Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."     

Characterization of mutations in severe methylenetetrahydrofolate reductase deficiency reveals an FAD-responsive mutation

Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to methionine. Severe MTHFR deficiency results in marked hyperhomocysteinemia and homocystinuria. Patients display developmental delay and a variety of neurological and vascular symptoms. Cloning of the human cDNA and gene has enabled the identification of 29 rare mutations in homocystinuric patients and two common variants [677C>T (A222V) and 1298A>C (E429A)] with mild enzymatic deficiency. Homozygosity for 677C>T or combined heterozygosity for both polymorphisms is associated with mild hyperhomocysteinemia. In this communication, we describe four novel mutations in patients with homocystinuria: two missense mutations (471C>G, I153M; 1025T>C, M338T), a nonsense mutation (1274G>A, W421X), and a 2-bp deletion (1553delAG). We expressed the 1025T>C mutation as well as two previously reported amino acid substitutions [983A>G (N324S) and 1027T>G (W339G)] and observed decreased enzyme activity at 10%, 36%, and 21% of control levels, respectively, with little or no effect on affinity for 5-methyltetrahydrofolate. One of these mutations, 983A>G (N324S), showed flavin adenine dinucleotide (FAD) responsiveness in vitro. Expression of these mutations in cis with the 677C>T polymorphism, as observed in the patients, resulted in an additional 50% decrease in enzyme activity. This report brings the total to 33 severe mutations identified in patients with severe MTHFR deficiency.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron-exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3'UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3'UTR for mutations in a large series of BRCA-mutation negative, population and clinic-based breast cancer cases, and controls. Fifteen novel BRCA1 3'UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.* 528G>C, c.* 718A>G, and c.* 1271T>C and four found in controls, c.* 309T>C, c.* 379G>A, c.* 823C>T, and c.* 264C>T, reduced 3'UTR activity (P < 0.02), whereas two variants found in cases, c.* 291C>T and c.* 1139G>T, increased 3'UTR activity (P < 0.01). Three case variants, c.* 718A>G, c.* 800T>C, and c.* 1340_1342delTGT, were predicted to create new miRNA binding sites and c.* 1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3'UTR reporter activity when coexpressed with the predicted targeting miRNA, miR-103. This is the most comprehensive identification and analysis of BRCA1 3'UTR variants published to date.     

Allelic heterogeneity of SMARD1 at the IGHMBP2 locus

Spinal Muscular Atrophy with Respiratory Distress (SMARD) is an autosomal recessive disorder characterized by neurogenic muscular atrophy due to progressive anterior horn cell degeneration and early life-threatening respiratory failure ascribed to diaphragmatic dysfunction. SMARD is clinically and genetically heterogeneous. SMARD type 1 is characterized by onset of respiratory failure within the first weeks of life and has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We report here the identification of nine novel IGHMBP2 mutations in five SMARD1 patients, including seven missense [ c.587A>G (p.Gln196Arg), c.647C>T (p.Pro216Leu), c.752T>C (p.Leu251Pro), c.1693G>A (p.Asp565Asn), c.1730T>C (p.Leu577Pro), c.1807C>T (p.Arg603Cys), c.1909C>T (p.Arg637Cys)] and two nonsense mutations [ c.1488C>A (p.Cys496X), c.2368C>T (p.Arg790X)]. Interestingly, 7 of 9 mutations occurred at highly conserved residues of the putative DNA helicase domain. The identification of novel IGHMBP2 variants will hopefully help diagnosing SMARD1 and contribute to a better functional characterization of IGHMBP2 gene product.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

High prevalence of mutations in LCAT in patients with low HDL cholesterol levels in The Netherlands: identification and characterization of eight novel mutations

Lecithin:cholesterol acyltransferase (LCAT) is crucial to the maturation of high-density lipoprotein (HDL). Homozygosity for LCAT mutations underlies rare disorders characterized by HDL-cholesterol (HDL-c) deficiency while heterozygotes have half normal HDL-c levels. We studied the prevalence of LCAT mutations in referred patients with low HDL-c to better understand the molecular basis of low HDL-c in our patients. LCAT was sequenced in 98 patients referred for HDL-c <5th percentile and in four patients referred for low HDL-c and corneal opacities. LCAT mutations were highly prevalent: in 28 of the 98 participants (29%), heterozygosity for nonsynonymous mutations was identified while 18 patients carried the same mutation (p.T147I). The four patients with corneal opacity were compound heterozygotes. All previously identified mutations are documented to cause loss of catalytic activity. Nine novel mutations-c.402G>T (p.E134D), c.403T>A (p.Y135N), c.964C>T (p.R322C), c.296G>C (p.W99S), c.736G>T (p.V246F), c.802C>T (p.R268C), c.945G>A (p.W315X), c.1012C>T (p.L338F), and c.1039C>T (p.R347C)--were shown to be functional through in vitro characterization. The effect of several mutations on the core protein structure was studied by a three-dimensional (3D) model. Unlike previous reports, functional mutations in LCAT were found in 29% of patients with low HDL-c, thus constituting a common cause of low HDL-c in referred patients in The Netherlands.     

Identification of novel SNPs in the interleukin 6 receptor gene (IL6R)

The human interleukin-6 receptor (IL6R) is responsible for signal transduction of IL6 that might have associations with immune diseases and various infectious diseases. We have sequenced all 10 exons including the putative splicing site to identify single nucleotide polymorphisms in IL6R. Seven novel single nucleotide polymorphisms (SNPs) were identified; one SNP in the promoter region (-183G>A), one synonymous SNP in exon 2 (24013G>A: Ala31Ala), one non-synonymous SNP in exon 9 (48892A>C: Asp358Ala) and four in introns (29753A>C, 42700T>C, 48869T>A and 59818C>T). The frequencies of each SNP in the Korean population (n=300) were 0.48 (-183 G>A), 0.13 (24013 G>A), 0.41 (29753A>C), 0.41 (42700T>C), 0.1 (48869T>A), 0.42 (48892A>C), and 0.07 (59818C>T), respectively. Haplotypes and their frequencies were estimated by the EM algorithm. Linkage disequilibrium coefficients (/D'/) between each SNP pair were also calculated. The information on SNPs and haplotypes in IL6R would be useful for genetic studies of this gene.     

Six novel alleles identified in Italian hereditary fructose intolerance patients enlarge the mutation spectrum of the aldolase B gene

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding.     

Identification and functional analysis of two novel mutations in the CBS gene in Polish patients with homocystinuria

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an inherited disorder of homocysteine transsulfuration, which manifests by neurological, vascular and connective tissue involvement. So far, 130 pathogenic mutations have been recognized in the CBS gene. We examined 10 independent alleles in Polish patients suffering from CBS deficiency, and we detected four already described mutations (c.1224-2A>C, c.684C>A, c.833T>C, and c.442G>A) and two novel mutations (c.429C>G and c.1039+1G>T). The pathogenicity of the novel mutations was demonstrated by expression in E.coli. This is the first published communication on mutations leading to CBS deficiency in Poland.     

The molecular basis of cystathionine beta-synthase (CBS) deficiency in UK and US patients with homocystinuria

The molecular basis of cystathionine beta-synthase (CBS) deficiency has been studied in 536 patient alleles with 130 different mutations described. To date, no study has reported on the incidence of any of the reported mutations in patients from the UK and the US. We developed a new antisense oligonucleotide (ASO) PCR/hybridization method to screen for 12 of the most frequent CBS mutations in 14 unrelated patients from the UK and 38 unrelated patients from the US, a total of 104 independent alleles. We determined 16/28 (57%) and 28/76 (37%) of the affected alleles in the UK and US patients, respectively. Four different mutations were identified in the UK patients (c.374G>A, R125Q; c.430G>A, E144K; c.833T>C, I278T; c.919G>A, G307S) and 8 mutations identified in the patients from the US (c.341C>T, A114V; c.374G>A, R125Q; c.785C>T, T262M; c.797G>A, R266K; c.833T>C, I278T; c.919G>A, G307S; g.13217A>C (del ex 12); c.1330G>A, D444N). The I278T was the predominant mutation in both populations, present in 8 (29%) of 28 independent alleles from the UK and in 14 (18%) of 76 independent alleles from the US. The incidence of the G307S mutation was 21% in the UK patients and 8% in the US patients. The spectrum of mutations observed in the patients from the UK and US is closer to that which is observed in Northern Europe and bears less resemblance to that observed in Ireland.     

Clustering of variations and haplotype analysis in the highly variable region of exon 11 of BRCA1 in Chinese women with sporadic breast cancer

Studies on mutations of BRCA1 gene in Oriental populations, especially in Chinese are sparse. To evaluate the contribution of BRCA1 mutations to sporadic breast cancer in Chinese, mutations in exon 11 from nucleotide positions 3533 to 3682(U14680), a highly variable region of BRCA1 gene, were screened by single-strand conformation polymorphism (SSCP) and DNA sequencing in 329 genomic DNAs from 95 Chinese women with sporadic breast cancer, 29 with benign breast disease and 50 controls. As results, 6 single nucleotide changes were found, and 5 of them (3545A>G, 3551G>T, 3607C>T, 3646T>A and 3661T>C) were newly discovered. The 3545A>G and 3607C>T were germline variations, and the other 3 were somatic variations. In addition, four new haplotypes were defined. Since these newly discovered nucleotide changes were only detected in patients, our results suggest that BRCA1 may also play a role in the development of sporadic breast cancer in Chinese populations.     

Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene

Antithrombin (AT) is a major physiological inhibitor of hemostasis. We report 22 novel antithrombin gene (SERPINC1) mutations associated with antithrombin deficiency in 17 French and five German families. They were all present at the heterozygous state. Nine missense mutations accounted for type I deficiency, defined by equally low antithrombin activity and antigen level. Most of them (7/9) affected highly conserved serpin residues and were associated with venous thrombosis occurring at a young age (before age 32). One splice site, one nonsense mutation, three small deletions and one insertion were also identified as a cause for type I antithrombin deficiency. Seven other missense mutations were identified in type II or unclassified AT deficiency; g.5270C>T (p.T147I, T115I) and g.5281A>T (p.I151F, I119F) change residues in the heparin binding region, g.13267C>G (p.P439A, P407A) and g.13271T>C (p.F440S, F408S) affect amino acids in the pleiotropic region, g.2372G>A (p.G25D, G-8D) changes a signal peptide amino acid, g.2456G>C (p.C53S, C21S) affects one of the three disulfide bonds of the protein, and g.7585A>T (p.M347K, M315K) changes a nonconserved residue on strand 2C.     

Clinical and molecular genetic analysis in Chinese patients with distal myopathy with rimmed vacuoles

Distal myopathy with rimmed vacuoles (DMRVs) is an autosomal recessive vacuolar myopathy that has been reported in different ethnic populations with the common mutations of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. We presented the clinical, pathological and genetic characteristics of eight Chinese DMRV patients from six unrelated families. Six previously reported Chinese DMRV patients from four unrelated families were also reviewed for comparison in GNE mutations. In the present eight patients with DMRV, direct sequencing analysis revealed one homozygous mutation of c.1760T>C (p.I587T) and seven compound heterozygous mutations in the GNE gene. The latter included two known mutations, c.1892C>T (p.A631V) and c.527A>T (p.D176V), and three novel mutations, c.1523T>C (p.L508S), c.103G>A (p.E35K) and c.153A>G (p.I51M). The allelic frequency of c.1523T>C (p.L508S) was 25% in the Chinese patients with DMRV. Our findings expand the genetic spectrum of DMRV and indicate that the common mutations of GNE gene in DMRV may be variable among different ethnic populations.     

Association of the osteoprotegerin gene polymorphisms with bone mineral density in postmenopausal women

OBJECTIVES: Osteoprotegerin (OPG) is a recently discovered member of the tumour necrosis factor receptor superfamily. It plays a crucial role in the control of bone resorption and its gene could therefore be a good candidate gene for osteoporosis. The aim of our work was to find polymorphisms in the OPG gene and to investigate their possible contribution to the genetic susceptibility to osteoporosis by testing for their association with bone mineral density (BMD). METHODS: The whole OPG gene coding region was screened for the presence of polymorphisms in a group of 60 osteoporotic women by single-strand conformation polymorphism analysis (SSCP) approach. Association of the discovered polymorphisms with bone mineral density was investigated in 136 Slovenian postmenopausal women. RESULTS: We detected eight OPG gene polymorphisms that were confirmed by direct DNA sequencing, deletion 4752_4753delCT and nucleotide substitutions 1181G>C, 1217C>T, 1284G>A, 4501C>T, 6893A>G, 6950A>C and 8738T>A. Nucleotide substitutions 1284G>A and 8738T>A have not been previously described. Polymorphisms 4752_4753delCT, 6893A>G and 6950A>C were in complete linkage and the same was true for 1217C>T and 4501C>T. The association with BMD was found only for polymorphism 1181G>C. Subjects with genotype 1181GG had significantly lower lumbar spine BMD than subjects displaying 1181GC. CONCLUSIONS: By our approach we detected eight polymorphisms in the OPG gene. According to our analysis polymorphism 1181G>C is associated with BMD and could therefore be considered as an element of genetic susceptibility to osteoporosis.     

Mutations in EDAR account for one-quarter of non-ED1-related hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of the eccrine sweat glands, hair, and teeth. The X-linked form of the disease, caused by mutations in the ED1 gene, represents the majority of HED cases. Autosomal-dominant and -recessive forms occur occasionally and result from mutations in at least two genes: EDAR and EDARADD. These different forms are phenotypically indistinguishable. To better assess the implication of the EDAR gene in HED, we screened for mutations in 37 unrelated HED families or sporadic cases with no detected mutations in the ED1 gene. We identified 11 different mutations, nine of which are novel variants, in two familial and seven sporadic cases. Seven of the 11 are recessive mutations (c.140G>A (p.Cys47Tyr), c.266G>A (p.Arg89His), c.329A>C (p.Asp110Ala), c.442T>C (p.Cys148Arg), c.1208C>T (p.Thr403Met), c.1302G>T (p.Trp434Cys) and c.528+1G>A), and the other four are probably dominant (c.1129C>T (p.Leu377Phe), c.1237A>C (p.Thr413Pro), c.1253T>C (p.Ile418Thr), and c.1259G>A (p.Arg420Gln)). Our study demonstrates that EDAR is implicated in about 25% of non-ED1 HED, and may account for both autosomal-dominant and -recessive forms. The correlation between the nature and location of EDAR mutations and their mode of inheritance is discussed. A genotype-phenotype relationship was evaluated, since such data could be helpful for genetic counseling.     

Clinical, immunological and molecular characteristics of 37 Iranian patients with X-linked agammaglobulinemia

BACKGROUND: X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency characterized by an early onset of recurrent bacterial infections, a profound deficiency of all immunoglobulin isotypes and a markedly reduced number of peripheral B lymphocytes. Eighty-five percent of the patients with this phenotype have mutations in Bruton's tyrosine kinase (BTK) gene. METHODS: To provide an informative outlook of clinical and immunological manifestations of XLA in Iran, 37 Iranian male patients with an age range of 1-34 years, followed over a period of 25 years, were studied. Twenty-four of the 37 patients were screened for BTK gene mutation using PCR-SSCP followed by direct sequencing. BTK protein expression assay was done by flow cytometry in 9 families. RESULTS: All patients first presented with infectious diseases, the most common of which were respiratory tract infections. Eighteen different mutations were identified, 13 of which were novel: IVS1+5G>C, 1896G>A, 349delA, 1618C>T, 1783T>C, 2084A>G, 1346delT, 1351delGAG, 587A>G, IVS14-1G>A, IVS3+2T>C, 1482G>A, 1975C>A. CONCLUSION: The fact that we found a great number of novel mutations in a relatively limited number of patients underlines the heterogeneity of BTK mutations in the Iranian population. The large number of new mutations indicates that extended studies in this region would be rewarding.     

Spectrum of novel mutations in the human PKLR gene in pyruvate kinase-deficient Indian patients with heterogeneous clinical phenotypes

Eighteen unrelated pyruvate kinase (PK)-deficient Indian patients were identified in the past 4 years with varied clinical phenotypes ranging from a mild chronic haemolytic anaemia to a severe transfusion-dependent disorder. We identified 17 different mutations in the PKLR gene among the 36 mutated alleles. Ten novel mutations were identified: 427G>A, 499C>A, 1072G>A, 1180G>T, 1216G>A, 1220A>G, 644delG, IVS5 (+20) C>A, IVS9 (+44) C>T, and IVS9 (+93) A>C. A severe syndrome was commonly associated with some mutations, 992A>G, 1436G>A, 1220A>G, 644delG and IVS9 (+93) A>C, in the PKLR gene. Molecular graphics analysis of human red blood cell PK (RPK), based on the crystal structure of human PK, shows that mutations located near the substrate or fructose 1,6-diphosphate binding site may change the conformation of the active site, resulting in very low PK activity and severe clinical symptoms. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. In particular, the 1216G>A and 1219G>A mutations significantly affect the interdomain interaction because they are located near the catalytic site in the A/B interface domains. The most frequent mutations in the Indian population appear to be 1436G>A (19.44%), followed by 1456C>T (16.66%) and 992A>G (16.66%). This is the first study to correlate the clinical profile with the molecular defects causing PK deficiency from India where 10 novel mutations that produce non-spherocytic haemolytic anaemia were identified.