Rare Genetic Disorders (RGDs) are clinical conditions with underlying genetic origins. Though RGDs are of low prevalence and individually rare, collectively they affect a considerable number of people in a highly populous country like India. The diagnosis of RGDs is challenging due to the lack of awareness and the genetic heterogeneity and variety of overlapping symptoms they present with, as well as the unavailability of accurate genetic tests. Where available, the cost of associated diagnostic and medical tests is beyond the reach of most people in our country.
Palliative treatment (where available) relies on obtaining a correct diagnosis of the disorder as early as possible. We are working towards developing diagnostic kits for RGDs that are cost effective, suitable for carrier and newborn screening, and specific for disease associated genetic mutations common among the Indian population.
Team
Gayatri R Iyer, Harvinder Kour Khera,
Runa Hamid, Shivranjani C Moharir
Activities:
- Development of Diagnostic Kits for Rare Genetic Disorders – Spinal Muscular Atrophy
- Development of digital PCR-based Diagnostic Assay for Spinal Muscular Atrophy (SMA)
- Development of Diagnostic Method for Monoamine Neurometabolic Disorders (mNMDs)
- Development of MS-PCR kits for Genomic Imprinting Disorders
- Comprehensive Genotyping for Diagnosis and Developing Pharmacogenomics Driven Management Protocol for Genomic Imprinting Disorders
- Molecular Investigation of Syndromic Cleft Lip and Palate for Appropriate Management and Genetic Counselling
The actual proportion of human genetic diseases caused due to copy number variations is unknown. With the advent of molecular techniques and whole genome sequencing-based approaches, the underlying cause of several genetic disorders can be unfolded. We are working towards identifying Indian population specific mutations and developing indigenous diagnostic tools and kits for population level screening. Initially, the target disorder is spinal muscular atrophy (SMA), with goals to later expand to other RGDs. The survival motor neuron genes (SMN1 and SMN2) are the causative genes for SMA with copy number variations and gene conversion events eventually leading to a degeneration of motor neurons.
Multiplex ligation-dependent probe amplification (MLPA) is a multiplex, semi-quantitative method for diagnostic testing of genetic disorders. The method is suitable for the identification of deletions or duplications over a broad range, from SNPs to chromosomal aneuploidies, given a suitable set of probes covering the entire target region. By coupling MLPA amplified probes with sequencing, one can include many hundreds of probes in a single reaction. The incorporation of an Next Generation Sequencing (NGS)-based detection approach would make the diagnostic strategy suitable for population level and carrier screening as multiplexing large number of samples for many disorders in a single assay would cut down the cost.
Investigators: Shivranjani C Moharir
Collaborators:
CSIR – Centre for Cellular and Molecular Biology (CCMB), Hyderabad
Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad
CureSMA India, Gurugram
Recent diagnostic advancements have shifted towards molecular testing, allowing for quick and accurate detection of homozygous SMN1 deletions. Alongside identifying SMN1 mutations, determining the copy number of SMN2 is essential, as SMN2 serves as a disease modifier. Quantitative assessment of SMN1 and SMN2 using multiplex ligation-dependent probe amplification (MLPA) is considered the gold standard for SMA genetic testing. However, other methods such as quantitative PCR and next-generation sequencing are also available.
The number of SMN2 copies correlates inversely with the severity of the symptoms. Knowledge of SMN2 copy numbers is crucial for the diagnosis as well as clinical trials. Digital PCR (dPCR), with its high sensitivity and accuracy, is a reliable method for quantifying SMN1 and SMN2 copy numbers over a wide range, providing valuable clinical insights. Under this project, we aim to demonstrate the feasibility and clinical relevance of a cost-effective dPCR-based assay to determine the mutations in SMN1 as well as the copy number of the SMN2 gene.
Investigators: Shivranjani C Moharir. Harvinder Kour Khera
Monoamine neurotransmitter disorders (mNMDs) are a group of rare genetic disorders (RGDs) resulting from defects in the biosynthetic pathway of key monoamine neurotransmitters, including dopamine, serotonin, epinephrine, and norepinephrine- with symptoms typically appearing from infancy to early childhood. Misdiagnosis of these disorders poses a significant challenge because there is an overlap of clinical features with other neurological disorders like encephalopathy, cerebral palsy, primary movement disorders, etc. These disorders are progressive and typically diagnosed through cerebrospinal fluid (CSF) analysis, an invasive procedure that carries risks of nerve damage, infection, and bleeding. Developing a blood-based diagnostic test that is rapid, minimally invasive, affordable, and predictive is essential for timely therapeutic interventions and effective management of these disorders.
We are developing a blood-based diagnostic method using Liquid Chromatography-Mass Spectrometry (LC-MS/MS) due to its capability for targeted analysis, allowing simultaneous detection of multiple metabolites within a specific biological pathway with high sensitivity and selectivity. This multiplexing approach enables a shift from a one-test-one-disorder model to a single test that can diagnose multiple monoamine disorders, significantly reducing overall diagnostic time and cost. We are also optimising this method using dried blood spots which will be convenient and minimally invasive for infants and young children.
Investigator: Runa Hamid
Collaborator:
Bangalore Child Neurology and Rehabilitation Centre, Bengaluru
Bangalore Baptist Hospital, Bengaluru
Indira Gandhi Institute of Child Health (IGICH), Bengaluru
Despite the advances and application of different genomic technologies over 5 decades, the etiopathology of more than 25% of rare genetic disorders associated with intellectual disability (ID), abnormal growth and behavior is unclear emphasizing the need for exploring novel and newer mechanisms of diagnosis.
One such group of disorders is disorders of genomic imprinting. About 150+ genes in the mammalian genome are exclusively expressed from either parent depending on the parent of origin. This is achieved by the epigenetic mechanism of DNA methylation and is known as genomic imprinting.
Methylation-specific polymerase chain reaction (MS-PCR) is a simple, rapid, cost-effective, low labor and equipment-intensive modality that can be easily deployed even in remote locations with robust performance. Isolated genomic DNA is first given a sodium bisulphite treatment that deaminates the unmethylated cytosines to uracil. The converted DNA sample now has differentiated methylated and unmethylated alleles in a single tube which can be amplified with the help of allele-specific primers A pilot study of 102 clinically suspected cases mentioned above showed about 28% to be confirmed as one of four known imprinting disorders by a simple methylation-based test indicating these disorders are not as rare as cited to be but require a timely referral and correct diagnostic modality to offer genetic counselling, plan prenatal diagnosis and facilitate appropriate management.
Towards this end we aim to develop a cost-effective diagnostic kit for imprinting disorders – Prader Willi, Angelman, Beckwith Wiedemann and Silver Russell Syndromes.
Investigator: Gayatri Iyer
Collaborators
Kamineni Academy of Medical Sciences and Research Centre, Hyderabad
JSS Medical College, Mysuru
Jaslok Hospital and Research Centre, Mumbai
Aster Hospitals, Bengaluru
Genomic imprinting is an epigenetic phenomenon wherein the gene expression is regulated without altering the underlying nucleotide sequence. The allele destined to be silenced is methylated at CpG clusters of crucial sites like promoters, transcription start sites and exons so that RNA polymerase cannot bind and thus preventing gene expression. Altered imprinting has been recognized as a cause for 13 established syndromes, four of these Prader Willi syndrome (PWS), Angelman syndrome (AS), Beckwith Wiedemann Syndrome (BWS) and Silver Russell Syndrome (SRS) are also reported in India where affected children have problems with over or undergrowth, behavioral disturbances, seizures, intellectual disability, cancer predisposition.
Genic polymorphisms govern the enzyme activity like absorption distribution, metabolism, and excretion of drugs constituting pharmacogenomics. Cytochrome P-450 superfamily group of liver enzymes are involved in metabolism of 70-80% of psychosomatic medications. PWS & AS individuals require long term medications, which can show side effects like neurosis, aggressive behavior, anxiety, depression, insomnia. Imprinting disorders as a group is more sensitive to psychotropics, requiring lower than typical doses. Polypharmacy, drug-drug interactions as well as hormonal therapies can influence outcomes. Performing pharmacogenomic profiling can enable clinicians to devise personalized therapy.
Imprinting disorder can arise due to several underlying mechanisms disturbing the methylated regions. Understanding the associated genotype – whether it is deletion, uniparental disomy or imprinting defects is not only crucial for appropriate genetic counselling, predicting recurrence risk and offering prenatal diagnosis but is also essential for devising customized surveillance plans for all four disorders. Similarly, Prader Willi Syndrome and Angelman Syndrome are required to be administered neurological medications from early childhood. Evaluating their pharmacogenomic profile and prescribing medications would be instrumental in achieving optimal therapeutic response as well as avoiding adverse drug events.
We are working on under-recognized imprinting disorders to aid timely cost-effective diagnosis, provide genetic counselling, plan prenatal diagnosis, facilitate appropriate management by developing pharmacogenomics guided pharmacotherapy with multidisciplinary management
Investigator: Gayatri R Iyer
Collaborator:
Kamineni Academy of Medical Sciences and Research Centre, Hyderabad
JSS Medical College, Mysuru
Jaslok Hospital and Research Centre, Mumbai
Aster Hospitals, Bengaluru
Angelman Foundation, India
Orofacial clefts, notably cleft lip (CL) and cleft palate (CP), are the most common craniofacial birth defects in humans and represent a substantial personal and societal burden. Clefts affect approximately 1 in 700 individuals globally. In India, the combined incidence of orofacial cleft lip and palate is 1 in 770 to 1000. Non-syndromic cleft lip or palate is a genetically complex disorder caused by the interaction of multiple genetic and environmental risk factors. Maternal diabetes, smoking, alcohol or nutrition deficits during pregnancy are a few of the major risk factors in addition to exposure to other teratogens. Most orofacial cleft cases lack additional features and are categorized as “non-syndromic”, i.e. about 70% of all CL/P cases are isolated incidences, however, the other 30 % are syndromic indicating they may additionally have features and ailments in addition to the congenital anomaly.
People with cleft lip and palate often require multidisciplinary care involving several surgical repairs commencing in the first year of life, orthodontic interventions for malocclusion, speech therapy, treatment of recurrent middle ear infections, and psychological interventions. In syndromic cases, additionally other systems like skeletal, neurological, cardiac also are involved affecting growth and development. Clefting is also associated with a higher risk of various cancer types, including breast, brain, and colon cancers.
Syndromic clefts can arise due to chromosomal aberrations like deletion in chr 22q.11 of Di George syndrome, or sequencing errors like Stickler, Treacher Collins syndrome or due to imprinting and epimutations in Beckwith Wiedemann syndrome. In a resource restricted country like India where the basic stratification of syndromic and non-syndromic clefts is not uniformly practiced, molecular diagnosis can prove to be a herculean task. In the absence of molecular diagnosis, proper genetic counselling for recurrence risk cannot be offered nor prenatal diagnosis can be facilitated adding on to the disease burden to the society.
Long read sequencing is a next generation contemporary technique that can be applied for detecting all three classes of molecular aetiologies of syndromic cleft lip and palate. This will facilitate syndrome specific management, enable effective surveillance of other systemic ailments, anticipatory guidance, and cross disciplinary expert referrals. Having molecular diagnosis will also facilitate prenatal diagnosis and prevent syndromic incidence.
We are developing a next generation sequencing based molecular test to identify syndromic cleft lip and palate for appropriate management and genetic counselling. This will help in improving the healthcare, family as well as societal outcomes in managing individuals with birth defect of cleft lip and palate.
Investigator: Gayatri R Iyer
Collaborator:
Kamineni Academy of Medical Sciences and Research Centre, Hyderabad
JSS Medical College, Mysuru