Short stature and dysmorphic features in Asian Indian siblings with DAAM2-associated steroid-resistant nephrotic syndrome: Expansion of the phenotypic spectrum or a blended phenotype?

Variants in more than 60 different genes, most of which code for podocyte-related proteins, have been found to be associated with monogenic forms of nephrotic syndrome (NS). Biallelic variants in DAAM2, a member of the formin family, were recently identified to cause autosomal recessive (AR) NS type 24 in four unrelated families with steroid-resistant nephrotic syndrome (SRNS). This case report represents only the fifth reported family of DAAM2-associated NS and the first from India, with two sibs who presented with a complex phenotype characterized by steroid-resistant nephrotic syndrome, short stature, dysmorphic facial features, deep-set toenails, myopia, increased thickness of the calvarium of the skull, and sloping ribs. Both sibs were found to have a homozygous likely pathogenic nonsense variant c.196C>T (p.Arg66Ter; NM_001201427.2) in exon 3 of the DAAM2 gene through whole exome sequencing. The dysmorphic features could possibly be part of the DAAM2-related phenotype which has hitherto not been reported or could represent a blended phenotype, with the extrarenal manifestations resulting from a yet to be identified coexisting genetic condition.

A novel homozygous synonymous splicing variant in SELENOI gene causes spastic paraplegia 81.

BACKGROUND: Hereditary spastic paraplegia 81 is a recently identified, rare autosomal recessive disease, caused by biallelic pathogenic variants in the SELENOI gene, with only two families reported to date. The features documented in the two previous affected families include sensorineural deafness, blindness, cleft palate, delayed motor development, regression of motor skills, impaired intellectual development, poor speech and language acquisition, spasticity, hyperreflexia, white matter abnormalities and cerebral and cerebellar atrophy. METHODS: In the present study, we performed exome sequencing analysis in a single family with two affected siblings to identify the genetic cause of complicated hereditary spastic paraplegia. The results were further confirmed by Sanger sequencing, cDNA analysis and 3D protein modelling. RESULTS: Exome sequencing identified a homozygous, synonymous variant in the SELENOI gene (NM_033505.4:c.126G>A:p.(Lys42Lys)) in both of the siblings. Sanger sequencing confirmed the heterozygous status in both parents consistent with the autosomal recessive inheritance. This variant has been found to disrupt normal splicing and lead to skipping of exon 2, causing in-frame deletion of SELENOI N-terminal 23 amino acids [NM_033505.4:c.57_126del:p.(Tyr20_Lys42del)] and further leading to structural changes in the protein. CONCLUSIONS: We report a novel homozygous synonymous variant in the SELENOI gene causing abnormal splicing in two patients affected with hereditary spastic paraplegia 81. This report further expands the phenotypic and genotypic spectrum of hereditary spastic paraplegia 81.

Long-range PCR amplification-based targeted enrichment & next generation sequencing: A cost-effective testing strategy for lysosomal storage disorders.

Background & objectives: Lysosomal storage disorders (LSDs) are genetic metabolic disorders which result from deficiency of lysosomal enzymes or defects in other lysosomal components. Molecular genetic testing of LSDs is required for diagnostic confirmation when lysosomal enzyme assays are not available or not feasible to perform, and for the identification of the disease causing genetic variants. The aim of this study was to develop a cost-effective, readily customizable and scalable molecular genetic testing strategy for LSDs. Methods: A testing method was designed based on the in-house creation of selective amplicons through long range PCR amplification for targeted capture and enrichment of different LSD genes of interest, followed by next generation sequencing of pooled samples. Results: In the first phase of the study, standardization and validation of the study protocol were done using 28 samples of affected probands and/or carrier parents (group A) with previously identified variants in seven genes, and in the second phase of the study, 30 samples of enzymatically confirmed or biopsy-proven patients with LSDs and/or their carrier parents who had not undergone any prior mutation analysis (group B) were tested and the sequence variants identified in them through the study method were validated by targeted Sanger sequencing. Interpretation & conclusions: This testing approach was found to be reliable, easily customizable and cost-effective for the molecular genetic evaluation of LSDs. The same strategy may be applicable, especially in resource poor settings, for developing cost-effective multigene panel tests for other conditions with genetic heterogeneity.

Ectodysplasin pathogenic variants affecting the furin-cleavage site and unusual clinical features define X-linked hypohidrotic ectodermal dysplasia in India.

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder caused by mutational inactivation of a developmental pathway responsible for generation of tissues of ectodermal origin. The X-linked form accounts for the majority of HED cases and is caused by Ectodysplasin (EDA) pathogenic variants. We performed a combined analysis of 29 X-linked hypohidrotic ectodermal dysplasia (XLHED) families (including 12 from our previous studies). In addition to the classical triad of symptoms including loss (or reduction) of ectodermal structures, such as hair, teeth, and sweat glands, we detected additional HED-related clinical features including facial dysmorphism and hyperpigmentation in several patients. Interestingly, global developmental delay was identified as an unusual clinical symptom in many patients. More importantly, we identified 22 causal pathogenic variants that included 15 missense, four small in-dels, and one nonsense, splice site, and large deletion each. Interestingly, we detected 12 unique (India-specific) pathogenic variants. Of the 29 XLHED families analyzed, 11 (38%) harbored pathogenic variant localized to the furin cleavage site. A comparison with HGMD revealed significant differences in the frequency of missense pathogenic variants; involvement of specific exons and/or protein domains and transition/transversion ratios. A significantly higher proportion of missense pathogenic variants (33%) localized to the EDA furin cleavage when compared to HGMD (7%), of which p.R155C, p.R156C, and p.R156H were detected in three families each. Therefore, the first comprehensive analysis of XLHED from India has revealed several unique features including unusual clinical symptoms and high frequency of furin cleavage site pathogenic variants.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Identification and characterization of 30 novel pathogenic variations in 69 unrelated Indian patients with Mucolipidosis Type II and Type III.

Mucolipidosis (ML) (OMIM 607840 & 607838) is a rare autosomal recessive inherited disorder that occurs due to the deficiency of golgi enzyme uridine diphosphate (UDP)- N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) responsible for tagging mannose-6-phosphate for proper trafficking of lysosomal enzymes to lysosomes. Variants in GlcNAc-phosphotransferase (GNPTAB (α, ß subunits) and GNPTG (γ subunits) are known to result in impaired targeting of lysosomal enzymes leading to Mucolipidosis (ML) Type II or Type III. We analyzed 69 Indian families of MLII/III for clinical features and molecular spectrum and performed in silico analysis for novel variants. We identified 38 pathogenic variants in GNPTAB and 5 pathogenic variants in GNPTG genes including missense, frame shift, deletion, duplication and splice site variations. A total of 26 novel variants were identified in GNPTAB and 4 in GNPTG gene. In silico studies using mutation prediction software like SIFT, Polyphen2 and protein structure analysis further confirmed the pathogenic nature of the novel sequence variants detected in our study. Except for a common variant c.3503_3504delTC in early onset MLII, we could not establish any other significant genotype and phenotype correlation. This is one of the largest studies reported till date on Mucolipidosis II/III in order to identify mutation spectrum and any recurrent mutations specific to the Indian ethnic population. The mutational spectrum information in Indian patients will be useful in better genetic counselling, carrier detection and prenatal diagnosis for patients with ML II/III.

Gaucher disease: single gene molecular characterization of one-hundred Indian patients reveals novel variants and the most prevalent mutation.

BACKGROUND: Gaucher disease is a rare pan-ethnic, lysosomal storage disorder resulting due to beta-Glucosidase (GBA1) gene defect. This leads to the glucocerebrosidase enzyme deficiency and an increased accumulation of undegraded glycolipid glucocerebroside inside the cells' lysosomes. To date, nearly 460 mutations have been described in the GBA1 gene. With the aim to determine mutations spectrum and molecular pathology of Gaucher disease in India, the present study investigated one hundred unrelated patients (age range: 1 day to 31 years) having splenomegaly, with or without hepatomegaly, cytopenia and bone abnormality in some of the patients. METHODS: The biochemical investigation for the plasma chitotriosidase enzyme activity and ß-Glucosidase enzyme activity confirmed the Gaucher disease. The mutations were identified by screening the patients' whole GBA gene coding region using bidirectional Sanger sequencing. RESULTS: The biochemical analysis revealed a significant reduction in the ß-Glucosidase activity in all patients. Sanger sequencing established 71 patients with homozygous mutation and 22 patients with compound heterozygous mutation in GBA1 gene. Lack of identification of mutations in three patients suggests the possibility of either large deletion/duplication or deep intronic variations in the GBA1 gene. In four cases, where the proband died due to confirmed Gaucher disease, the parents were found to be a carrier. Overall, the study identified 33 mutations in 100 patients that also covers four missense mutations (p.Ser136Leu, p.Leu279Val, p.Gly383Asp, p.Gly399Arg) not previously reported in Gaucher disease patients. The mutation p.Leu483Pro was identified as the most commonly occurring Gaucher disease mutation in the study (62% patients). The second common mutations identified were p.Arg535Cys (7% patients) and RecNcil (7% patients). Another complex mutation Complex C was identified in a compound heterozygous status (3% patients). The homology modeling of the novel mutations suggested the destabilization of the GBA protein structure due to conformational changes. CONCLUSIONS: The study reports four novel and 29 known mutations identified in the GBA1 gene in one-hundred Gaucher patients. The given study establishes p.Leu483Pro as the most prevalent mutation in the Indian patients with type 1 Gaucher disease that provide new insight into the molecular basis of Gaucher Disease in India.

Spectrum of ARSA variations in Asian Indian patients with Arylsulfatase A deficient metachromatic leukodystrophy.

Metachromatic leukodystrophy due to Arylsulfatase A enzyme deficiency is an autosomal recessive disorder caused by biallelic variations in ARSA gene. Till date 186 variations have been reported in ARSA gene worldwide, but the variation spectrum in India is not known. The aim of this study was to identify the variation profile in Indian patients presenting with features of Arylsulfatase A deficient metachromatic leukodystrophy. We sequenced the ARSA gene in 51 unrelated families and identified 36 variants out of which 16 were novel. The variations included 23 missense, 3 nonsense, and 6 frameshift variants (3 single-base deletions and 3 single-base duplications), 1 indel, one 3 bp deletion, and 2 splice site variations. The pathogenicity of the novel variations was inferred with the help of mutation prediction softwares like MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using in silico methods. The most common variation was c.931 C > T(p.Arg311*), found in 11.4% (14 out of 122 alleles) of the tested individuals. To the best of our knowledge, this study is the first of its kind in India with respect to the size of the cohort and the molecular diagnostic method used and one of the largest cohorts of metachromatic leukodystrophy studied till date.

Prenatal screening for genetic disorders: Suggested guidelines for the Indian Scenario.

Prenatal testing is the best strategy for reducing the burden of genetic disorders and congenital disabilities that cause significant postnatal functional impairment. Universal prenatal screening is advisable for common genetic disorders and congenital anomalies such as Down syndrome, beta-thalassaemia and neural tube defects. Several prenatal-screening tests are now available for Down syndrome, but knowledge about the appropriate timing of the test and the need for pre- and post-test counselling may not be updated among the primary care physicians. There is also a considerable degree of confusion regarding the prenatal screening test to be chosen in each case, due to the availability of a number of new and advanced screening techniques. At present, there is no nation-wide consensus regarding the nature and timing of these prenatal-screening protocols. Due to the absence of any definite guidelines and the additional lacunae in the awareness regarding the appropriate prenatal screening in the country, the optimum benefits of these screening protocols are not reaching the population. This review focuses on the various prenatal screening and diagnostic tests that are available for common genetic conditions and congenital disabilities and attempts to outline the most cost-effective and gestational age-appropriate strategies for prenatal screening for the Indian healthcare set-up. The recommendations suggested would serve as a source guide for formulating prenatal-screening guidelines for reducing the incidence of common genetic disorders and congenital disabilities in India.

Clinical and mutation profile of multicentric osteolysis nodulosis and arthropathy.

​Multicentric osteolysis nodulosis and arthropathy (MONA) is an infrequently described autosomal recessive skeletal dysplasia characterized by progressive osteolysis and arthropathy. Inactivating mutations in MMP2, encoding matrix metalloproteinase-2, are known to cause this disorder. Fifteen families with mutations in MMP2 have been reported in literature. In this study we screened thirteen individuals from eleven families for MMP2 mutations and identified eight mutations (five novel and three known variants). We characterize the clinical, radiographic and molecular findings in all individuals with molecularly proven MONA from the present cohort and previous reports, and provide a comprehensive review of the MMP2 related disorders.

Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc.

Novel mutations of the arylsulphatase B (ARSB) gene in Indian patients with mucopolysaccharidosis type VI.

BACKGROUND & OBJECTIVES: Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disorder caused by deficient enzymatic activity of N-acetyl galactosamine-4-sulphatase resulting from mutations in the arylsulphatase B (ARSB) gene. The ARSB gene is located on chromosome 5q11-q13 and is composed of eight exons. More than hundred ARSB mutations have been reported so far, but the mutation spectrum of MPS VI in India is still unknown. Hence, the aim of the present study was to identify the mutational spectrum in patients with MPS VI in India and to study the genotype-phenotype association and functional outcomes of these mutations. METHODS: Molecular characterization of the ARSB gene by Sanger sequencing was done for 15 patients (aged 15 months to 11 yr) who were enzymatically confirmed to have MPS VI. Age of onset, clinical progression and enzyme activity levels in each patient were studied to look for genotype-phenotype association. Haplotype analysis performed for unrelated patients with the recurring mutation W450C, was suggestive of a founder effect. Sequence and structural analyses of the ARSB protein using standard software were carried out to determine the impact of detected mutations on the function of the ARSB protein. RESULTS: A total of 12 mutations were identified, of which nine were novel mutations namely, p.D53N, p.L98R, p.Y103SfsX9, p.W353X, p.H393R, p.F166fsX18, p.I220fsX5, p.W450L, and p.W450C, and three were known mutations (p.D54N, p.A237D and p.S320R). The nine novel sequence variants were confirmed not to be polymorphic variants by performing sequencing in 50 unaffected individuals from the same ethnic population. INTERPRETATION & CONCLUSIONS: Nine novel mutations were identified in MPS VI cases from India in the present study. The study also provides some insights into the genotype-phenotype association in MPS VI.

Mutations in the human UBR1 gene and the associated phenotypic spectrum.

Johanson-Blizzard syndrome (JBS) is a rare, autosomal recessive disorder characterized by exocrine pancreatic insufficiency, typical facial features, dental anomalies, hypothyroidism, sensorineural hearing loss, scalp defects, urogenital and anorectal anomalies, short stature, and cognitive impairment of variable degree. This syndrome is caused by a defect of the E3 ubiquitin ligase UBR1, which is part of the proteolytic N-end rule pathway. Herein, we review previously reported (n = 29) and a total of 31 novel UBR1 mutations in relation to the associated phenotype in patients from 50 unrelated families. Mutation types include nonsense, frameshift, splice site, missense, and small in-frame deletions consistent with the hypothesis that loss of UBR1 protein function is the molecular basis of JBS. There is an association of missense mutations and small in-frame deletions with milder physical abnormalities and a normal intellectual capacity, thus suggesting that at least some of these may represent hypomorphic UBR1 alleles. The review of clinical data of a large number of molecularly confirmed JBS cases allows us to define minimal clinical criteria for the diagnosis of JBS. For all previously reported and novel UBR1 mutations together with their clinical data, a mutation database has been established at LOVD.

Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients.

GALNS mutations in Indian patients with mucopolysaccharidosis IVA.

Mucopolysaccharidosis IV A (Morquio syndrome A, MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS). The mutation spectrum in this condition is yet to be determined in Indians. We aimed to analyze the mutations in the GALNS gene in Asian Indians with MPS IVA. All the exons and the adjacent intronic regions of the gene were amplified and sequenced in sixty-eight unrelated Indian families. We identified 136 mutant alleles comprising of 40 different mutations. We report twenty-two novel mutations that comprise of seventeen missense (p.Asn32Thr, p.Leu36Arg, p.Pro52Leu, p.Pro77Ser, p.Cys79Arg, p.His142Pro, p.Tyr191Asp, p.Asn204Thr, p.Gly188Ser, p.Phe216Ser, p.Trp230Cys, p.Ala291Ser, p.Gly317Arg, p.His329Pro, p.Arg386Ser, p.Glu450Gly, p.Cys501Ser), three splice-site variants (c.120+1G>C, c.1003-3C>G, c.1139+1G>A), one nonsense mutation (p.Gln414*) and one frameshift mutation (p.Pro420Leufs*440). Eighteen mutations have been reported earlier. Among these p.Ser287Leu (8.82%), p.Phe216Ser (7.35%), p.Asn32Thr (6.61%) and p.Ala291Ser (5.88%) were the most frequent mutations in Indian patients but were rare in the mutational profiles reported in other populations. These results indicate that the Indian patients may have a distinct mutation spectrum compared to those of other populations. Mutant alleles in exon 1, 7 and 8 accounted for 44.8% of the mutations, and sequencing of these exons initially may be a cost-effective approach in Asian Indian patients. This is the largest study on molecular analysis of patients with MPS IVA reported in the literature, and the first report from India.

Novel mutations in PRG4 gene in two Indian families with camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome.

BACKGROUND & OBJECTIVES: Camptodactyly--arthropathy-coxa vara-pericarditis (CACP) syndrome is an autosomal recessive disorder caused by mutations in the PRG4 (proteoglycan 4) gene. Hallmarks of the syndrome include congenital or early-onset camptodactyly and arthropathy with synovial hyperplasia, progressive coxa vara deformity and non-inflammatory pericardial effusions. Till date only around 25 pathogenic mutations have been reported in this gene and none have been reported from India. We report here the mutations in the PRG4 gene in three patients of CACP from two unrelated families from India. METHODS: Molecular genetic studies were done for the three patients with the CACP syndrome, from two unrelated Indian families, through sequence analysis of all coding exons and the exon-intron boundaries of the PRG4 gene. RESULTS: Two novel frame-shift deletion mutations leading to premature protein termination were found. One patient was identified to be homozygous for a 2 base pair deletion in exon 6 (c.2645_2646delGA) and the two affected siblings from the other family were found to be homozygous for a 4 base pair deletion in exon 6 (c.2883_2886delAAGA). CONCLUSIONS: This is perhaps the first report of PRG4 mutations from India. Further mutation studies in Indian CACP cases will help to determine the mutation spectrum of the PRG4 gene in the Indian population and also help to further elucidate the molecular pathology and the genotype-phenotype correlation of this rare disease.

Etiologic Spectrum of Pediatric-Onset Leukodystrophies and Genetic Leukoencephalopathies: The Five-Year Experience of a Tertiary Care Center in Southern India.

BACKGROUND: White matter (WM) disorders with a genetic etiology are classified as leukodystrophies (LDs) and genetic leukoencephalopathies (GLEs). There are very few studies pertaining to the etiologic spectrum of these disorders in the Asian Indian population. METHODS: This study was conducted over a period of five years from January 2016 to December 2020, in the medical genetics department of a tertiary care hospital in southern India. A total of 107 patients up to age 18 years, with a diagnosis of a genetic WM disorder confirmed by molecular genetic testing and/or metabolic testing, were included in the study and categorized into LD or GLE group as per the classification suggested by the Global Leukodystrophy Initiative consortium in 2015. RESULTS: Forty-one patients were diagnosed to have LDs, and 66 patients had GLEs. The two most common LDs were metachromatic LD (16 patients) and X-linked adrenoleukodystrophy (seven patients). In the GLE group, lysosomal storage disorders were the most common (40 patients) followed by mitochondrial disorders (nine patients), with other metabolic disorders and miscellaneous conditions making up the rest. The clinical presentations, neuroimaging findings, and mutation spectrum of the patients in our cohort are discussed. CONCLUSIONS: This is one of the largest cohorts of genetic WM disorders reported till date from the Asian Indian population. The etiologies and clinical presentations identified in our study cohort are similar to those found in other Indian studies as well as in studies based on other populations from different parts of the world.

Functional characterization of thyroid peroxidase missense variants causing thyroid dyshormonogenesis in Asian Indian population.

INTRODUCTION: Thyroid dyshormonogenesis (TDH) is a sub-group of congenital hypothyroidism with recessive inheritance resulting from disease causing variants in thyroid hormone biosynthesis pathway genes, like DUOX2, TG, TPO, SLC5A5, SLC26A4, IYD, DUOXA2, SLC26A7 and SECISBP2. Thyroid peroxidase (TPO) is a crucial enzyme involved in thyroid hormone biosynthesis and is one of the frequently mutated genes in patients with TDH. The purpose of the study is to describe the insilico and functional characterization of novel variants in TPO gene identified in patients with thyroid dyshormonogenesis. METHODS: We performed exome sequencing in Indian patients with TDH. In the current study, we describe the results of patients with TPO gene mutations. Exome sequencing results were further analysed by sanger sequencing, computational studies and invitro functional studies such as immunofluorescence and enzyme assay. RESULTS: We identified nine biallelic disease-causing variants in the TPO gene in twelve patients from nine unrelated Indian families. Eight of the nine variants were novel. No recurrent variants were identified. Computational analysis of six missense variants showed that these amino acid substitutions caused changes in non-covalent interactions with the adjacent residues that may affect the TPO protein structure and function. In-vitro experimental data using immunofluorescence assay showed that these variants did not affect the plasma membrane localization of the TPO protein but caused significant loss of TPO enzymatic activity compared to wild type. CONCLUSION: Our study revealed multiple novel pathogenic variants in TPO gene in Indian patients, thereby expanding the genotype spectrum. Functional studies helped us to reveal the pathogenicity of the missense variants.

Eye of the Tiger: Looking Beyond Neurodegeneration with Brain Iron Accumulation Disorders.

The "eye-of-the-tiger" sign in brain magnetic resonance imaging (MRI) is typically associated with neurodegeneration with brain iron accumulation disorders, especially pantothenate kinase-associated neurodegeneration. However, very similar neuroimaging findings may be seen in other neurodegenerative disorders involving the basal ganglia. We report here a patient with fucosidosis who had MRI brain findings closely resembling the "eye-of-the-tiger" sign.