Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development

Aim Congenital hypopituitarism has an incidence of 1:3500–10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism. Methods Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel. Results We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A]. Conclusions Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism.

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Massively Parallel Sequencing for Genetic Diagnosis of Hearing Loss

Otolaryngology ◽

10.1177/0194599815591156 ◽

2015 ◽

Vol 153 (2) ◽

pp. 175-182 ◽

Cited By ~ 68

Author(s):

A. Eliot Shearer ◽

Richard J. H. Smith

Keyword(s):

Hearing Loss ◽

Massively Parallel Sequencing ◽

Genetic Diagnosis ◽

Massively Parallel ◽

Parallel Sequencing

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Non-ocular Stickler Syndrome With a Novel Mutation in COL11A2 Diagnosed by Massively Parallel Sequencing in Japanese Hearing Loss Patients

Annals of Otology Rhinology & Laryngology ◽

10.1177/0003489415575044 ◽

2015 ◽

Vol 124 (1_suppl) ◽

pp. 111S-117S ◽

Cited By ~ 6

Author(s):

Yoh-ichiro Iwasa ◽

Hideaki Moteki ◽

Mitsuru Hattori ◽

Ririko Sato ◽

Shin-ya Nishio ◽

...

Keyword(s):

Hearing Loss ◽

Clinical Symptoms ◽

Massively Parallel Sequencing ◽

Novel Mutation ◽

Genetic Diagnosis ◽

Analysis Data ◽

Massively Parallel ◽

Stickler Syndrome ◽

Parallel Sequencing ◽

First Case

Objectives: This study aims to document the clinical features of patients with COL11A2 mutations and to describe the usefulness of massively parallel sequencing. Methods: One thousand one hundred twenty (1120) Japanese hearing loss patients from 53 ENT departments nationwide participated in this study. Massively parallel sequencing of 63 genes implicated in hearing loss was performed to identify the genetic causes in the Japanese hearing loss patients. Results: A novel mutation in COL11A2 (c.3937_3948delCCCCCAGGGCCA) was detected in an affected family, and it was segregated in all hearing loss individuals. The clinical findings of this family were compatible with non-ocular Stickler syndrome. Orofacial features of mid-facial hypoplasia and slowly progressive mild to moderate hearing loss were also presented. Audiological examinations showed favorable auditory performance with hearing aid(s). Conclusion: This is the first case report of the genetic diagnosis of a non-ocular Stickler syndrome family in the Japanese population. We suggest that it is important to take both genetic analysis data and clinical symptoms into consideration to make an accurate diagnosis.

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OR27-01 Combining Clinical and Genetic Approaches in Diagnosing a Large Brazilian Cohort of Patients with 46,XY Differences/Disorders of Sex Development (DSD)

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1508 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Nathalia Lisboa Rosa Almeida Gomes ◽

Rafael Loch Batista ◽

Mirian Yumie Nishi ◽

Antonio Marcondes Lerario ◽

Thatiana Evilen Silva ◽

...

Keyword(s):

Molecular Diagnosis ◽

Massively Parallel Sequencing ◽

Genetic Diagnosis ◽

Disorders Of Sex Development ◽

Androgen Insensitivity Syndrome ◽

Unknown Etiology ◽

Androgen Synthesis ◽

Sex Development ◽

Liquid Chromatography Tandem Mass ◽

Phenotype Heterogeneity

Abstract Background: It is recommended a multidisciplinary approach consisted of clinical, hormonal and genetic workups for diagnosing 46,XY DSD. However, no previous study has quantified how useful is this combined approach. Objectives: To retrospectively review the clinical and genetic findings for diagnosing a large cohort of patients with 46,XY DSD from a single Brazilian center. Methods: 247 non-syndromic 46,XY DSD individuals (159 sporadic and 88 familial cases from 39 families) were studied. Clinical and hormonal data were collected from medical files. Testosterone (T), androstenedione (A) were measured by immunoradiometric or immunofluorimetric assays and dihydrotestosterone (DHT) by RIA after celite chromatography or by liquid chromatography tandem mass spectrometry; T/DHT and T/A ratios were calculated. Analysis of sensitivity (SE), specificity (SP) of T/DHT was performed, being the molecular diagnosis considered the gold standard for diagnosing SRD5A2 deficiency. A T/A>0.8 was considered indicative of 17ß-HSDB3 deficiency. The patients were clinically classified into four subgroups: 1) androgen insensitivity syndrome (AIS), 2) gonadal dysgenesis (GD); 3) defects in androgen synthesis (DAS) and 4) DSD of unknown etiology. Molecular studies were performed by Sanger sequencing and/ or massively parallel sequencing (MPS). Results: The median age at first visit was 14 years (range 0.1 to 59 years). The molecular diagnosis was established in 96.5% of the cases with AIS (n=28/29), in 96% of the subjects with DAS (n=46/48), in 36% of the patients with GD (n=21/57) and in 26.7% (n=15/56) with DSD of unknown etiology. The best cut-off for T/DHT in basal state and hCG stimulated was 12.5 (SE=100%; SP=78.57%) and 24 (SE=87.5%; SP=95.7%) respectively. A T/A<0.8 was observed in 13/16 (81%) of the patients with molecular diagnosis of 17ß-HSDB3 deficiency and also in 1/49 patients with other diagnose. Classification according to the phenotype matched with the genetic diagnosis in most cases. The molecular evaluation allowed that 16% (9/56) of the patients that were classified as DSD of unknow etiology had a definitive diagnosis, including six GD cases, two individuals with SRD5A2 deficiency and one with 17ß-HSDB3 deficiency. A clear AIS phenotype of five patients allowed us to consider and prove the pathogenicity of two synonymous and one promoter region variants as the cause of AIS. The combination of clinical and molecular diagnosis led to an increase in 8% the diagnosis in a total of 116 index-cases (58.5%) with a molecular diagnosis. Conclusion: Considering the phenotype heterogeneity, pitfalls of the hormonal assessment and number of genes involved, it is reasonable to consider MPS as a first test for diagnosing patients with 46,XY DSD. However, the combination of clinical and molecular diagnosis is more accurate than either strategies alone in diagnosing 46,XY DSD.

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Impacts of massively parallel sequencing for genetic diagnosis of neuromuscular disorders

Acta Neuropathologica ◽

10.1007/s00401-012-1072-7 ◽

2012 ◽

Vol 125 (2) ◽

pp. 173-185 ◽

Cited By ~ 29

Author(s):

Nasim Vasli ◽

Jocelyn Laporte

Keyword(s):

Massively Parallel Sequencing ◽

Neuromuscular Disorders ◽

Genetic Diagnosis ◽

Massively Parallel ◽

Parallel Sequencing

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USH2 Caused by GPR98 Mutation Diagnosed by Massively Parallel Sequencing in Advance of the Occurrence of Visual Symptoms

Annals of Otology Rhinology & Laryngology ◽

10.1177/0003489415574070 ◽

2015 ◽

Vol 124 (1_suppl) ◽

pp. 123S-128S ◽

Cited By ~ 5

Author(s):

Hideaki Moteki ◽

Hidekane Yoshimura ◽

Hela Azaiez ◽

Kevin T. Booth ◽

A. Eliot Shearer ◽

...

Keyword(s):

Hearing Loss ◽

Massively Parallel Sequencing ◽

Usher Syndrome ◽

Genetic Diagnosis ◽

Massively Parallel ◽

Parallel Sequencing ◽

Visual Symptoms ◽

Targeted Genomic Enrichment ◽

Usher Syndrome Type

Objective: We present 2 patients who were identified with mutations in the GPR98 gene that causes Usher syndrome type 2 (USH2). Methods: One hundred ninety-four (194) Japanese subjects from unrelated families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known nonsyndromic hearing loss genes were used to identify the genetic causes of hearing loss. Results: We identified causative mutations in the GPR98 gene in 1 family (2 siblings). The patients had moderate sloping hearing loss, and no progression was observed over a period of 10 years. Fundus examinations were normal. However, electroretinograms revealed impaired responses in both patients. Conclusion: Early diagnosis of Usher syndrome has many advantages for patients and their families. This study supports the use of comprehensive genetic diagnosis for Usher syndrome, especially prior to the onset of visual symptoms, to provide the highest chance of diagnostic success in early life stages.

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Noninvasive Prenatal Diagnosis of Duchenne Muscular Dystrophy: Comprehensive Genetic Diagnosis in Carrier, Proband, and Fetus

Clinical Chemistry ◽

10.1373/clinchem.2014.236380 ◽

2015 ◽

Vol 61 (6) ◽

pp. 829-837 ◽

Cited By ~ 29

Author(s):

Seong-Keun Yoo ◽

Byung Chan Lim ◽

Jiyoung Byeun ◽

Hee Hwang ◽

Ki Joong Kim ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Recombination Event ◽

Massively Parallel Sequencing ◽

Genetic Diagnosis ◽

Maternal Plasma ◽

Massively Parallel ◽

Carrier Status ◽

Parallel Sequencing ◽

Sequencing Platform ◽

Noninvasive Prenatal Diagnosis

Abstract BACKGROUND Noninvasive prenatal diagnosis of monogenic disorders using maternal plasma and targeted massively parallel sequencing is being investigated actively. We previously demonstrated that comprehensive genetic diagnosis of a Duchenne muscular dystrophy (DMD) patient is feasible using a single targeted sequencing platform. Here we demonstrate the applicability of this approach to carrier detection and noninvasive prenatal diagnosis. METHODS Custom solution-based target enrichment was designed to cover the entire dystrophin (DMD) gene region. Targeted massively parallel sequencing was performed using genomic DNA from 4 mother and proband pairs to test whether carrier status could be detected reliably. Maternal plasma DNA at varying gestational weeks was collected from the same families and sequenced using the same targeted platform to predict the inheritance of the DMD mutation by their fetus. Overrepresentation of an inherited allele was determined by comparing the allele fraction of 2 phased haplotypes after examining and correcting for the recombination event. RESULTS The carrier status of deletion/duplication and point mutations was detected reliably through using a single targeted massively parallel sequencing platform. Whether the fetus had inherited the DMD mutation was predicted correctly in all 4 families as early as 6 weeks and 5 days of gestation. In one of these, detection of the recombination event and reconstruction of the phased haplotype produced a correct diagnosis. CONCLUSIONS Noninvasive prenatal diagnosis of DMD is feasible using a single targeted massively parallel sequencing platform with tiling design.

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Pre-implantation genetic diagnosis for a family with Usher syndrome through targeted sequencing and haplotype analysis

10.1101/460378 ◽

2018 ◽

Author(s):

Haining Luo ◽

Chao Chen ◽

Yun Yang ◽

Yuan Yuan ◽

Wanyang Wang ◽

...

Keyword(s):

Molecular Diagnosis ◽

Genomic Dna ◽

Haplotype Analysis ◽

Massively Parallel Sequencing ◽

Multiple Displacement Amplification ◽

Usher Syndrome ◽

Genetic Diagnosis ◽

Massively Parallel ◽

Parallel Sequencing ◽

Targeted Capture

AbstractObjectiveOur objective was to investigate the applicability of targeted capture massively parallel sequencing in developing personalized pre-implantation genetic diagnosis (PGD) assay.MethodsOne couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. The genomics DNA (gDNA) was extracted from the peripheral blood and underwent in vitro fertilization (IVF)-PGD. Prenatal molecular diagnosis was performed in the 20th week of gestation and the chromosomal anomaly was analyzed.ResultsCustomized capture probe targeted at USH2A gene and 350kb flanking region were designed for PGD. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A HMM-based haplotype analysis was performed to deduce embryo’s genotype by using SNPs identified in each sample. Four embryos were diagnosed as free of father’s rare mutation, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGD and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby’s genomic DNA sample. The auditory test showed that the hearing was normal.ConclusionTargeted capture massively parallel sequencing (MPS) is an effective and convenient strategy to develop customized PGD assay.Key pointsGenetic counseling session was conducted with a family having Usher patient who was molecularly diagnosed, and a healthy baby was born with the help of successful PGD assay. This is of vast importance in management plans since it is the first report of PGD in Usher syndrome based on targeted capture MPS.

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