very low density lipoprotein receptor gene causes autosomal recessive cerebellar hypoplasia with cerebral gyral simplification. Am J Hum Genet 77(3):477-83. Chang JC, Kan YW. 1979. beta 0 thalassemia, a nonsense mutation in man. Proc Natl Acad Sci U S A 76(6):2886-9. Chouery E, Pangrazio A, Frattini A, Villa A, Van Wesenbeeck L, Piters E, Van Hul W, Coxon FP, Schouten T, Helfrich M and others. 2010. A new familial sclerosing bone dysplasia. J Bone Miner Res 25(3):676-80. Cooper DN, Chen JM, Ball EV, Howells K, Mort M, Phillips AD, Chuzhanova N, Krawczak M, KehrerSawatzki H, Stenson PD. 2010. Genes, mutations, and human inherited disease at the dawn of the age of personalized genomics. Hum Mutat 31(6):631-55.

This article is protected by copyright. All rights reserved.

22

Cooper GM, Stone EA, Asimenos G, Program NCS, Green ED, Batzoglou S, Sidow A. 2005. Distribution and intensity of constraint in mammalian genomic sequence. Genome Res 15(7):901-13. Davydov EV, Goode DL, Sirota M, Cooper GM, Sidow A, Batzoglou S. 2010. Identifying a high fraction of the human genome to be under selective constraint using GERP++. PLoS Comput Biol 6(12):e1001025. El-Shanti H, Al-Salem M, El-Najjar M, Ajlouni K, Beck J, Sheffiled VC, Stone EM. 1999. A nonsense mutation in the retinal specific guanylate cyclase gene is the cause of Leber congenital amaurosis in a large inbred kindred from Jordan. J Med Genet 36(11):862-5. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, Zhang S and others. 2006. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38(11):1310-5. Genomes Project C. 2010. A map of human genome variation from population-scale sequencing. Nature 467(7319):1061-73. Haack TB, Gorza M, Danhauser K, Mayr JA, Haberberger B, Wieland T, Kremer L, Strecker V, Graf E, Memari Y and others. 2014. Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening. Mol Genet Metab 111(3):342-52. Hamamy H. 2012. Consanguineous marriages : Preconception consultation in primary health care settings. J Community Genet 3(3):185-92. Hamamy H, Antonarakis SE, Cavalli-Sforza LL, Temtamy S, Romeo G, Kate LP, Bennett RL, Shaw A, Megarbane A, van Duijn C and others. 2011. Consanguineous marriages, pearls and perils: Geneva International Consanguinity Workshop Report. Genet Med 13(9):841-7. Harville HM, Held S, Diaz-Font A, Davis EE, Diplas BH, Lewis RA, Borochowitz ZU, Zhou W, Chaki M, MacDonald J and others. 2010. Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping. J Med Genet 47(4):262-7.

This article is protected by copyright. All rights reserved.

23

Kingsmore S. 2012. Comprehensive carrier screening and molecular diagnostic testing for recessive childhood diseases. PLoS Curr:e4f9877ab8ffa9. Kobayashi K, Nakahori Y, Miyake M, Matsumura K, Kondo-Iida E, Nomura Y, Segawa M, Yoshioka M, Saito K, Osawa M and others. 1998. An ancient retrotransposal insertion causes Fukuyamatype congenital muscular dystrophy. Nature 394(6691):388-92. Kumar P, Henikoff S, Ng PC. 2009. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4(7):1073-81. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754-60. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078-9. Lorenz-Depiereux B, Bastepe M, Benet-Pages A, Amyere M, Wagenstaller J, Muller-Barth U, Badenhoop K, Kaiser SM, Rittmaster RS, Shlossberg AH and others. 2006. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 38(11):1248-50. Makrythanasis P, Antonarakis SE. 2012. High-throughput sequencing and rare genetic diseases. Mol Syndromol 3(5):197-203. Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, Beck AE, Tabor HK, Cooper GM, Mefford HC and others. 2010. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 42(9):790-3. Orkin SH, Alter BP, Altay C, Mahoney MJ, Lazarus H, Hobbins JC, Nathan DG. 1978. Application of endonuclease mapping to the analysis and prenatal diagnosis of thalassemias caused by globin-gene deletion. N Engl J Med 299(4):166-72. Pasutto F, Sticht H, Hammersen G, Gillessen-Kaesbach G, Fitzpatrick DR, Nurnberg G, Brasch F, Schirmer-Zimmermann H, Tolmie JL, Chitayat D and others. 2007. Mutations in STRA6 cause This article is protected by copyright. All rights reserved.

24

a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation. Am J Hum Genet 80(3):550-60. Pruitt KD, Tatusova T, Maglott DR. 2007. NCBI reference sequences (RefSeq): a curated nonredundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35(Database issue):D61-5. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ and others. 2007. PLINK: a tool set for whole-genome association and populationbased linkage analyses. Am J Hum Genet 81(3):559-75. Rafiq MA, Kuss AW, Puettmann L, Noor A, Ramiah A, Ali G, Hu H, Kerio NA, Xiang Y, Garshasbi M and others. 2011. Mutations in the alpha 1,2-mannosidase gene, MAN1B1, cause autosomalrecessive intellectual disability. Am J Hum Genet 89(1):176-82. Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. 2010. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 7(8):575-6. Sheridan E, Wright J, Small N, Corry PC, Oddie S, Whibley C, Petherick ES, Malik T, Pawson N, McKinney PA and others. 2013. Risk factors for congenital anomaly in a multiethnic birth cohort: an analysis of the Born in Bradford study. Lancet 382:1350-9. . Stenson PD, Mort M, Ball EV, Shaw K, Phillips AD, Cooper DN. 2014. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet. 133:1-9. Stoll C, Alembik Y, Roth MP, Dott B. 1999. Parental consanguinity as a cause for increased incidence of births defects in a study of 238,942 consecutive births. Ann Genet 42(3):133-9. Stoltenberg C, Magnus P, Skrondal A, Lie RT. 1999. Consanguinity and recurrence risk of birth defects: a population-based study. Am J Med Genet 82(5):423-8.

This article is protected by copyright. All rights reserved.

25

Tokunaga S, Hashiguchi A, Yoshimura A, Maeda K, Suzuki T, Haruki H, Nakamura T, Okamoto Y, Takashima H. 2012. Late-onset Charcot-Marie-Tooth disease 4F caused by periaxin gene mutation. Neurogenetics 13(4):359-65. Tucker EJ, Hershman SG, Kohrer C, Belcher-Timme CA, Patel J, Goldberger OA, Christodoulou J, Silberstein JM, McKenzie M, Ryan MT and others. 2011. Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation. Cell Metab 14(3):428-34. Wang K, Li M, Hakonarson H. 2010. ANNOVAR: functional annotation of genetic variants from highthroughput sequencing data. Nucleic Acids Res 38(16):e164. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z and others. 2013. Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 369(16):1502-11. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z. 2009. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25(21):2865-71. Yoshida A, Kobayashi K, Manya H, Taniguchi K, Kano H, Mizuno M, Inazu T, Mitsuhashi H, Takahashi S, Takeuchi M and others. 2001. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 1(5):717-24. Zhang Q, Bethmann C, Worth NF, Davies JD, Wasner C, Feuer A, Ragnauth CD, Yi Q, Mellad JA, Warren DT and others. 2007. Nesprin-1 and -2 are involved in the pathogenesis of Emery Dreifuss muscular dystrophy and are critical for nuclear envelope integrity. Hum Mol Genet 16(23):2816-33.

This article is protected by copyright. All rights reserved.

26

Table 1. Overview of the patient collection

Country of origin

Phenotype

Family_1

Lebanon

Family_2

Lebanon

Sclerosing bone dysplasia Syndromic ID/DD

Family_3

Jordan

Syndromic ID/DD

Family_4

Lebanon

Family_5

Lebanon

Syndromic ID + Neurological disorder Neurological disorder

Family_6

Egypt

Family_7

Affected individuals per family

Age of affected ‡ individuals

Closest relationship between parents of probands

2

39/36

1st cousins

2

34/33

1st cousins double 1st cousins double 2nd cousins 1st cousins

3

‡‡

1

2

41/31

2

2/1

Syndromic ID/DD

2

12/3

Egypt

Syndromic ID/DD

2

7/3

Family_8

Egypt

3

4/3/1.5

Family_9

Greece

1

1

1st cousins

Family_10

Greece

Skeletaldysplasia Non-syndromic ID/ DD Non-syndromic ID/ DD

1st cousins 1st cousins, 2nd cousins 1st cousins

3

35/28/1.5

4th cousins

Family_11

Egypt

Syndromic ID/DD

2

10/5

Family_12

Egypt

Syndromic ID/DD

2

2/1

double 1st cousins 1st cousins

Family_13

Egypt

Syndromic ID/DD

3

9/1.5/1.5

1st cousins

Family_14

Egypt

Syndromic ID/DD

2

20/10

1st cousins

Family_15

Jordan

Syndromic ID/DD

3

17/11/9

1st cousins

Family_16

Greece

Skeletaldysplasia

3

11/8/5

1st cousins

Family_17

Egypt

Skeletaldysplasia

2

19/15

1st cousins

Family_18

Iraq

Thrombocytopenia

3

9/7/6

1st cousins

Family_19

Morocco

Cardiac malformation

3

10/6/3

1st cousins

Family_20

Jordan

Syndromic ID/DD

2

19/5

1st cousins

3

1.5

‡‡

2

10/2

2

24/5

1st cousins 2nd cousins once removed 1st cousins

3

19/17/6

1st cousins

2

4/1.5

1st cousins

Family_21

UAE

Family_22

Greece

Family_23

Jordan

Family_24

Switzerland

Family_25

Jordan

Syndromic ID/DD Non-syndromic ID/ DD Syndromic ID/DD Non-syndromic ID/ DD Syndromic ID/DD

Family_26

Jordan

Syndromic ID/DD

3

7/4/2

1st cousins

Family_27

Tunisia

2

14/2.5

1st cousins

Family_28

Jordan

2

13/10

1st cousins

Family_29

Jordan

Syndromic ID/DD Non-syndromic ID/ DD Visual impairment

2

3/0.5

1st cousins

Family_30

Iraq

Syndromic ID/DD

3

33/29/20

1st cousins

This article is protected by copyright. All rights reserved.

27

Family_31

Jordan

Syndromic ID/DD

3

8/6/5

Family_32

Jordan

Syndromic ID/DD

3

15/13/7

double 1st cousins 1st cousins

Family_33

Jordan

Regression / ID

2

7/4

1st cousins

Family_34

Jordan

Syndromic ID/DD

3

24/15/1.5

2nd cousins

Family_35

Egypt

Syndromic ID/DD

2

13/5

1st cousins

Family_36

Egypt

2

15/9

1st cousins

Family_37

Egypt

2

11/10

1st cousins

Family_38

Egypt

2

7/1

1st cousins

Family_39

Egypt

2

6/5

1st cousins

Family_40

Egypt

2

11/2

1st cousins

Family_41

Egypt

2

5/3

1st cousins

Family_42

Egypt

Syndromic ID/DD Non-syndromic ID/ DD Non-syndromic ID/ DD Non-syndromic ID/ DD Syndromic ID/DD Non-syndromic ID/ DD Syndromic ID/DD

3

4/3/3

1st cousins

Family_43

Jordan

Syndromic ID/DD

3

19/11/5

1st cousins

Family_44

Jordan

Microphthalmia

3

15/14/4

1st cousins

Family_45

Jordan

Syndromic ID/DD

2

6/5

1st cousins

Family_46

Jordan

Syndromic ID/DD

2

4/1

1st cousins

Family_47

Jordan

Syndromic ID/DD

2

9/7

1st cousins

Family_48

Egypt

Syndromic ID/DD

2

17/10

1st cousins

Family_49

Egypt

2

6/3

1st cousins

Family_50

Egypt

Skeletal dysplasia Progressive hypotonia

2

20/19

1st cousins



age in years, ‡‡two of the affected individuals are deceased

This article is protected by copyright. All rights reserved.

28

Table 2. Overview of the variants identified by exome sequencing in known pathogenic genes Family ID

Gene (OMI M #)

Exon

Variant

dbSNP, frequenc ‡ y

Disease (OMIM #)

Variant literature

Family_1

DMP1 (6009 80)

exon 2

NM_004407:c.1A>G: p.(Met1Val)

rs104893 834, NA

 Hypophosphatemic rickets (241520)

Family_1 2

ARFGE F2 (6053 71)

exon 20

NM_006420.2:c.277 6C>T: p.(Arg926*)

NA

 Periventricular heterotopia with microcephaly, autosomal recessive (608097)

Feng et al(Feng, et al., 2006), Lorenz-Deperieux et al(LorenzDepiereux, et al., 2006) novel

Family_1 3

FKTN (6074 40)

exon 4

NM_006731:c.218T> C: p.(Phe73Ser)

NA

novel

Family_2 6

SEPSE CS (6138 11) GUCY2 D (6001 79) BBS4 (6003 74) SYNE1 (6084 41)

exon 11

NM_016955:c.1466A >T: p.(Asp489Val)

rs145703 544, 0.022%

 Dilated Cardiomyopathy IX (611615)  Muscular dystrophydystroglycanopathy type (brain -eye anomalies) A4 (253800)  Muscular dystrophydystroglycanopathy type (without mental retardation) B4 (613152)  Muscular dystrophydystroglycanopathy type (limb-girdle) C4 (611588)  Pontocerebellar hypoplasia type 2D (613811)

exon 13

NM_000180:c.2563C >T: p.(Gln855*)

NA

El-Shanti et al(ElShanti, et al., 1999)

exon 4

NM_033028:c.1573C>G

NA

 Cone-rod dystrophy 6 (601777)  Leber congenital amaurosis 1 (204000)  Bardet-Biedl syndrome 4 (209900)

exon 142

NM_033071:c.25597 dup: p.(Ser8533Phefs*2)

NA

Family_2 9

Family_3 0 Family_3 1

This article is protected by copyright. All rights reserved.

 Emery-Dreifuss muscular dystrophy 4, autosomal dominant (612998)  Spinocerebellar ataxia, autosomal recessive 8 (610743)

in

Harville et al(Harville, et al., 2010) Novel

29

 Muscular dystrophydystroglycanopathy (congenital with brain and eye anomalies), type A3 (253280)  Muscular dystrophydystroglycanopathy (congenital with mental retardation), type B3 (613151)  Muscular dystrophydystroglycanopathy (limbgirdle), type C3 (613157)  Combined oxidative phosphorylation deficiency 15 (614947)

Yoshida et al(Yoshida, et al., 2001)

NA

 Mental retardation, autosomal recessive 15 (614202)

novel

NM_016360.3:c.421 C>T: p.(Arg141*)

NA

novel

exon 20

NM_005609.2:c.244 7G>A: p.(Arg816His)

exon 7

NM_181882.2:c.309 9del: p.(Glu1034Argfs*5)

rs139230 055, 0.000439 rs139230 055, 0.000439

 Leigh syndrome due to mitochondrial complex IV deficiency  McArdle disease (232600)

Family_3 2

POMG NT1 (6068 22)

exon 18

NM_017739.3:c.153 9+1G>A

rs138642 840, 0.0879%

Family_3 ‡‡ 6

MTFM T (6117 66) MAN1 B1 (6043 46) TACO1 (6129 58) PYGM (6084 55) PRX (6057 25)

exon 1

NM_139242.3:c.17G >C: p.(Arg6Pro)

NA

exon 13

NM_016219.4:c.199 0del: p.(Thr664Argfs*64)

exon 3

Family_3 7 Family_3 ‡‡‡ 8 Family_3 ‡‡‡ 9

Family_4 3 Family_4 4

Family_4 6

Family_4 8

Family_4 9

TUSC3 (6013 85) STRA6 (6107 45)

exon 4

NM_006765.3:c.544 A>T: p.(Ile182Phe)

NA

exon 19

NM_022369.3:c.193 1C>T: p.(Thr644Met)

rs118203 960, 0.00022

ALDH3 A2 (6095 23) RNASE T2 (6129 44) MMP2 (1203 60)

exon 4

NM_000382.2:c.628 G>A: p.(Gly210Arg)

NA

exon 27

NM_003730.4:c.115 dup: p.(Met39Asnfs*7)

NA

exon 4

NM_004530.4:c.538 G>A: p.(Asp180Asn)

NA

This article is protected by copyright. All rights reserved.

novel

 Charcot-Marie-Tooth disease, type 4F (614895)  Dejerine-Sottas disease, autosomal recessive (145900)  Mental retardation, autosomalrecessive 7 (611093)  Microphthalmia, isolated, with coloboma 8 (601186)  Microphthalmia, syndromic 9 (601186)  Sjogren-Larsson syndrome (270200)

 Leukoencephalopathy, cystic, withoutmegalencephaly (612951)  Torg-Winchester syndrome(259600)

novel

Pautto et al(Pasutto, et al., 2007) novel

novel

novel

30



the variant’s name in dbSNP followed by the reported frequency.

carries two pathogenic variants.

‡‡

This patient possibly

‡‡‡

These variants only partially account for the patient’s

phenotype.

This article is protected by copyright. All rights reserved.

31

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.

Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic ...
720KB Sizes 0 Downloads 4 Views