Next generation sequencing: window to a new era of molecular diagnostics
Deepti Saxena Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow Email:saxenadrdeepti@gmail.com
The advent of next generation sequencing has changed the scenario of diagnostic methodology. This new technique has
been used in various areas ranging from the identification of new genes for single gene disorders, diseases with locus/
phenotypic heterogeneity, discovery of cancer causing genomic variations and understanding the pathogenicity of various
microorganisms in human diseases.
1 Using single cell sequencing data to model the evolutionary history of a tumor1
Routine sequencing methods detect average signals generated by different cells within a tumor tissue. Single cell
sequencing is a recently developed technology that detects genetic variation within a single cell and sequencing of different
cells from a tumor can identify the cellular heterogeneity within a tumor. The analysis of mutations detected in different
cells can identify the earliest mutation that is present in all cells and those that are present in further sub – clones. This
helps to understand the sequential series of mutational events that resulted in a heterogeneous tumor. The major
drawback of this technology is the high error rate. Kim and Simon have proposed a new statistical method that
determines the order of mutation between any two sites and also reduces the error rate. They also provide a method to
estimate the proportion of time from the earliest mutation event of the tumor to the most recent common ancestor
(MRCA) of the cells sequenced. Based on the pair-wise mutation orders of all sites, the authors have constructed a
mutation tree using the minimal spanning tree algorithm. The data was obtained by single cell exome
sequencing of 58 cells from a patient with essential thrombocythemia (ET) and two tissue sequencing data
(one normal and one tumor tissue). The process of carcinogenesis involves multistage progression of tumor
clones, so identifying the order of mutation in various genes helps in better understanding of the tumor
progression.
2 Whole-Exome Sequencing Identified KCNJ11 as the Thirteenth MODY Gene2
“Maturity onset diabetes of the young” is a heterogeneous disorder characterized by autosomal dominant mode of
inheritance, onset before 25 years of age and a primary defect in the pancreatic β-cell function. Till now, 12 different types
of MODY caused due to mutation in different genes have been identified and together they account for around 70% of the
MODY cases and the remaining 30% are categorized as MODY-X. Bonnefond et al have done whole exome sequencing of
four individuals (3 affected and one unaffected) from a large French MODY-X family. After analysis of the data they
identified 324 variants of interest (present in all 3 affected individuals, not present in the unaffected individual and
not reported in the database dbSNP130). The authors then assessed the status of these variants in an
additional 23 members of the family and in 406 controls aged above 47 years with normal fasting blood sugar
levels. Among variants that were not present in 406 controls, only one variant (c.679G>A; p.Glu227Lys)
located in the KCNJ11 gene, at a heterozygous state was found to be present in all eight relatives with overt
non autoimmune diabetes. Linkage analysis also suggested that KCNJ11 p.Glu227Lys mutation is causal
for MODY in the analyzed pedigree. This mutation in KCNJ11 is already known to be associated with
neonatal diabetes mellitus and screening is currently indicated by guidelines in all patients who present with
diabetes diagnosed before 6 to 12 months of age. As affected carriers of this mutation can be treated with
sulfonylureas instead of insulin, this study suggests that molecular diagnosis of MODY should include KCNJ11
testing.
3 Lifting the lid on unborn lethal Mendelian phenotypes3
Hanan et al have shown, for the first time, the use of exome sequencing in identification of the causative genetic variation
in embryonic lethal disorders. They studied a consanguineous couple with history of two fetal losses due to non-immune
fetal hydrops. Fetal karyotypes in both the pregnancies were normal. In the current pregnancy, fetal hydrops was detected
at 19 weeks gestational age with an otherwise normal ultrasound. So, considering consanguinity, the causal mutation was
considered to be most likely homozygous. Exome sequencing of the fetus identified 4400 homozygous variants and out of
them 440 were present within the autozygome. Out of these 440, 2 (R254C in CHRNA1 and P825S in SAP130) were
novel, i.e not present in dbSNP and 250 controls. The mutation in CHRNA1 was particularly significant as this
gene had been previously associated with multiple pterygium and fetal akinesia syndrome. The other was
considered unlikely to be contributory, as mice homozygous for a transposon insertion in this gene lack similar
phenotype. Thus, this study highlights the role of NGS in identification of the genetic cause of unborn lethal
phenotypes.
4 Actionable Diagnosis of Neuroleptospirosis by NGS4
Approximately 50% cases of meningoencephalitis remain undiagnosed despite extensive clinical and laboratory evaluation.
Because more than 100 different infectious agents can cause meningoencephalits, conventional diagnostic methods such as
culture, PCR and serological tests can miss the diagnosis. The authors have proven the use of NGS for the identification
of the infectious etiology in a 14 year old boy having severe combined immunodeficiency who presented with fever,
headache, hydrocephalus and status epilepticus with history of minor illnesses for last 1 year. The workup for infectious
diseases was negative. MRI of the head showed persistent hyperintensities in the basal ganglia and basilar
leptomeningitis extending into the cerebral hemispheres. Biopsy of the right frontal lobe was performed and
histologic examination revealed inflamed leptomeninges with a granulomatous infiltrate. Immunohistochemical
testing and electron microscopy did not identify fungi, bacteria, or viruses. NGS of the cerebrospinal fluid
and serum was done and bioinformatics analysis was done for the detection of all known pathogens which
identified 475 of 3,063,784 reads of leptospira infection in CSF but not in serum. He was started on targeted
antimicrobial therapy and gradually improved. Identification of leptospira infection was later confirmed by
targeted PCR and Sanger sequencing. So, next generation sequencing has proven helpful in many clinical
situations and its role has expanded from the identification of genes for known as well as uncharacterized
Mendelian disorders to understanding the molecular genetic basis of cancers, common disorders and microbial
infections.
References
1. Kim and Simon. BMC Bioinformatics 2014; 15: 27.
2. Bonnefond A, et al. PLoS ONE 2012; 7: e37423.
3. Hanan E., et al. Genet Med 2013; 15: 307-9.
4. Michael R. Wilson, et al. N Eng J Med 2014; 370: 2408-17.