Determining Cost-Optimal Next-Generation Sequencing Panels for Rare Disease and Pharmacogenomics Testing.

BACKGROUND: Multi-gene panel sequencing using next-generation sequencing (NGS) methods is a key tool for genomic medicine. However, with an estimated 140 000 genomic tests available, current system inefficiencies result in high genetic-testing costs. Reduced testing costs are needed to expand the availability of genomic medicine. One solution to improve efficiency and lower costs is to calculate the most cost-effective set of panels for a typical pattern of test requests. METHODS: We compiled rare diseases, associated genes, point prevalence, and test-order frequencies from a representative laboratory. We then modeled the costs of the relevant steps in the NGS process in detail. Using a simulated annealing-based optimization procedure, we determined panel sets that were more cost-optimal than whole exome sequencing (WES) or clinical exome sequencing (CES). Finally, we repeated this methodology to cost-optimize pharmacogenomics (PGx) testing. RESULTS: For rare disease testing, we show that an optimal choice of 4-6 panels, uniquely covering genes that comprise 95% of the total prevalence of monogenic diseases, saves $257-304 per sample compared with WES, and $66-135 per sample compared with CES. For PGx, we show that the optimal multipanel solution saves $6-7 (27%-40%) over a single panel covering all relevant gene-drug associations. CONCLUSIONS: Laboratories can reduce costs using the proposed method to obtain and run a cost-optimal set of panels for specific test requests. In addition, payers can use this method to inform reimbursement policy.

Lipoprotein Lipase Deficiency.

OBJECTIVES: To analyse the clinical and molecular spectrum of Lipoprotein Lipase (LPL) deficiency and to highlight the effect of a cost-effective indigenous diet for management of this disorder. METHODS: This is a single-centre retrospective study. Fifteen patients from 14 kindreds with severe hypertriglyceridemia (more than 1000 mg/dl) were evaluated for a period of 12.5 y at Amrita Institute of Medical Sciences, Kerala, India. RESULTS: Thirteen of 15 patients were referred after incidental detection of lipemic plasma, 1/15 had chylothorax in the neonatal period and 1/15 had pancreatitis. The mean age of presentation was 7 mo (ranging from 2 d to 4 y), and 20% of the patients had a positive history of consanguinity. Hepatomegaly (15/15), splenomegaly (9/15) and lipemia retinalis (14/15) were common findings. Lipemia retinalis was a useful non-invasive diagnostic tool. All the patients were subjected to diet modification and followed up at regular intervals. Fourteen of 15 complied with the diet, resulting in a dramatic improvement in the fasting lipid profile; only 1/15 developed pancreatitis. Genetic screening analysis was offered to 14/15 patients (1/15 was lost to follow-up); six different variants were identified, of which two were novel variants. CONCLUSIONS: Lipemic serum, chylothorax and recurrent pancreatitis in children should raise the suspicion of Lipoprotein Lipase deficiency. Early diagnosis and prompt initiation of a stringent fat-restricted diet are the keys to success for the management of LPL deficiency and prevention of pancreatitis.

Assessing Utility of Clinical Exome Sequencing in Diagnosis of Rare Idiopathic Neurodevelopmental Disorders in Indian Population.

BACKGROUND: Neurological diseases are phenotypically and genotypically heterogeneous. Clinical exome sequencing (CES) has been shown to provide a high diagnostic yield for these disorders in the European population but remains to be demonstrated for the Indian population. OBJECTIVE: The study aimed to understand the utility of clinical exome sequencing for the diagnosis of neurodevelopmental disorders. MATERIALS AND METHODS: A cohort of 19 idiopathic patients with neurological phenotypes, primarily intellectual disability and developmental delay, were recruited. CES covering 4620 genes was performed on all patients. Candidate variants were validated by Sanger sequencing. RESULTS: CES in 19 patients provided identified 21 variants across 16 genes which have been associated with different neurological disorders. Fifteen variants were reported previously and 6 variants were novel to our study. Eleven patients were diagnosed with autosomal dominant de novo variants, 7 with autosomal recessive and 1 with X-linked recessive variants. CES provided definitive diagnosis to 10 patients; hence, the diagnostic yield was 53%. CONCLUSION: Our study suggests that the diagnostic yield of CES in the Indian population is comparable to that reported in the European population. CES together with deep phenotyping could be a cost-effective way of diagnosing rare neurological disorders in the Indian population.

Lethal Cenani Lenz syndrome in two consecutive pregnancies: Further extension of phenotype from Maldives.

Cenani Lenz syndrome is a rare autosomal recessive disorder associated with variable degree of limb malformations, dysmorphism, and renal agenesis. It is caused due to pathogenic variants in the LRP4 gene, which plays an important role in limb and renal development. Mutations in the APC gene have also been occasionally associated with CLS. The phenotypic spectrum ranges from mild to very severe perinatal lethal type depending on the type of variant. We report a pathogenic variant, c.2710 del T (p.Trp904GlyfsTer5) in theLRP4 gene, in a fetus with lethal Cenani Lenz syndrome with antenatal presentation of tetraphocomelia and symmetrical involvement of hands and feet.

Targeted sequencing of the DMD locus: A comprehensive diagnostic tool for all mutations.

Background & objectives: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the. DMD: gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies. Methods: The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation. Results: In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene. Interpretation & conclusions: Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.

Homozygous sequestosome 1 (SQSTM1) mutation: a rare cause for childhood-onset progressive cerebellar ataxia with vertical gaze palsy.

Mutations in sequestosome 1 (SQSTM1) gene are associated with neurodegenerative diseases, such as frontotemporal dementia and amyotrophic lateral sclerosis. Recently, mutation in SQSTM1 was also found to cause a progressive childhood-onset cerebellar ataxia. We describe here a case of progressive childhood-onset cerebellar ataxia with vertical supra nuclear gaze palsy with no family history and a normal magnetic resonance imaging (MRI) of brain. The clinical exome sequencing in this patient showed a homozygous mutation in SQSTM1. This case highlights the importance of next-generation sequencing in the diagnosis of inherited ataxia syndromes. SQSTM1 mutation should be considered in the differential diagnosis in a patient with both cerebellar ataxia and ophthalmological manifestations.

Chronic Mucocutaneous Candidiasis in an Adolescent Boy Due to a Novel Mutation in TRAF3IP2.

BACKGROUND: IL-17-mediated signaling is crucial in defense against fungi and bacteria. Defective Th17 immunity has been implicated in a group of disorders called chronic mucocutaneous candidiasis (CMC). TRAF3IP2 is an adaptor protein involved in downstream signaling for IL-17 receptors. CASE: An 18-year-old boy, product of consanguineous wedlock, presented with history of repeated episodes of oral thrush and recurrent pneumonia from first year of life. On examination, he was wasted and had oral thrush and abnormal dentition; grade 2 clubbing and respiratory system examination revealed coarse crepitations. On evaluation, HIV status was negative and basic immunological screen was unrewarding. Genetic testing by next-generation sequencing showed a novel homozygous mutation in TRAF3IP2 gene not reported to date. The defect is likely to cause ACT1 deficiency. He was started on antibiotic and antifungal prophylaxis and remains well on follow-up. CONCLUSION: We describe an adolescent boy with recurrent oral candidiasis and bronchiectasis due to a novel mutation in TRAF3IP2 gene, not reported to date. This is also the only second report of CMC due to ACT1 deficiency.

1q42.12q42.2 Deletion in a Child with Midline Defects and Hypoplasia of the Corpus Callosum.

Chromosome 1q42.12q42.2 deletions are documented as "disease causing" and show overlapping phenotypes depending on the genes involved in the deletion. In this report, we detected a 5.8-Mb deletion encompassing the chromosome 1q42.12q42.2 region in a 4-year-old boy with hypoplastic corpus callosum, epilepsy, developmental delay, microcephaly, cataract, cleft palate, and skeletal changes. The deletion was de novo. Genotype-phenotype correlations suggest that the major features of 1q42.12q42.2 microdeletion were attributed to the genes with a high probability of loss-of-function intolerance score in this deletion, namely LBR, ENAH, ACBD3, LIN9, ITPKB, CDC42BPA, ARF1, TAF5L, GALNT2, SPRTN, and EGLN1 along with GNPAT.

Multi-gene testing in neurological disorders showed an improved diagnostic yield: data from over 1000 Indian patients.

BACKGROUND: Neurological disorders are clinically heterogeneous group of disorders and are major causes of disability and death. Several of these disorders are caused due to genetic aberration. A precise and confirmatory diagnosis in the patients in a timely manner is essential for appropriate therapeutic and management strategies. Due to the complexity of the clinical presentations across various neurological disorders, arriving at an accurate diagnosis remains a challenge. METHODS: We sequenced 1012 unrelated patients from India with suspected neurological disorders, using TruSight One panel. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. RESULTS: We were able to detect mutations in 197 genes in 405 (40%) cases and 178 mutations were novel. The highest diagnostic rate was observed among patients with muscular dystrophy (64%) followed by leukodystrophy and ataxia (43%, each). In our cohort, 26% of the patients who received definitive diagnosis were primarily referred with complex neurological phenotypes with no suggestive diagnosis. In terms of mutations types, 62.8% were truncating and in addition, 13.4% were structural variants, which are also likely to cause loss of function. CONCLUSION: In our study, we observed an improved performance of multi-gene panel testing, with an overall diagnostic yield of 40%. Furthermore, we show that NGS (next-generation sequencing)-based testing is comprehensive and can detect all types of variants including structural variants. It can be considered as a single-platform genetic test for neurological disorders that can provide a swift and definitive diagnosis in a cost-effective manner.

Mutation Spectrum of GNE Myopathy in the Indian Sub-Continent.

BACKGROUND: GNE myopathy is an adult onset recessive genetic disorder that affects distal muscles sparing the quadriceps. GNE gene mutations have been identified in GNE myopathy patients all over the world. Homozygosity is a common feature in GNE myopathy patients worldwide. OBJECTIVES: The major objective of this study was to investigate the mutation spectrum of GNE myopathy in India in relation to the population diversity in the country. MATERIALS AND METHODS: We have collated GNE mutation data of Indian GNE myopathy patients from published literature and from recently identified patients. We also used data of people of Indian subcontinent from 1000 genomes database, South Asian Genome database and Strand Life Science database to determine frequency of GNE mutations in the general population. RESULTS: A total of 67 GNE myopathy patients were studied, of whom 21% were homozygous for GNE variants, while the rest were compound heterozygous. Thirty-five different mutations in the GNE gene were recorded, of which 5 have not been reported earlier. The most frequent mutation was p.Val727Met (65%) found mainly in the heterozygous form. Another mutation, p.Ile618Thr was also common (16%) but was found mainly in patients from Rajasthan, while p.Val727Met was more widely distributed. The latter was also seen at a high frequency in general population of Indian subcontinent in all the databases. It was also present in Thailand but was absent in general population elsewhere in the world. CONCLUSION: p.Val727Met is likely to be a founder mutation of Indian subcontinent.

Clinical and genetic analysis of Indian patients with NDP-related retinopathies.

PURPOSE: NDP-related retinopathies are a group of X-linked disorders characterized by degenerative and proliferative changes of the neuroretina, occasionally accompanied with varying degrees of mental retardation and sensorineural hearing loss. NDP is the predominant gene associated with NDP-related retinopathies. The purpose of this study was to report the clinical and genetic findings in three unrelated patients diagnosed with NDP-related retinopathies. METHODS: The patients underwent complete ophthalmic examination followed by genetic analyses. NDP gene was screened by direct sequencing approach. Targeted resequencing of several other ocular genes was carried out in patient samples that either indicated NDP gene deletion or tested negative for NDP mutation. Gene quantitation analysis was performed using real-time PCR. RESULTS: The whole NDP gene was deleted in patient I, while a missense NDP mutation, c.205T>C, was identified in patient II, and both had classical Norrie disease ocular phenotype (with no other systemic defects). Patient III who was diagnosed with familial exudative vitreoretinopathy did not show any mutation in the known candidate genes as well as in other ocular genes tested. CONCLUSIONS: The patient with whole NDP gene deletion did not exhibit any apparent extraocular defects (like mental retardation or sensorineural hearing loss) during his first decade of life, and this is considered to be a notable finding. Our study also provides evidence emphasizing the need for genetic testing which could eliminate ambiguities in clinical diagnosis and detect carrier status, thereby aiding the patient and family members during genetic counseling.

Genetic studies in a patient with X-linked retinoschisis coexisting with developmental delay and sensorineural hearing loss.

BACKGROUND: In this study, we present a juvenile retinoschisis patient with developmental delay, sensorineural hearing loss, and reduced axial tone. X-linked juvenile retinoschisis (XLRS) is a retinal dystrophy, most often not associated with systemic anomalies and also not showing any locus heterogeneity. Therefore it was of interest to understand the genetic basis of the condition in this patient. MATERIALS AND METHODS: RS1 gene screening for XLRS was performed by Sanger sequencing. Whole genome SNP 6.0 array analysis was carried out to investigate gross chromosomal aberrations that could result in systemic phenotype. In addition, targeted next generation sequencing (NGS) was employed to determine any possible involvement of X-linked syndromic and non-syndromic mental retardation genes. This NGS panel consisted of 550 genes implicated in several other rare inherited diseases. RESULTS: RS1 gene screening revealed a pathogenic hemizygous splice site mutation (c.78+1G>T), inherited from the mother. SNP 6.0 array analysis did not indicate any significant chromosomal aberrations that could be disease-associated. Targeted resequencing did not identify any mutations in the X-linked mental retardation genes. However, variations in three other genes (NSD1, LARGE, and POLG) were detected, which were all inherited from the patient's unaffected father. CONCLUSIONS: Taken together, RS1 mutation was found to segregate with retinoschisis phenotype while none of the other identified variations were co-segregating with the systemic defects. Hereby, we infer that the multisystemic defects harbored by the patient are a rare coexistence of XLRS, developmental delay, sensorineural hearing loss, and reduced axial tone reported for the first time in the literature.

Identification of Novel Mutations in ABCA4 Gene: Clinical and Genetic Analysis of Indian Patients with Stargardt Disease.

Stargardt disease (STGD) is the leading cause of juvenile macular degeneration associated with progressive central vision loss, photophobia, and colour vision abnormalities. In this study, we have described the clinical and genetic features of Stargardt patients from an Indian cohort. The next generation sequencing was carried out in five clinically confirmed unrelated patients and their family members using a gene panel comprising 184 retinal specific genes. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation. Genetic analysis revealed ABCA4 mutations in all of the five unrelated patients. Among these, four patients were found with compound heterozygous mutations and another one had homozygous mutation. All the affected individuals showed signs and symptoms consistent with the disease phenotype. We report two novel ABCA4 mutations in Indian patients with STGD disease, which expands the existing spectrum of disease-causing variants and the understanding of phenotypic and genotypic correlations. Screening for causative mutations in patients with STGD using panel of targeted gene sequencing by NGS would be a cost effective tool, might be helpful in confirming the precise diagnosis, and contributes towards the genetic counselling of asymptomatic carriers and isolated patients.

Non-syndromic hearing impairment in India: high allelic heterogeneity among mutations in TMPRSS3, TMC1, USHIC, CDH23 and TMIE.

Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.

Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss.

In a study of 530 individuals with non-syndromic, sensorineural hearing loss, we identified 18 mutations at connexin 26 (Cx26), four of which are novel (-23G>T, I33T, 377_383dupTCCGCAT, W172R) and the remaining 14 (ivs1+1G>A, M1V, 35delG, W24X, I35S, V37I, R75W, W77X, 312del14, E120del, Q124X, Y136X, R143W, R184P) being mutations previously described. To gain insight into functional consequences of these mutations, cellular localization of the mutant proteins and their ability to permit lucifer yellow transfer between cells was studied in seven of them (W24X, I33T, I35S, R75W, E120del, W172R and R184P). I35S and R184P showed impaired trafficking of the protein to the plasma membrane. I33T, R75W, E120del and W172R showed predominantly membrane localization but did not form functional gap junction channels. Surprisingly, W24X, a protein-truncating mutation, apparently permits formation of a full-length protein, perhaps due to a stop codon read-through mechanism. These results provide further evidence that Cx26 mutations affect gap junction activity by mis-regulation at multiple levels.