Identification of Candidate Genes In Neurogenetic Disorders By Whole Exome Sequencing and Modeling In Zebrafish

ABSTRACT IDENTIFICATION OF CANDIDATE GENES IN NEUROGENETIC DISORDERS BY WHOLE EXOME SEQUENCING AND MODELING IN ZEBRAFISH AYŞEGÜL OZANTÜRK Doctor of Philosophy, Department of Biology, Molecular Biology Section Supervisor: Prof. Dr. R. Köksal Özgül September 2015, 171 pages Application of whole exome and whole genome sequencing on affected individuals with unknown diseases has been using successfully to understand the cause of human inherited disorders. It became a diagnostic approach in medicine for undiagnosed families. By combining these high throughput-sequencing methods with functional studies that conducted on model organism accelerate the succes rate. The aim of this study is to identify candidate genes in patients with unknown conditions by applying whole exome sequencing and test pathogenicity of the determined alleles on zebrafish. This study includes six undiagnosed families with neurogenetic/neurocognitive clinical features. All families have two affected individuals with the same or similar neurological findings. The most common symptom of the affected individuals is intellectual disability. Three families have anatomical abnormalities; one family has cerebellar atrophy and two have craniofacial dysmorphology. First, all families, parents and affected individuals, were subjected to whole exome sequencing to list candidate genes that might be responsible for observed phenotypes. After defining the candidates, all selected alleles were tested on zebrafish to reveal their contribution to the disease phenotypes. Zebrafish is a relatively new organism to model human genetics disorders. There is a high amount of genomic sequence similarity, 70 %, between human and zebrafish. iv Using these molecular approaches genetic causes of the disorders in three families were identified; Family 1, has two affected sibs, M-11-1496 and M-11-1497, who have intellectual disability, ambiguous genitale, and craniofacial dysmorphology, is a carrier of two CNVs which are located in 16q (13,5 Mb duplication) and 5p (7,5 Mb deletion). Clinical features of M-11-1472 and M-11-1473 in Family 2, failure to thrive, microcephaly, vision impairment and hearing loss, axial hypotony, coreic movement, were associated with Pontocerebellar hypoplasia type 2B which caused by a novel mutation, p.T364R, in TSEN2 gene. Family 3 has two affected sibs with dysmorphic features, bigger and low set ears, hypertelorism is associated with ATRX syndrome which was resulted in a novel mutation, p.D217G, in ATRX gene. Family 4 has two affected males with severe intellectual disability, autism spectrum disorders. Exome sequencing identified 5 candidate genes, ANKRD30A, TCEB3, PLIN2, VPS26B, METTL4 in this family. There was no association between these candidate genes and clinical features of affected males. Family 5 has two affected sibs, M-11-1500 and M-11-1501, with intellectual disability, autism spectrum disorders. 11 candidate genes identified by whole exome sequencing. This family was excluded from the functional study since there are too many candidate alleles need to tested on zebrafish. Family 6 has a clear candidate, CELSR3, in which functional study showed the severe phenotype that might be related neurological symptoms seen in patient M-11-1476 and M-11-1477. By combining genomics and functional aspects, we were able to define molecular causes of neurologic phenotypes in three families and a likely pathogenic variation in one family. Our results provided molecular diagnosis for these families. Therefore, these applied combined molecular approaches such as whole exome sequencing and zebrafish modeling are powerful methodologies to elucidate the molecular pathology of diseases and especially useful in identification of rare and unknown diseases.ÖZET NÖROGENETİK HASTALIKLARDA EKZOM DİZİLEME ANALİZİ İLE ADAY GENLERİN BELİRLENMESİ VE ZEBRAFİSH MODELLERİNİN OLUŞTURULMASI AYŞEGÜL OZANTÜRK Doktora, Biyoloji Bölümü, Moleküler Biyoloji AbD Tez Danışmanı: Prof. Dr. R. Köksal Özgül Eylül 2015, 171 sayfa Tanısı konulamayan nörogenetik hastalıklarda uygulanan tüm ekzom/genom dizileme yöntemleri ile bu kalıtsal hastalıkların moleküler patolojilerinin belirlenmesinde başarılı sonuçlar elde edilmekte ve klasik yöntemlerle tanısı aydınlatılmamış hastalara moleküler genetik yaklaşımlarla tanı konulabilmektedir. Yüksek işlem hacimli genomik yöntemlerle model organizmalar üzerinde yapılan fonksiyonel çalışmaların birarada kullanılması bu hastalarda nadir görülen sendromik fenotiplerin moleküler nedenlerini açıklamada başarı oranını yükseltmektedir. Bu tez kapsamında, tanısı klasik yöntemler ile aydınlatılamamış ailelerde ekzom dizileme yöntemi ile hastalık fenotipinden sorumlu olabilecek aday genlerin belirlenmesi ve bu genlerin zebrafish üzerinde modellenmesi amaçlanmaktadır. Çalışmaya nörogenetik/nörokognitif bulgulara sahip altı farklı aile dahil edilmiştir. Her ailede aynı veya benzer klinik bulgulara sahip iki etkilenmiş birey bulunmaktadır. Çalışmaya dahil edilen ailelerde baskın ortak fenotip motor mental geriliktir. İki ailede ise yapısal bozukluk olan serebellar atrofi (Aile 2) ve kraniyofasiyal anomali (Aile 1, Aile 3) görülmüştür. Çalışmanın ilk aşamasında, tüm aileler için, ebeveynler ve hasta kardeşlerde ekzom dizileme analizi yapıldı. Bu yöntem ile hastalık fenotipinden sorumlu olabilecek aday genler belirlendi. İkinci aşama fonksiyonel çalışmaları içermektedir. Bu basamakta, saptanan aday genlerin/varyasyonların zebrafish üzerinde patojeniteleri test edilerek, ii hastalık fenotipine olan etkileri aydınlatıldı. Zebrafish insan hastalıklarının modellenmesi için kullanılan yeni bir model organizmadır. Zebrafish genomu ile insan genomu % 70 benzerlik göstermektedir.Ekzom dizileme analizi sonucunda üç ailede hastalık fenotipiyle ilişkili olan genetik faktörler belirlenmiştir. Aile 1’de birey M-11-1496 ve M-11-1497’de gözlenen zeka geriliği, dismorfik yüz yapısı ve genital anomali bulguları bu bireylerde saptanan iki büyük kopya sayısı değişikliği (CNV) ile ilişkilendirildi. Bu CNV’ler 16. kromozomun kısa kolunda 13.5 Mb’lık bir duplikasyon ve 5. kromozomun uzun kolunda 7.5 Mb’lık bir delesyondur. Aile 2’de birey M-11-1472 ve M-11-1473’te gözlenen gelişme geriliği, mikrosefali, ağır zeka geriliği, görme ve işitme kaybı, aksiyel hipotoni, koreik hareketler TSEN2 geninde saptanan p.T364R yeni mutasyonu ile ilişkilendirilerek hastalık fenotipinin pontoserebellar hipopilazi tip 2B olduğu aydınlatıldı. Aile 3’de birey M-11-1960 ve M-11-1465’te gözlenen kaba yüz görünümü, hipertelorizm, büyük ve düşük kulak kepçeleri, hafif zeka geriliği ATRX geninde saptanan p.D217G yeni mutasyonu ile ilişkilendirilerek hastalık fenotipinin alfa-talasemi/mental gerilik/hipotonik yüz sendromu olduğu sonucuna varıldı. Aile 4’te birey M-11-1447 ve M-11-1448’de ağır zeka geriliği, otistik bulgular ve ekolali rapor edilmiştir. Bu ailede ANKRD30A, TCEB3, PLIN2, VPS26B, METTL4, hastalıktan sorumlu aday genler olarak saptandı ancak hastalarda görülen nörolojik bulgularla ilişkileri aydınlatılamadı. Aile 5’te birey M-11-1500 ve M-111501’de görülen zeka geriliği ve otistik bulguları ekzom verilerinden elde edilen on bir aday gen ile ilişkisi açıklanamadı. Bu aile fonksiyonel çalışmaya dahil edilmedi. Bir ailede (Aile 6) bulunan iki hastada gözlenen nörolojik fenotip, zeka geriliği ve otizm bulgularının, hasta bireylerde saptanan mutasyonun zebrafishde yapılan fonksiyonel çalışması sonucu saptanan tek aday gen olan CELSR3 geninin hastalıktan sorumlu olduğu gösterildi. Bu tez çalışmasının sonucunda, ekzom dizileme ve fonksiyonel analizler birarada kullanılarak zebrafish modelleri üzerinde hastalıktan sorumlu mutasyonların etkileri gösterildi. Elde edilen sonuçlar, insanda görülen patolojik fenotiplerin aydınlatılması ve özellikle nadir ve bilinmeyen hastalıkların tanısının konulması için ekzom dizileme ve fonksiyonel analizlerin etkin bir şekilde kullanılabilecek iyi bir hayvan modeli ve güçlü moleküler yaklaşımlar olduğunu göstermektedir

A T(5;16) Translocation Is The Likely Driver Of A Syndrome With Ambiguous Genitalia, Facial Dysmorphism, Intellectual Disability, And Speech Delay

Genetic studies grounded on monogenic paradigms have accelerated both gene discovery and molecular diagnosis. At the same time, complex genomic rearrangements are also appreciated as potent drivers of disease pathology. Here, we report two male siblings with a dysmorphic face, ambiguous genitalia, intellectual disability, and speech delay. Through quad-based whole-exome sequencing and concomitant molecular cytogenetic testing, we identified two copy-number variants (CNVs) in both affected individuals likely arising from a balanced translocation: a 13.5-Mb duplication on Chromosome 16 (16q23.1 → 16qter) and a 7.7-Mb deletion on Chromosome 5 (5p15.31 → 5pter), as well as a hemizygous missense variant in CXorf36 (also known as DIA1R). The 5p terminal deletion has been associated previously with speech delay, whereas craniofacial dysmorphia and genital/urinary anomalies have been reported in patients with a terminal duplication of 16q. However, dosage changes in either genomic region alone could not account for the overall clinical presentation in our family; functional testing of CXorf36 in zebrafish did not induce defects in neurogenesis or the craniofacial skeleton. Notably, literature and database analysis revealed a similar dosage disruption in two siblings with extensive phenotypic overlap with our patients. Taken together, our data suggest that dosage perturbation of genes within the two chromosomal regions likely drives the syndromic manifestations of our patients and highlight how multiple genetic lesions can contribute to complex clinical pathologies.PubMe

The phenotypic and molecular genetic spectrum of Alström syndrome in 44 Turkish kindreds and a literature review of Alström syndrome in Turkey.

Alström syndrome (ALMS) is an autosomal recessive disease characterized by multiple organ involvement, including neurosensory vision and hearing loss, childhood obesity, diabetes mellitus, cardiomyopathy, hypogonadism, and pulmonary, hepatic, renal failure and systemic fibrosis. Alström Syndrome is caused by mutations in ALMS1, and ALMS1 protein is thought to have a role in microtubule organization, intraflagellar transport, endosome recycling and cell cycle regulation. Here, we report extensive phenotypic and genetic analysis of a large cohort of Turkish patients with ALMS. We evaluated 61 Turkish patients, including 11 previously reported, for both clinical spectrum and mutations in ALMS1. To reveal the molecular diagnosis of the patients, different approaches were used in combination, a cohort of patients were screened by the gene array to detect the common mutations in ALMS1 gene, then in patients having any of the common ALMS1 mutations were subjected to direct DNA sequencing or next-generation sequencing for the screening of mutations in all coding regions of the gene. In total, 20 distinct disease-causing nucleotide changes in ALMS1 have been identified, eight of which are novel, thereby increasing the reported ALMS1 mutations by 6% (8/120). Five disease-causing variants were identified in more than one kindred, but most of the alleles were unique to each single patient and identified only once (16/20). So far, 16 mutations identified were specific to the Turkish population, and four have also been reported in other ethnicities. In addition, 49 variants of uncertain pathogenicity were noted, and four of these were very rare and probably or likely deleterious according to in silico mutation prediction analyses. ALMS has a relatively high incidence in Turkey and the present study shows that the ALMS1 mutations are largely heterogeneous; thus, these data from a particular population may provide a unique source for the identification of additional mutations underlying Alström Syndrome and contribute to genotype-phenotype correlation studies. J Hum Genet 2015 Jan; 60(1):1-9