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European Journal of Medical Genetics 58 (2015) 471e478

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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg

Clinical research

Identification of mutations, genotypeephenotype correlation and prenatal diagnosis of maple syrup urine disease in Indian patients Deepti Gupta a, b, Sunita Bijarnia-Mahay a, *, Renu Saxena a, Sudha Kohli a, Ratna Dua-Puri a, Jyotsna Verma a, E. Thomas a, Yosuke Shigematsu c, Seiji Yamaguchi d, Roumi Deb b, Ishwar Chander Verma a a

Center of Medical Genetics, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi, India Amity Institute of Biotechnology, Amity University, Noida, U.P., India Department of Health Science, Faculty of Medical Sciences, University of Fukui, Matsuoka, Japan d Department of Pediatrics, Shimane University School of Medicine, Izumo, Shimane, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2015 Received in revised form 3 August 2015 Accepted 3 August 2015 Available online 7 August 2015

Maple syrup urine disease (MSUD) is caused by mutations in genes BCKDHA, BCKDHB, DBT encoding E1a, E1b, and E2 subunits of enzyme complex, branched-chain alpha-ketoacid dehydrogenase (BCKDH). BCKDH participates in catabolism of branched-chain amino acids (BCAAs) e leucine, isoleucine and valine in the energy production pathway. Deficiency or defect in the enzyme complex causes accumulation of BCAAs and keto-acids leading to toxicity. Twenty-four patients with MSUD were enrolled in the study for molecular characterization and genotypeephenotype correlation. Molecular studies were carried out by sequencing of the 3 genes by Sanger method. Bioinformatics tools were employed to classify novel variations into pathogenic or benign. The predicted effects of novel changes on protein structure were elucidated by 3D modeling. Mutations were detected in 22 of 24 patients (11, 7 and 4 in BCKDHB, BCKDHA and DBT genes, respectively). Twenty mutations including 11 novel mutations were identified. Protein modeling in novel mutations showed alteration of structure and function of these subunits. Mutations, c.1065 delT (BCKDHB gene) and c.939G > C (DBT gene) were noted to be recurrent, identified in 6 of 22 alleles and 5 of 8 alleles, respectively. Two-third patients were of neonatal classical phenotype (16 of 24). BCKDHB gene mutations were present in 10 of these 16 patients. Prenatal diagnoses were performed in 4 families. Consanguinity was noted in 37.5% families. Although no obvious genotype ephenotype correlation could be found in our study, most cases with mutation in BCKDHB gene presented in neonatal period. Large number of novel mutations underlines the heterogeneity and distinctness of gene pool from India. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Maple syrup urine disease BCKDHA BCKDHB DBT Mutation Genotypeephenotype Prenatal diagnosis Modeling Genetics Indian

1. Introduction Maple syrup urine disease (MSUD, OMIM 248600) is an autosomal recessive inherited metabolic disorder caused by deficiency of mitochondrial enzyme complex, branched-chain alpha-ketoacid dehydrogenase (BCKD) (Chuang and Shih, 2001). This enzyme complex is required for catalyzing the oxidative decarboxylation of branched chain keto-acids (BCKAs) that are derived from essential branched chain amino acids (BCAAs); leucine, isoleucine and valine. The metabolic block at this step results in the accumulation

* Corresponding author. E-mail address: [email protected] (S. Bijarnia-Mahay). http://dx.doi.org/10.1016/j.ejmg.2015.08.002 1769-7212/© 2015 Elsevier Masson SAS. All rights reserved.

of BCAAs and their BCKAs in the body leading to severe symptoms of encephalopathy, seizures, developmental delay or even infantile death, if untreated (Chuang et al., 2006). BCKD is a multimeric mitochondrial enzyme complex composed of four subunits namely E1a, E1b, E2, and E3, around a cubic core of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunit which binds the multiple subunits of BCKD decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) (Aevarsson et al., 2000). Two regulatory subunits; BCKD kinase and BCKD phosphatase are also attached to the complex. E1 is a thiamine dependent decarboxylase subunit, and is heteromeric, comprising of two a (E1a) and two b (E1b) subunits (a2b2). Based on the genes involved, the types of MSUD are: e Type 1A (OMIM 608348) in case of mutations in BCKDHA gene encoding E1a subunit; Type 1B (OMIM 248611) for

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mutations in BCKDHB gene which encode E1b subunit and Type II (OMIM 248610) caused by mutations in DBT gene encoding E2 subunit. E3 component is common to BCKD, pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex and an impaired E3 activity results in combined ketoacid dehydrogenase deficiencies. Five types of clinical phenotypes based on age of onset, severity of clinical presentation, and response to thiamine have been reported (Chuang, 1998). MSUD is rare in most populations with incidence of 1 in 150,000 live births in the general population and high incidence in some populations like the Mennonites (incidence is 1 in 176) (Danner and Doering, 1998). The incidence in India is currently not known in the absence of universal newborn screening program, but analysis of high risk cases show that it is one of the common metabolic disorders observed in clinical practice (Nagaraja et al., 2010). We report here a clinical and genetic study which includes mutation analysis of genes, BCKDHA, BCKDHB and DBT in 24 unrelated Indian patients of MSUD.

(NM_001918.3) to determine all the mutations and variations.

2. Patient data

3.4. Molecular modeling

This study enrolled twenty-four Indian patients diagnosed with MSUD, referred for genetic testing at our center, from year 2010e2014. Spectrum of clinical symptoms, age of onset, family history of consanguinity and of any similar disease was noted. Parents were specifically asked for if there was a blood relationship between them that they were aware of. A four generation pedigree was used to rule out or rule in consanguinity. Biochemical testing (elevated BCAAs) on Tandem Mass Spectrometry (TMS) and/or abnormal metabolites (elevated BCKAs, 2-keto isocaproate, 3hydroxy butyrate, 2-hydroxy isovalerate, 2-hydroxy caproate, 2keto 3-methyl valerate) on a urinary organic acid profile using Gas Chromatography Mass Spectrometry (GCeMS) was performed to make the diagnosis. All biochemically proven cases were enrolled for molecular studies. Patients were categorized into two groups according to age of presentation as ‘neonatal classic’ or ‘intermediate’ presenting in infancy or later. A clinical follow up data was also gathered, wherever possible (Table 1).

Crystal structure with PDB ID e 1DTW was used as a template to perform computational modeling. The source organism for 1DTW is human. It has a resolution of 2.7 Å, obtained by X-ray diffraction method. The initial few amino acids of both subunits of BCKDH enzyme complex (1e45 for alpha chain, 1e50 for beta-chain) have not been crystallized. Therefore, the length of the crystallized alpha subunit (1DTW_A) is only 400 amino acids instead of 445 amino acids (46e445 amino acids of reference protein P12694), and that of beta subunit (1DTW_B) is 342 amino acids (51e392 amino acids for reference protein P21953) (Aevarsson et al., 2000). Swiss-Prot PDB Viewer software was used to visualize the effect of novel variations on protein structure (Guex and Peitsch, 1997).

3. Methods For molecular study, blood samples were collected from patients after obtaining an informed consent from their parents. Since the probands were either young (less than 5 years of age) or intellectually disabled, informed consent was obtained from proband's parents. Samples from parents of 18 children were also collected. 3.1. Isolation of DNA and PCR Genomic DNA was extracted using salt precipitation method (Miller et al., 1988). Coding regions as well as flanking exoneintron boundaries of genes (BCKDHA- 9 exons, BCKDHB- 10 exons, and DBT- 11 exons) were amplified by PCR. Primers for PCR were designed using web primer software (Supplementary Table 1 for the list of primer sequences and PCR amplification conditions). 3.2. Sequencing of PCR products Amplified PCR products were checked on agarose gel and purified using multiscreen ®HTS Millipore vacuum manifold (Millipore, Massachusetts, USA). These purified products were then subjected to bidirectional sequencing on 3500 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA). Sequences were analyzed by blasting them against the genomic sequences of BCKDHA (NM_000709.3), BCKDHB (NM_183050.2) and DBT

3.3. Classification/annotation of novel variations In-silico tools such as PolyPhen2 (Adzhubei et al., 2010), SIFT (Kumar et al., 2009), Mutation Taster (Schwarz et al., 2014), and MutPred (Li et al., 2009) software's were used to predict the effect of novel missense variations. The novel variations were also analyzed in 100 control alleles to rule out it being a polymorphism. These mutations were also checked in the 1000 genome project. Nonsense variations giving rise to truncated protein were considered pathogenic. Implication of splicing mutations was predicted using the BDGP site (Reese et al., 1997). I-mutant 2.0 based on the principle of free energy (DDG), was used to predict the stability/ instability of the protein (Capriotti et al., 2005). Conservation of the aminoacid residues was checked using polyphen-2 and Mutation Taster software. Inheritance of mutations was confirmed in cases where parents samples were available.

4. Results Of 24 patients enrolled in the study, 16 were males and 8 were females. Sixteen (63.6%) probands, presented in the neonatal period and were categorized into classical MSUD type. Remaining 8 (33.4%) patients presented in infancy (1month to 1year of age) or later, and were the intermediate type. Consanguinity was observed in 9 (37.5%) of 24 cases. Amongst these, 5 were from Muslim families and 3 were from South Indian Hindu community. Main clinical features in the patients were seizures (in 17), acute encephalopathy (in 15), developmental delay (in 12), and failure to thrive (in 3). A family history of similar affection was present in 11(52.5%) of 21 families. Information was not available for 3 families. A follow up data was obtained in 21 of 24 patients where information was available. Twelve (57.2%) patients were alive and on protein restricted diet, while the remaining nine (42.8%) died. Amongst surviving children, one is doing well after liver transplantation, 3 are in infancy with satisfactory outcome so far, and the other 8 patients have varying degrees of intellectual disability. Amongst deceased patients, majority died in early infancy, one child (in intermediate group) died at 5 years of age following a febrile illness (Table 1). 4.1. Molecular studies Of 24 patients (48 alleles), mutations were identified in 43 (89.5%) alleles; 21 patients had mutations on both alleles and in one patient, mutation could be identified in only one allele. Mutation could not be identified in 2 neonates. Twenty different mutations were identified; 10 in BCKDHB gene, 8 in BCKDHA and 2 in DBT gene.

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Table 1 Patient profiles of clinical phenotype, age of onset, biochemical parameters and their outcome. Patient Sex Age of Age at Family Clinical ID onset diagnosis history subtype

Clinical symptoms

TMS/GCeMS findings Leu þ IleLeu þ IleVal- 436 Leu þ IleVal- 315 Leu þ IleVal- 275 Leu þ Ile-

GCeMS organic Follow up outcome acids

1235 Val- 591 *MSUD pattern Died at 5 yr 2640 MSUD pattern Died at 6 mo

e e

1306

MSUD pattern

Died (age- NA)

e

1810

NA

Died (age- NA)

e

On protein restricted diet Died (age- NA)

5 yr

On protein restricted diet, improved on diet, non-compliant Died at 10 mo

8 yr

P1 P2

M M

1.5 yr 2.5 yr 1 mo 6 mo

Yes Yes

P3

M

Infant 2 yr

No

P4

F

13 d

Yes

Intermediate Moderate dev del, seizures, Classic Moderate dev del, seizures episodic encephalopathy, Intermediate Severe dev del, seizures, hypoglycemia Classic Seizures, NE

P5

M

Infant 9 mo

No

Intermediate Mild to moderate dev del

P6

M

5d

11 d

Yes

Classic

NE, seizures

P7

F

7d

10 d

No

Classic

NE, seizures, dev del

P8

M

Neo

3 mo

No

P9 P10

M M

Neo 10 d

NA 10 mo

No No

P11

F

6 mo

2.5 yr

Yes

P12

M

15 d

20 d

NA

P13

M

8d

9d

NA

P14

F

2 mo

2 mo

Yes

P15

M

2 mo

1.5 yr

Yes

P16

M

5d

7d

No

NE, seizures, partially treated Leu þ Ilewith dietary protein restriction Classic NE, seizures NA Classic Dev del, seizures Leu- 893 Ile- 269 Val- 512 Intermediate Dev del, no seizures, NA macrocephaly Classic NE, seizures, poor feeding, Leu þ Ilelethargic Val- 236 Classic NE, dev del Leu þ IleVal- 442 Intermediate Dev del, seizures, MRI changes Leu þ Iles/o MSUD Val- 343 Intermediate Dev del, epileptic Leu þ Ileencephalopathy Val- 315 Classic NE Leu þ Ile-

P17

F

8d

16 d

Yes

Classic

P18

F

9 mo

10 mo

Yes

P19

F

6 mo

1.5 yr

Yes

NE, episodes of seizures, lethargy, refusal to feed Intermediate Seizures, refusal to feed, global dev del, hyperammonemia (112ug/dl) Intermediate Dev del

P20

M

5d

21 d

No

Classic

NE, seizures

P21

M

7d

12 d

No

Classic

NE, seizures

MSUD pattern Leu- 1706 Ile- 194 Val- 189 Leu þ Ile- 2198 Val- 386 MSUD pattern

P22

M

7d

10 d

Yes

Classic

NE, neutropenia, seizures

Leu þ Ile- 2755 Val- 599 MSUD pattern

P23

M

5d

15 d

No

Classic

P24

F

14 d

17 d

NA

Classic

Seizures, excessive crying, refusal to feed, convulsions NE, seizures

Leu þ Ile- 4792 Val- 671.19 Leu þ Ile- 2654 Val- 363

13 d

Classic

Current age of child

1958 Val- 686 MSUD pattern

Leu þ Ile- 2663 NA Val- 331 Leu þ Ile- 2730 Val- 364 MSUD pattern

2234 Val- 399 MSUD pattern

e

Death

MSUD pattern MSUD pattern

Died (age- NA) On protein restricted diet

Death 8 yr

MSUD pattern

4.5 yr NA

2175

MSUD pattern

On protein restricted diet NA

3596

NA

NA

NA

2783

MSUD pattern

2 yr

517

NA

On protein restricted diet On protein restricted diet Received Liver transplantation. Normal development NA

2850 Val- 760 MSUD pattern

4 yr 7 yr

Leu þ Ile- 4546 Val- 774 Leu þ Ile- 813 Val- 508

MSUD pattern

on fosium, carnitine, biotin

1 yr

Leu þ Ile- 519 Val- 425

MSUD pattern

On protein restricted diet On protein restricted diet

14 yr

6 mo

MSUD pattern

On protein restricted diet On protein restricted diet Died (age- NA)

Death

NA

Died at 18 d

Death

NA

NA

5 mo

4 mo

Abbreviations: d-days; yr-year; mo-month; Neo-neonate; infant-infancy; NA-not available; Leu-leucine, Ile-Ilecine; Val-valine; Dev del-Developmental delay; NE-Neonatal encephalopathy; TMS-acylcarnitine analysis using tandem mass spectrometry, GCMS-urinary organic acid analysis using Gas Chromatography mass spectrometry. Normal ranges on TMS: Leu þ Ile A (p.Gly345Arg) in DBT gene were identified in 3 (5 alleles) and 2 (2 alleles) patients each. None of the patients were known to be related, and were not from same community. 4.2. In-silico analysis for novel variations 4.2.1. BCKDHA gene Four novel mutations were identified. Non-sense variation, p.Lys400Ter was considered pathogenic as it results in premature truncation of protein. The truncated protein is expected to undergo nonsense mediated decay. Mutation p.Asp282His is predicted to affect the stability of protein as bonding pattern of His237 with the

474

Table 2 Mutations in BCKDHA, BCKDHB and DBT gene and bioinformatics tools used. Age of onset

Gene

Exon

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19

1.5 yr 1 mo Infant 13 d Infant 5d 7d Neo Neo 10 d 6 mo 15 d 8d 2 mo 2 mo 5d 8d 9 mo 6 mo

BCKDHA BCKDHA BCKDHA DBT DBT BCKDHA BCKDHB BCKDHB BCKDHB BCKDHB BCKDHA BCKDHB BCKDHA BCKDHA DBT BCKDHB BCKDHB DBT BCKDHB

9 6 7 7 9 7 3, 5 9 2 7 3, 4, 4 7 4, 1 7, 5,

P20 P21 P22 P23 P24

5d 7d 7d 5d 14 d

BCKDHB BCKDHB BCKDHB

10 3, 10 1

10

10 9

10 9 9

Mutation (protein change)

Consanguinity

Allele 1

Allele 2

c.1234G > A (Val412Met) c.844G > Ca (Asp282His) c.940C > T (Arg314Ter) c.939G > C (Lys313Asn) c.1033G > Aa (Gly345Arg) c.979G > A (Glu327Lys) c.293T > Ga (Val98Gly) c.554 C > Ta (Pro185Leu) c.1022T > Aa (Ile341Asn) c.197-2A > Ga (splice site) c.868G > A (Gly290Arg) c.293T > Ga (Val98Gly) c.476 G > Aa (Arg159Gln) c.470A > Ca (Gln157Pro) c.939G > C (Lys313Asn) c.401T > A (Ile134Asn) c.3G > Aa (Met1?) c.939G > C (Lys313Asn) c.548G > C (Arg183Pro)

c.1234G > A (Val 412Met) c.844G > Ca (Asp282His) c.940C > T (Arg314Ter) c.939G > C (Lys313Asn) No mutation was identified c.979G > A (Glu327Lys) c.1065 delT (Pro356Leufs*34) c.554 C > Ta (Pro185Leu) c.1022T > Aa (Ile341Asn) c.197e2 A > Ga (splice site) c.868G > A (Gly290Arg) c.1065delT (Pro356Leufs*34) c.1198 A > Ta (Lys400Ter) c.470A > Ca (Gln157Pro) c.939G > C (Lys313Asn) c.1065delT (Pro356Leufs*34) c.3G > Aa (Met1?) c.1033G > A (Gly345Arg) c.964A > G (Thr322Ala)

c.1065delT (Pro356Leufs*34) c.293T > Ga (Val98Gly) exon 1a deletion No mutation was identified No mutation was identified

c.1065delT (Pro356Leufs*34) c.1065delT (Pro356Leufs*34) exon 1a deletion

Prediction software analysis Polyphen2, SIFT, mutation taster

b

I-mutant score

MutPred scores

Yes Yes No No Yes Yes No No No No Yes No No Yes No No Yes No No

e Disease causing e e Disease causing e Disease causing Disease causing Disease causing e e Disease causing Disease causing Disease causing e e d Disease causing Disease causing e

e 1.15 e e 1.46 e 3.95 0.86 3.43 e e 3.95 1.21 1.26 e e Decrease 1.46 e

e 0.452

No No Yes Yes No

e Disease causing e

e 3.95 e

Abbreviations: d-days; yr-year; mo-month; Neo-neonate; infant-infancy. Bioinformatics tools were not used for reported mutation. NA e Not available, () means not done. a Novel mutations highlighted in bold. b I-Mutant DDG value < 0 means decrease stability, DDG > 0 means increased stability of protein structure. c Mutpred scores, >0.75 indicates probability of deleterious mutation. d Polyphen 2 called this change as benign.

Conservation

Reference

e Conserved e e Conserved e Conserved Conserved Conserved e e Conserved Conserved Conserved e e Conserved Conserved e

Henneke et al., 2003 This Study Rodriguez-Pombo et al., 2006 Quental et al., 2008 This study Rodriguez-Pombo et al., 2006 This study, Bashyam et al., 2012 This study This study This study Gorzelany et al, 2009, Flaschker et al., 2007 This study, Bashyam et al., 2012 This study This study Quental et al., 2008 Bashyam et al., 2012 This study Quental et al., 2008, This study Edelmann et al., 2001, Bashyam et al., 2012 Bashyam et al., 2012 This study, Bashyam et al., 2012 This study

c

0.913

0.915 0.794

0.837 0.965 0.711

0.869 0.913

0.837

e Conserved e

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Patient ID

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Fig. 1. Computational analysis for novel mutations using PDB viewer. A) Bonds of Asp 237 with neighboring amino acid (Arg 252, Ile 31) is shown in wild type while modified bond and bond length of His 237 with neighboring amino acids is shown in mutant type. B) Bonds between Gln 112 with the cofactor TDP at 403 position and Arg at 114 with surrounding amino acids (Thr 88, His 291) and TDP is shown in wild type protein while it gets abolished in mutant type. C) Bonds of Val 48 with surrounding amino acids (Thr 57, Gly 51 and Asn 71) is shown in wild type protein while altered bond and bond length is shown in mutant type. D) Van der waal's interaction of wild type (Pro135) amino acid with surrounding amino acids is shown in wild type protein while altered interactions with Leu at 135 is shown in mutant protein. E) Ile 291 does not form any bond with surrounding atoms as shown in wild type but mutated Asn 291 forms bond with Phe 287, Thr 294 and Val 295, as shown in mutant type of protein. (Blue indicates Nitrogen, Red indicates oxygen of the atom, white is the carbon chain, yellow is phosphate group. White and blue balls indicate van der waal interaction. Amino acid position is according to crystal structure. Dashed lines are the strong H-bond with bond length displayed in purple. Position and 3 letter code of amino acids is mentioned in red. Amino acid position is according to crystal structure Orange arrow indicates amino acid in wild and mutant type and green arrow shows altered bonds. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

surrounding amino acids gets altered Fig. 1A. Mutations, p.Gln157Pro and p.Arg159Gln are predicted to prevent the proper folding of the protein as its bonding with cofactor ThDP (Thiamine diphosphate) is predicted to be abolished (Fig. 1B). 4.2.2. BCKDHB gene Six novel mutations were identified. a) Novel variation, p.Met1?, was considered pathogenic as the initiation codon no longer codes for methionine. The highly conserved ‘G’ of ATG is substituted by ‘A’. Due to mutation in the start codon, translational process does not start at the expected position and requires an alternative downstream initiation codon (Wolf et al., 2011). The next potential start site of the gene, in out of frame sequence, would comprise of only 27 amino acids. The next in-frame methionine codon is present 70 amino acids downstream from the original start codon. If it is used it is expected to lead to a truncated protein without Nterminal domain and hence unlikely to be functional (Star-Orf server and NCBI Orf finder). b) Deletion of exon 1 is predicted to be pathogenic because it disrupts the initiation of translation, and is expected to result in truncated protein as entire exon is deleted. Homozygous deletion of exon 1 was confirmed by using alternate primers as well as by multiplexing exon 1 and 5 of this gene, where

exon 5 served as a control fragment. (Refer to Supplementary File for further details) c) Splice site mutation (c.197-2A > G) was predicted to be pathogenic by BDGP software as it is expected to lead to a loss of an acceptor site. PDB viewer software analysis predicted pathogenicity for following missense mutations: d) Mutation p.Val98Gly is predicted to cause instability of the protein as bonding with surrounding amino acids is altered (Fig. 1C). e) Mutation p.Pro185Leu; is predicted to destabilize the protein as total potential energy of the protein is predicted to increase from 25053 KJ/Mol to 5690 KJ/ Mol as a result of substitution of a more hydrophobic residue, Pro (3.01 KJ/Mol) to leucine (7.11 KJ/Mol) (Fig. 1D). f) Mutation p.Ile341Asn may lead to improper folding of the protein as Ile (hydrophobic) is substituted by Asn (hydrophilic) residue, which would alter hydrophobic interactions with surrounding residues (Fig. 1E). These hydrophobic interactions are reported to be favorable for protein folding and maintaining the folded structure of protein (Matthews, 2001). 4.2.3. DBT gene One novel mutation, p.Gly345Arg was identified. In-silico analysis using Polyphen-2, mutation taster, SIFT and MutPred,

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showed this mutation to be pathogenic (Modeling was not done as crystal structure was not available for DBT). 5. Prenatal diagnoses Prenatal diagnoses were carried out in four families, based on mutation analysis on chorionic villous sampling. One fetus was noted to be affected, 2 were carriers and 1 was normal. Pregnancy with affected fetus was terminated after counseling. All unaffected babies are doing well after birth (Supplementary table 2). 6. Discussion In this study, clinical and mutational profile of 24 patients is presented. Two-thirds of the cohort presented with a classical phenotype of MSUD in neonatal period. Although five types of clinical presentations have been reported in MSUD (Chuang, 1998), the present study had patients with only classic or intermediate type. No patients with either ‘thiamine responsive’ or ‘intermittent MSUD’ were identified. The present study observed a skewed gender proportion, with males being more in number as compared to females. The skewed proportion only reflects the gender bias skewed towards males for most disorders in medical care, and the excess of males does not truly reflect the males being more affected with the disease. In the current study, consanguinity was observed in 37.5% families. The increased prevalence of consanguinity amongst children with MSUD is significant, as it is much higher than observed in the general population in India, of 11.9% ranging from 0.5 to 30.8% (Bittles, 2002). Consanguinity was however noted majorly in Muslims and South Indian Hindus where prevalence is stated to be high (Bittles, 2002). The high prevalence of homozygous mutations (62.5%) even in non-consanguineous families (46.6%) is noted in the current study, similar to that reported in other genetic disorders (Dalal et al., 2012; Ankala et al., 2015). The social structure of Indian population is such that marriages are arranged between individuals belonging to one community, but separated by family names or gotras (sub-castes), thus avoiding consanguinity. This, however, is akin to endogamy, explaining the homozygosity noted in various genetic disorders (Dalal et al., 2012). In the present study, mutations in BCKDHB gene accounted for half of the cases, while the other half was shared between BCKDHA (32%) and DBT gene (18%). This is in contrast with the reported literature where involvement of BCKDHA and BCKDHB genes is noted to be similar (Nellis and Danner, 2001; Bashyam et al., 2012; Narayanan et al., 2013). Mutations in DBT gene observed in the present study (18%) is similar to the frequency observed by others (20%) (Strauss et al., 2013). 6.1. Genotypeephenotype correlation In BCKDHA gene, previously known genotypes p.Gly290Arg and p.Val412Met have been reported to be associated with severe intermediate phenotype of MSUD (Chuang et al., 1995; RodriguezPombo et al., 2006; Flaschker et al., 2007). The current study also shows patients with homozygous genotypes, p.Gly290Arg and p.Val412Met to be having intermediate phenotype. However, the correlation could not be established for two other mutations, p.Glu327Lys and p.Arg314Ter. Patient in the present cohort (with p.Glu327Lys) had classic presentation while with the same mutation has been reported to be associated with intermediate phenotype in compound heterozygous state (Rodriguez-Pombo et al., 2006). Similarly, genotype, p.Arg314Ter which has been reported to cause classic neonatal phenotype (in compound heterozygous

form with another mutation), was noted to be associated with intermediate phenotype in homozygous form in the present study (Rodriguez-Pombo et al., 2006). In the study, we could not establish any genotypeephenotype correlation in our patients with MSUD. Majority of our cases (66.6%) represented the neonatal classical type of MSUD. All the three genes are implicated in classical neonatal and intermediate types of MSUD. In contrast with study from Flaschker et al., most of our neonatal classic patients (10 of 14 cases) had mutations in BCKDHB gene (Flaschker et al., 2007). Most of the intermediate type of MSUD patients with mutations in DBT gene have been shown to be thiamine responsive but no such response was obtained in our study (Chuang et al., 1997, 2004; Brodtkorb et al., 2010). In this study, homozygous p.Pro185Leu genotype in BCKDHB gene was detected in one patient with classic phenotype. Although this variant is present in the 1000 genome project (rs148905512), it was considered a novel mutation in the present study based on software analysis and its absence in 100 normal control alleles. A large proportion of novel mutations [11(55%) of 20] were identified in the study. The higher number of novel mutations highlights the heterogeneity in the Indian population, and the new mutations correlate with the distinctness of gene pool (Dalal et al., 2012). Amongst the novel mutations, one large deletion (entire exon 1) was noted in homozygous form in BCKDHB gene. Large deletions have never been reported in BCKDHB gene so far to the best of our knowledge. Novel mutation, p.M1? present in the initiation codon in the BCKDHB gene was considered to be deleterious, as mutation at the start site has been reported as pathogenic in other genes (Ramalho et al., 2009). Mutations could not be identified in 2 neonates, and in one allele of an infant. Large deletions or duplications in coding exons and deep intronic mutations could not be detected by the Sanger sequencing method used in the study. Large intronic and exonic deletions in DBT gene have been previously reported in HGMD database (Stenson et al., 2009). There were no founder or common genotypes noted except for one, c.1065delT (p.Pro356Leufs*34) in BCKDHB gene, which was observed in six of 48 alleles (5 patients). This mutation has been reported in one case (Bashyam et al., 2012). Two more genotypes were recurrent, c.939G > C (p.Lys313Asn) and c.1033G > A (p.Gly345Arg) in DBT gene, identified in 3 (5 alleles) and 2 (2 alleles) patients each. We noted mutational hot spot in exon 6 and 7 (8 of 14 alleles, 57% of mutations) of BCKDHA gene, similar to that reported in HGMD database. HGMD database lists 60% mutations in exons 4, 5 and 6 in BCKDHB gene. However, we noted only 36% mutations in exon 4 and 5, and none in exons 6. Exon 10 harbored 54.5% of mutations in BCKDHB gene (Stenson et al., 2009). This study reports prenatal diagnoses in 4 families performed using molecular methods. There have been very few studies reporting prenatal diagnosis for MSUD, and none are from India. Prenatal diagnosis in a single case, based on mutation analysis, was reported from Thailand (Tammachote et al., 2009). Prior to mutation analysis, prenatal diagnosis was performed by BCKA decarboxylase enzyme activity in chorionic villi or cultured amniocytes which was cumbersome and was not easily available (Potashnik et al., 1987). In conclusion, this study reports 20 different mutations, of which 11 (55%) were novel, and all were localized on both N-terminal and C-terminal of E1a, E1b, and E2 subunit disrupting assembly of the a2b2 complex. The high proportion of novel mutations, underlines the heterogeneity in Indian population and distinctness of the gene pool. Two mutations, c.1065delT in BCKDHB gene (6/22 alleles) and c.939G > C (p.Lys313Asn) in DBT gene (5/8

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alleles), are noted to be recurrent in the present study. No exact genotype phenotype correlation could be established, although most of our neonatal cases had mutations in BCKDHB gene and intermediate cases had mutation in BCKDHA and DBT gene. Information of gene mutations in families led to prenatal diagnosis in 4 cases. Thus, mutation identification helps in early and accurate diagnosis and is required for prevention of this burdensome disorder by prenatal diagnosis in families at risk. Acknowledgments The authors wish to acknowledge the contribution of our colleagues at Center of Medical Genetics e Dr Udhaya Kotecha, Dr Pratima Dash, Dr Swasti Pal, Dr Prahlad Balakrishnan, Dr Sireesha Movva, Dr Pratibha Bhai, Dr Sudhisha Dubey, Mr. Prashant Bhaskar, Mr Naresh Kashyap, Mr Rajendra Mishra; colleagues from other institutions for referring cases e Dr Bijoy Balakrishnan and Dr Riyas PK (Edappal Hospital, Kerala), Dr Rachna Gupta (Apollo hospital, Indore), Dr Sheela Nampoothiri (Amrita Institute of Medical Genetics, Cochin), Dr Chaitanya Datar (Sahaydri Medical Genetics and Tissue Engineering Facility, Pune), Dr Pritesh Pandya, (Sanjeevani Hospital, Rajkot), Dr Suvasini Sharma (Kalawati Saran Children's Hospital, New Delhi), Dr Jaspreet Kaur & Dr Sharad Sharma (Cocoon Hospital, Jaipur), Dr Neelam Kler, Dr Satish Saluja, Dr Pankaj Garg and Dr Arun Soni (Neonatology, SGRH, New Delhi). We would also like to thank our technical staff- Jyoti Singh, Deepika Babbar, Devender Prasad, Geetika Jhingan, Sandeepika Sharma, Azad Singh and other lab mates. The study was partially funded by Research Development Board of Sir Ganga Ram Hospital (SGRH). Our sincere thanks to all the patients and their families for their kind support. URLs used http://www.yeastgenome.org/cgi-bin/web-primer. http://www.fruitfly.org/seq_tools/splice.html. http://folding.biofold.org/i-mutant/i-mutant2.0.html. http://www.expasy.org/spdbv/. http://star.mit.edu/orf/runapp_html.html. http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi. Conflicts of interest All the authors declare that there is no conflict of interest with regards to preparation and submission of the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2015.08.002. References Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P., Kondrashov, A.S., Sunyaev, S.R., 2010. A method and server for predicting damaging missense mutations. Nat. Methods 7 (4), 248e249. http://genetics. bwh.harvard.edu/pph2/ (accessed 10.02.15.). Aevarsson, A., Chuang, J.L., Wynn, R.M., Turley, S., Chuang, D.T., Hol, W.G.J., 2000. Crystal structure of human branchred chain alpha ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. Structure 8 (3), 277e291. Ankala, A., Tamhankar, P.M., Valencia, C.A., Rayam, K.K., Kumar, M.M., Hegde, M.R., 2015. Clinical applications and implications of common and founder mutations in Indian subpopulations. Hum. Mutat. 36 (1), 1e10. Bittles, A.H., 2002. Endogamy, consanguinity and community genetics. J. Genet. 81 (3), 91e98. Bashyam, M.D., Chaudhary, A.K., Sinha, M., Nagarajaram, H.A., Devi, A.R., Bashyam, L., Reddy, E.C., Dalal, A., 2012. Molecular genetic analysis of MSUD from India reveals mutations causing altered protein truncation affecting the Ctermini of E1a and E1b. J. Cell Biochem. 113 (10), 3122e3132.

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