Publications

The T-box4 (TBX4) gene (OMIM *601719) belongs to the T-box family of transcription regulators that share a conserved homology domain and are expressed at specific sites during various stages of embryonic development. Tbx4 has been found to be a crucial transcriptional regulator in embryonic hindlimb development in animal models. Monoallelic variants in the TBX4 gene are reported to be associated with skeletal defects of the pelvis and lower limbs. We report here a fetus with a novel multiple malformation syndrome associated with sacrococcygeal agenesis, bilateral lower limb aplasia, hypoplastic left heart, bilateral lung hypoplasia, hydroureteronephrosis, and nonimmune fetal hydrops, found to have a homozygous nonsense variant in the TBX4 gene. We propose that biallelic variants in the TBX4 gene are associated with a severe syndromic phenotype of sacrococcygeal agenesis and lower limb reduction defects.

Abstract
Background
Studies evaluating next‐generation sequencing (NGS) for retinal disorders may not reflect clinical practice. We report results of retrospective analysis of patients referred for clinical testing at two institutions (US and India).

Methods
This retrospective study of 131 patients who underwent clinically validated targeted NGS or exome sequencing for a wide variety of clinical phenotypes categorized results into a definitive, indeterminate, or negative molecular diagnosis.

Results
A definitive molecular diagnosis (52%) was more common in the India cohort (62% vs. 39%, p = .009), while an indeterminate molecular diagnosis occurred only in the US cohort (12%). In the US cohort, a lower diagnostic rate in Hispanic, non‐Caucasians (23%) was seen compared to Caucasians (57%). The India cohort had a high rate of homozygous variants (61%) and different frequency of genes involved compared to the US cohort.

Conclusion
Despite inherent limitations in clinical testing, the diagnostic rate across the two cohorts (52%) was similar to the 50%–65% diagnostic rate in the literature. However, the diagnostic rate was lower in the US cohort and appears partly explained by racial background. The high rate of consanguinity in the Indian population is reflected in the high rate of homozygosity for pathogenic mutations and may have implications for population level screening and genetic counseling. Clinical laboratories may note diagnostic rates that differ from the literature, due to factors such as heterogeneity in racial background or consanguinity rates in the populations being tested. This information may be useful for post‐test counseling.

Abstract In this case report, we described a 15-year-old boy who presented with intermittent episodes of ataxia and diplopia since 6.5 years of age. Extensive workup done over several years was negative. Brain biopsy showed a neuroinflammatory disorder, and hence, differential diagnosis of chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids, central nervous system (CNS) lymphoma, and small vessel CNS vasculitis were considered. A final diagnosis of familial hemophagocytic lymphohistiocytosis was made when the patient developed episodes of prolonged fever with pancytopenia much later in the course of illness and genetic workup revealed pathogenic mutations in the PRF1 gene.

Retinoblastoma is a rare pediatric tumor of the retina, caused by the homozygous loss of the Retinoblastoma 1 (RB1) tumor suppressor gene. Previous microarray studies have identified changes in the expression profiles of coding genes; however, our understanding of how non-coding genes change in this tumor is absent. This is an important area of research, as in many adult malignancies, non-coding genes including LNC-RNAs are used as biomarkers to predict outcome and/or relapse. To establish a complete and in-depth RNA profile, of both coding and non-coding genes, in Retinoblastoma tumors, we conducted RNA-seq from a cohort of tumors and normal retina controls. This analysis identified widespread transcriptional changes in the levels of both coding and non-coding genes. Unexpectedly, we also found rare RNA fusion products resulting from genomic alterations, specific to Retinoblastoma tumor samples. We then determined whether these gene expression changes, of both coding and non-coding genes, were also found in a completely independent Retinoblastoma cohort. Using our dataset, we then profiled the potential effects of deregulated LNC-RNAs on the expression of neighboring genes, the entire genome, and on mRNAs that contain a putative area of homology. This analysis showed that most deregulated LNC-RNAs do not act locally to change the transcriptional environment, but potentially function to modulate genes at distant sites. From this analysis, we selected a strongly down-regulated LNC-RNA in Retinoblastoma, DRAIC, and found that restoring DRAIC RNA levels significantly slowed the growth of the Y79 Retinoblastoma cell line. Collectively, our work has generated the first non-coding RNA profile of Retinoblastoma tumors and has found that these tumors show widespread transcriptional deregulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease (COVID-19) that has resulted in a global pandemic. It is a highly contagious positive strand RNA virus and its clinical presentation includes severe to critical respiratory disease that appears to be fatal in ~3-5% of the cases. The viral spike (S) coat protein engages the human angiotensin-converting enzyme2 (ACE2) cell surface protein to invade the host cell. The SARS-CoV-2 S-protein has acquired mutations that increase its affinity to human ACE2 by ~10-15-fold compared to SARS-CoV S-protein, making it highly infectious. In this study, we assessed if ACE2 polymorphisms might alter host susceptibility to SARS-CoV-2 by affecting the ACE2 S-protein interaction. Our comprehensive analysis of several large genomic datasets that included over 290,000 samples representing >400 population groups identified multiple ACE2 protein-altering variants, some of which mapped to the S-protein-interacting ACE2 surface. Using recently reported structural data and a recent S-protein-interacting synthetic mutant map of ACE2, we have identified natural ACE2 variants that are predicted to alter the virus-host interaction and thereby potentially alter host susceptibility. In particular, human ACE2 variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R are predicted to increase susceptibility. The T92I variant, part of a consensus NxS/T N-glycosylation motif, confirmed the role of N90 glycosylation in immunity from non-human CoVs. Other ACE2 variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y are putative protective variants predicted to show decreased binding to SARS-CoV-2 S-protein. Overall, ACE2 variants are rare, consistent with the lack of selection pressure given the recent history of SARS-CoV epidemics, however, are likely to play an important role in altering susceptibility to CoVs.

Abstract THOC6 is a newly described causal gene for an autosomal recessive intellectual disability (ID) – Beaulieu Boycott Innes syndrome (BBIS) (OMIM # 613680). It is characterized by ID with dysmorphic facies, genitourinary, cardiac anomalies, and dentition problems. Here, we report the first two siblings of BBIS from the Indian subcontinent with previously unreported skeletal anomalies such as Sprengel shoulder, calcaneo valgus deformity, radioulnar dysostosis, and overlapping toes. Whole exome sequencing (WES) identified previously reported three missense variants (p.Trp100Arg, p.Val234Leu, p.Gly275Asp) in THOC6. THOC6 is a subunit of TRanscription and EXport (TREX) complex involved in mRNA transcription, processing, and nuclear export of spliced mRNAs and has a potential role in neurodevelopment. Till date, only 12 patients with BBIS have been reported. This report reviews the phenotypic and genetic data of known BBIS cases in addition to the new phenotypic features, thereby expanding the phenotype of this rare syndrome.

Author contributions Dr. Poornima Narayanan Nambiar: study design, acquisition of data, and drafting of manuscript. Dr. Santha Kumar S: acquisition of data and drafting of manuscript. Dr. Ramshekhar Menon: study design and critical revision of the manuscript. Dr. Sruthi. and S. Nair: critical revision of manuscript. Dr. Soumya Sundaram: study concept, design, and critical revision of manuscript. Dr. GK Madhavilatha: acquisition of data.

How satellite cells and their progenitors balance differentiation and self-renewal to achieve sustainable tissue regeneration is not well understood. A major roadblock to understanding satellite cell fate decisions has been the difficulty of studying this process in vivo. By visualizing expression dynamics of myogenic transcription factors during early regeneration in vivo, we identify the time point at which cells undergo decisions to differentiate or self-renew. Single-cell RNA sequencing reveals heterogeneity of satellite cells, including a subpopulation enriched in Notch2 receptor expression, during both muscle homeostasis and regeneration. Furthermore, we reveal that differentiating cells express the Dll1 ligand. Using antagonistic antibodies, we demonstrate that the DLL1 and NOTCH2 signaling pair is required for satellite cell self-renewal. Thus, differentiating cells provide the self-renewing signal during regeneration, enabling proportional regeneration in response to injury while maintaining the satellite cell pool. These findings have implications for therapeutic control of muscle regeneration.