First Report of an α Chain Variant [Hb Coombe Park (HBA2: c.382A>G)] from India, Coinherited with a Novel SERPINC1 Gene Mutation: A Double Whammy?

Coinheritance of a high oxygen affinity structural hemoglobin (Hb) variant along with a thrombophilia marker is a rare occurrence. This may lead to a multi fold increase in the risk of thrombosis in patients. We report here a first case of Hb Coombe Park (HBA2: c.382A>G; p.Lys128Glu) from India, coinherited with a novel mutation (c.839C>G; p.Ser280Ter) on the SERPINC1 gene. This coinheritance has not been reported before. Though the patient is presently asymptomatic, identification of these variants will help in genetic counseling and to decide the future course of action in case of any clinical complications.

Detection of a novel mutation in NLRP3/CIAS1 gene in an Indian child with Neonatal-Onset Multisystem Inflammatory Disease (NOMID).

Neonatal-Onset Multisystem Inflammatory Disease (NOMID) or Chronic Infantile Neurologic Cutaneous Articular (CINCA) syndrome is a monogenic autoinflammatory disorder characterized by urticarial skin rash, fever, chronic meningitis and joint manifestations. Here we report a case of an Indian male child who presented at the age of 9 months with fever, respiratory distress, urticarial skin rash, arthritis, and neuroregression. Suspecting NOMID/CINCA syndrome, the child's blood was sent to the Jaslok Hospital and Research Centre for mutation analysis of the CIAS1/NLRP3 gene. The DNA was screened for mutations in exon 3 of CIAS1/NLRP3 gene by automated Sanger sequencing. DNA sequencing showed a novel heterozygous c.1813A➔G, p.R605G mutation in exon 3 of CIAS1/NLRP3 gene (ref no NM_001243133.1). His parents tested negative for this mutation. We therefore identified a novel de novo mutation in this family in the CIAS1/NLRP3 gene responsible for the child's clinical features.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient.

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

Molecular pathology of rare bleeding disorders (RBDs) in India: a systematic review.

BACKGROUND: Though rare in occurrence, patients with rare bleeding disorders (RBDs) are highly heterogeneous and may manifest with severe bleeding diathesis. Due to the high rate of consanguinity in many caste groups, these autosomal recessive bleeding disorders which are of rare occurrence in populations across the world, may not be as rare in India. OBJECTIVES: To comprehensively analyze the frequency and nature of mutations in Indian patients with RBDs. METHODS: Pubmed search was used (www.pubmed.com) to explore the published literature from India on RBDs using the key words "rare bleeding disorders", "mutations", "India", "fibrinogen", "afibrinogenemia", "factor II deficiency", "prothrombin" "factor VII deficiency", "factor V deficiency", "factor X deficiency", "factor XI deficiency", "combined factor V and VIII deficiency", "factor XIII deficiency", "Bernard Soulier syndrome" and "Glanzmanns thrombasthenia" in different combinations. A total of 60 relevant articles could be retrieved. The distribution of mutations from India was compared with that of the world literature by referring to the Human Gene Mutation Database (HGMD) (www.hgmd.org). RESULTS: Taken together, 181 mutations in 270 patients with different RBDs have been reported from India. Though the types of mutations reported from India and their percentage distribution with respect to the world data are largely similar, yet much higher percentage of small deletions, duplication mutations, insertions, indels were observed in this analysis. Besides the identification of novel mutations and polymorphisms, several common mutations have also been reported, which will allow to develop a strategy for mutation screening in Indian patients with RBDs. CONCLUSION: There is a need for a consortium of Institutions working on the molecular pathology of RBDs in India. This will facilitate a quicker and cheaper diagnosis of RBDs besides its utility in first trimester prenatal diagnosis of the affected families.

Hb Koln [ß98(FG5) [GTG → ATG, Val → Met]: the first report from India.

BACKGROUND: The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies. AIM: To identity the cause of mild anemia, reticulocytosis, and hepatosplenomegly in a case of non-spherocytic hemolytic anemia. MATERIALS AND METHODS: A 34-year-old female patient originating from Maharashtra, western India presented with mild anemia and jaundice which had persisted since childhood. Investigations included a complete blood count, screening for red cell membrane protein defects, Hb analysis by high-performance liquid chromatography (HPLC) and cellulose acetate electrophoresis (pH 8.9), heat instability test and DNA sequencing. RESULTS: Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window (9.8%) with a retention time of 4.90 minutes. Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A2 and Hb S. The heat instability test was positive. DNA analysis of α globin genes showed absence of both deletional and non- deletional α thalassemia. DNA sequencing of the ß globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln. CONCLUSION: Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.

Hemoglobin Fontainebleau [a21(B2)Ala>Pro]: The second report from India.

Structural hemoglobin (Hb) variants are mainly due to point mutations in the globin genes resulting in single amino acid substitutions. Until date, about 200 alpha chain variants have been identified and they are usually detected during the hemoglobinopathy screening programs. Under a community control program for hemoglobinopathies, which involved screening of antenatal cases followed by prenatal diagnosis if indicated. Here, we report a rare alpha globin gene variant Hb Fontainebleau [a21(B2)Ala>Pro] detected in the heterozygous condition in a 35-year-old pregnant lady screened during this program. This is the second report of this alpha globin variant from India. Unlike the earlier case from India where Hb Fontainebleau was reported in a neonate who was also a carrier of Hb Sickle and had no clinical problems, this case presented with a bad obstetric history associated with the secondary infertility. However, the presence of the variant and the obstetric complications may be unrelated.

Variable presentation of HB H disease due to homozygosity for the rare polyadenylation signal A T(Indian) (AATAAA>AATA- -) mutation in four Indian families.

The aim of this study was to identify the molecular defects leading to the variable clinical and hematological presentation of four patients with Hb H disease. Investigations included a complete blood count, high performance liquid chromatography (HPLC) analyses, cellulose acetate electrophoresis (pH 8.9), heat stability test, α genotyping by multiplex gap polymerase chain reaction (gap-PCR) to screen for the eight common α-globin gene deletions and DNA sequencing to detect the other deletional and nondeletional α-globin gene mutations. Two patients aged 15 and 5.5 years had a mild clinical presentation. The first patient aged 3 years had a severe presentation requiring regular transfusions. This patient also had an enlarged spleen and had to undergo splenectomy. The third patient, aged 5 years, also had severe anemia, had been transfused once and had a spleen of 4.5 cms. The hemoglobin (Hb) levels in the four patients ranged from 4.2 to 8.2 g/dL and they all had reticulocytosis (10.0 to 31.0%). Cellulose acetate electrophoresis at pH 8.9 showed a fast moving band that ranged from 18.0 to 25.9%. All the four patients were homozygous for the polyadenylation signal A (polyA) T(Indian) (AATAAA>AATA-) mutation. This mutation has been seen in Eastern India but not from Maharashtra and Uttar Pradesh where our patients originated.

SRY sequence in maternal plasma: Implications for non-invasive prenatal diagnosis: First report from India.

AIM: The presence of circulatory cell-free fetal DNA in maternal plasma has found new applications in non-invasive risk-free prenatal diagnosis. MATERIALS AND METHODS: We made use of a size separation approach along with real time polymerase chain reaction (PCR) to evaluate the use of fetal DNA in the detection of the sex of the fetus. Cell-free fetal DNA was isolated from the plasma of 30 women (10-20 weeks gestation) using a size separation approach. We made use of Taq Man Chemistry and real time PCR using primers and probes for GAPDH and SRY. RESULTS: Only 24 cases could be studied as there was no amplification in six cases. Fetal sex was accurately determined in all of the 24 cases wherein 19 women were carrying male fetuses and five women were carrying female fetuses. An increase in the amount of fetal DNA was observed with an increase in the gestational age. CONCLUSIONS: Real time PCR analysis is a highly sensitive and accurate tool for non-invasive prenatal diagnosis, allowing detection of the sex of the fetus as early as 10 weeks of gestation. Non-invasive prenatal diagnosis eliminates the risk of fetal loss associated with the invasive procedure.

First case of Hb Fontainebleau with sickle haemoglobin and other non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders.

OBJECTIVE: To evaluate the significance of non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders. METHODS: 1534 newborn babies were screened in the last 2 years for sickle cell disease using a targeted screening approach. Investigations included a complete blood count, high performance liquid chromatography analysis, cellulose acetate electrophoresis (pH 8.9), heat stability test, restriction digestion and Amplified Refractory Mutation System for confirmation of sickle haemoglobin (Hb S), α genotyping by multiplex PCR and DNA sequencing. RESULTS: Three non-deletional α gene variants, Hb Fontainebleau, Hb O Indonesia and Hb Koya Dora, were identified in heterozygous condition in newborns. This is the first report of Hb Fontainebleau in association with Hb S. The baby had anaemia at birth (Hb 11.4 g/dl) with no cyanosis, icterus or need for transfusion. She had occipital encephalocoele and was operated on day 24 to remove the mass. The baby diagnosed with Hb O Indonesia in combination with Hb S also had a low haemoglobin level of 12.7 g/dl. CONCLUSION: Newborn screening for sickle cell disorders also enabled us to identify three α globin chain variants. Two babies who inherited Hb Fontainebleau and Hb O Indonesia along with Hb S had reduced Hb levels at birth and need to be followed up.

Hb H disease due to homozygosity for a rare alpha2-globin variant, Hb Sallanches.

We report a 6-year-old child with Hb H disease due to homozygosity for Hb Sallanches [alpha104(G11)Cys-->Tyr], an unstable alpha2 chain variant. This child presented with a hemolytic anemia of intermediate severity and had never been transfused. This variant often remains undetected in the heterozygous state.

Effect of hydroxyurea on the transfusion requirements in patients with severe HbE-beta-thalassaemia: a genotypic and phenotypic study.

BACKGROUND: Haemoglobin E (HbE)-beta-thalassaemia has a very variable clinical presentation. The management of severe cases that are often transfusion dependent is similar to that of cases of beta-thalassaemia major; however, this is often not possible in India because of its high cost and the lack of availability of safe blood at many places. Thus there was a need for a drug such as hydroxyurea, which is known to reduce the transfusion requirements of patients with thalassaemia intermedia. This study was undertaken to evaluate the response of Indian patients with HbE-beta-thalassaemia to hydroxyurea. MATERIALS AND METHODS: 11 patients with HbE-beta-thalassaemia receiving regular transfusion plus two less frequently transfused patients were selected for hydroxyurea therapy. Clinical and haematological evaluation was performed before and after treatment for 2 years. Molecular studies included beta-globin genotype, beta-globin gene haplotype, Xmn I polymorphism and alpha-genotyping. RESULTS: Four clinically severe patients became transfusion independent (responders) after hydroxyurea therapy, four patients showed a reduction in their transfusion requirements (partial responders), and three patients were non-responders. Responders showed a statistically significant increase in Hb, mean corpuscular volume, mean cell Hb, fetal Hb and F cells with a reduction in their transfusion requirements. A reduction in serum ferritin concentration was also seen in responders and non-responders. CONCLUSIONS: Genetic markers such as Xmn I polymorphism and alpha-gene deletions were not always beneficial for the response to hydroxyurea therapy. Thus many more markers and a larger cohort need to be studied to predict the response in these patients.

Molecular diversity of hemoglobin H disease in India.

This study was undertaken to evaluate the variable clinical expression of hemoglobin (Hb) H disease in India. For the study, alpha genotyping was done in 8 patients with Hb H disease using multiplex polymerase chain reaction and DNA sequencing. The study revealed that 4 genotypes (- -(SEA)/ -alpha(3.7), - -(SA)/-alpha(3.7), - -(SEA)/-alpha(3.7 Sallanches), - -alpha(3.7)/-alpha(3.7 Sallanches)) were responsible for Hb H disease, the alpha+ thalassemia mutation (-alpha(3.7) deletion) being the most common defect. The nondeletional mutation Hb Sallanches (alpha 2 codon 104 G --> A) was seen in 3 cases. Two unique and novel genotypes leading to Hb H disease were characterized (- -(SEA)/-alpha(3.7 Sallanches) and -alpha(3.7)/-alpha(3.7 Sallanches)). Because a majority of patients with Hb H disease do not have severe manifestations, prenatal diagnosis is usually unwarranted in India.

Response to hydroxyurea in beta thalassemia major and intermedia: experience in western India.

BACKGROUND: The clinical and hematological response to hydroxyurea was evaluated in beta thalassemia patients in western India with variable clinical severity and correlated with genetic factors. MATERIALS AND METHODS: Seventy-nine patients-[38-beta thalassemia intermedia-(group I), 41-beta thalassemia major-(group II)] on hydroxyurea therapy were followed-up for 20-24months. RESULTS: Among the frequently transfused patients in group I, 58% became transfusion independent and 16% showed a 50% reduction in transfusions after therapy which correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. Forty-one percent of patients in group I had associated alpha-thalassemia and 72.7% were XmnI (+/+). beta thalassemia chromosomes among the responders of group I (41%) were linked to haplotype (- + + - + + - - +) as against haplotype (+ - - - - - - - +) being more common among the non-responders. Response was not linked to the beta thalassemia mutations. Thirty-two percent of group II patients showed a 50% reduction in their transfusion requirements after therapy which also correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. A significant decrease in serum ferritin was seen in both groups. 19% of patients could not tolerate the drug. CONCLUSIONS: In group I, clinical response to hydroxyurea was better in patients with alpha-thalassemia, XmnI (+/+) and a higher mean fold increase in gamma mRNA expression. In group II, only one-third of patients showed a partial response.

Hematological and molecular analysis of novel and rare beta-thalassemia mutations in the Indian population.

A variety of mutations causing beta-thalassemia (beta-thal) have been seen in the Indian subcontinent. We report eight families in whom two novel mutations [codon 16 (C>T), IVS-II-613 (C>T)] and three rare mutations [codons 22/23/24 (-7 bp) (-AAGTTGG), -87 (C>A), codon 15 (-T)] were encountered among 375 beta-thal heterozygotes. They were referred to us for molecular characterization or prenatal diagnosis during a period of 2 years. Haplotyping was also done for linkage analysis.

Hydroxyurea in sickle cell disease--a study of clinico-pharmacological efficacy in the Indian haplotype.

There is clinical variability in the presentation of sickle cell disease among Indians. Vaso-occlusive crisis is common among non-tribal patients. Hydroxyurea, induces fetal hemoglobin (HbF) synthesis and reduces the clinical severity of sickle cell disease but individual patients have a variable response. This study was undertaken to investigate the efficacy and safety of hydroxyurea in Indians with severe manifestations where the beta(s) gene is linked to the Arab-Indian haplotype and is associated with higher HbF levels. Seventy-seven patients (29 adult sickle homozygous, 25 pediatric sickle homozygous, 23 adult sickle beta-thalassemia) selected for hydroxyurea therapy were evaluated for clinical, hematological, biochemical and genetic parameters and were followed for 24 months. Ninety-eight point seven percent of the sickle chromosomes were linked to the Arab-Indian haplotype, 27% of patients had associated alpha thalassemia and 65% were Xmn I +/+. Seventy-eight percent of the patients had no further crises after starting hydroxyurea. This effect was accompanied by a significant increase in HbF (p<0.001), but this increase was variable in individual cases. There was also an increase in gamma gene mRNA expression in the few cases so studied. Hemoglobin levels increased significantly (p<0.001) resulting in the cessation of blood transfusions. Leucopoenia was observed in one patient. Hydroxyurea was effective in reducing the clinical severity in Indian patients who initially had higher HbF levels and the presence of ameliorating factors, such as alpha-thalassemia and the Xmn I polymorphism. Hydroxyurea therapy with careful monitoring can thus change the quality of life of Indians with sickle cell disease.

A novel Ser123Pro substitution in the MIDAS domain of integrin 3 associated with variant Glanzmann's thrombasthenia in an Indian patient.

We report a novel 465T-->C (S123P) mutation in exon 3 of the GPIIIalpha gene in a patient with type III or variant Glanzmann's thrombasthenia (GT). Though this mutation did not affect fibrinogen binding to GPIIb-IIIalpha in activated platelets, it interfered with the platelet aggregation in a manner similar to GT.

Glanzmann's thrombasthenia: updated.

Glanzmann's thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen, epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmann's thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmann's thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb-IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic. However, there may be several laboratories in the developing world, which have no access to molecular biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.

Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease.

Glanzmann's thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA-1) system is important. Studies have shown that, in the normal population, the HPA-1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty-one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA-1 to HPA-6, using PCR-ASA (polymerase chain reaction-allele-specific amplification), and for GPIIb-IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA-1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 micro mol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 micro mol/l ADP. Seven out of 37 patients with HPA-1a/1a phenotype showed 1-5% fibrinogen binding and GPIIb-IIIa receptors. The two HPA-1b/1b patients showed 34.6% and 32% fibrinogen binding and > 10% GPIIb-IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA-1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.