Introduction

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme deficiency affecting more than 400 million people worldwide (WHO Working Group 1989) with a frequency of >5% in Asia Pacific region (Howes et al. 2013). The prevalence of G6PD deficiency, first reported in 1963, varied from 2 to 27% in different population groups; however, there is limited information about its molecular basis among the diverse Indian populations (Kumar et al. 2016; Verma et al. 2020). G6PD is a housekeeping enzyme and is known to protect red blood cells (RBCs) from the harmful effects of reactive oxygen species (Al-Musawi et al. 2012). It is expressed in all tissues, but clinical manifestations of its deficiency are seen almost exclusively in RBCs. Since mature RBCs lack the citric acid cycle, pentose–phosphate pathway is the only NADPH-generation process therein and thus G6PD deficiency invariably has serious clinical consequences including neonatal jaundice and acute haemolytic anaemia but not all neonates with G6PD deficiency develop features of hemolysis (Bizzarro et al. 2004) related to drugs known to exacerbate hemolysis, infection or ingestion of fava beans among other food. In India, malaria is a national health problem and many programmes have been initiated to control its incidence. As a result, anti-malarial drugs are increasingly used, sometimes indiscriminately that can lead to sudden haemolysis in cases of G6PD-deficient individuals, and hence it is of great concern in populations where this deficiency is high.

Neonatal hyperbilirubinaemia remains an important reason to initiate newborn screening for G6PD deficiency to prevent kernicterus and its subsequent neurodevelopmental sequelae, as also highlighted recently (Tiwari 2017). G6PD deficiency may be either profound or partial; and according to the level of enzyme activity and severity of the symptoms, it has been grouped by WHO into five different classes, namely (i) enzyme deficiency with chronic nonspherocytic hemolytic anaemia; (ii) severe enzyme deficiency (<10% activity); (iii) moderate to mild deficiency (10–60% activity); (iv) very mild or no enzyme deficiency (~60% activity); and (v) increased enzyme activity (>2× normal activity) (Beutler 2008). These variations in severity are determined by specific mutations in G6PD (Xq28;154547586-154531390) (https://www.ncbi.nlm.nih.gov/gene/2539). The G6PD gene is about 20 kb in length comprising 13 exons and 12 introns (Martini et al. 1986). More than 200 mutations have been identified globally in G6PD-deficient individuals (Minucci et al. 2012). Almost all reported mutations are missense changes and the general absence of large deletions, frameshift and nonsense mutations support the concept of G6PD expression being critical for survival.

Thus, we aimed at establishing the clinical and mutation profiles for G6PD by carrying out a systematic screening of neonates for enzyme deficiency, as a part of a large prospective newborn screening study across several hospitals in the state of Delhi, India.

Methodology

Sample recruitment

This cross-sectional study with institutional ethical committee approval from the participating centres was conducted from November 2014 to 2016 under the larger prospective newborn screening programme by dried blood spot (DBS) sampling. All the newborns enrolled at the collaborating centres were screened for G6PD activity level at 24–48 h of life at Lok Nayak Hospital and Maulana Azad Medical College, Delhi. G6PD activity levels were measured by the semiautomated platform using Victor 2D in the first year of study and later by a completely automated platform using Genomic Screening Processor (GSP) and values <2.2 U/g Hb and <16 IU/dL respectively were considered as screen positives. All the putative screen positives were confirmed by quantitative test by spectrophotometric analysis using RBC lysates prepared from the respective neonate samples collected during the follow-up and only neonates thus confirmed were considered for genetic screening. Only a subset of these parents consented for additional sampling for mutation analysis were finally recruited for this study.

Demographic and clinical data

All the relevant data including demographic information containing gender, ethnicity, consanguinity, gestational age, birth weight and presence of cephalohematoma and other risk factors contributing to hyperbilirubinaemia were recorded for the study cohort.

Genetic analysis

Genomic DNA was extracted from peripheral blood samples of neonates (n=570) confirmed to be G6PD deficient using the screening protocol detailed above. To start with, G6PD exons known to encompass most commonly reported mutations along with ~100 bp flanking regions were amplified by PCR using primers designed using Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/); and checked on 2% agarose gel. Mutations were identified by polymerase chain reaction (PCR)-sequencing and/or PCR-restriction fragment length polymorphism and confirmed by Sanger sequencing using a commercial facility (Scigenom, India) and data analysed against NCBI reference sequence of G6PD (NC_000023.11) using DNASTAR (https://www.dnastar.com/).

Results

As mentioned in the sample recruitment section above, a total of ~200,000 neonates were screened at a multiple care centre in Delhi. All the putative screen positives were confirmed by quantitative test by spectrophotometric analysis using RBC lysates prepared from the respective neonates during the follow-up; and only they were called for genetic screening. A total of 570 neonates from a larger set, whose parents or caregivers consented to this study were finally included in the mutation screening. All these newborns are of north Indian origin based on self-reporting and mother tongue; with 399 (70%) being hindus and 171 (30%) muslims. Demographic details are presented in table 1.

Table 1 Demographic variables and clinical parameters of G6PD deficient neonates (n=570).
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Mutation profile of G6PD deficient neonates

All the 570 G6PD deficient neonates screened for mutations in G6PD were accounted for by seven different mutations, namely Mediterranean, Orissa, Kerala-Kalyan, Jammu, Chattam, Mahidol and Nilgiri/Coimbra. Of these, Mediterranean was the most common with 318 neonates (55.8%) followed by Orissa (n=187; 32.8%), Kerala-Kalyan (n=25), Jammu (n=24), Mahidol (n=14), Chattam (n=1), Nilgiri/Coimbra (n=1) (figure 1).

Figure 1
figure 1

Distribution of seven mutations among 570 G6PD deficient neonates.

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Genotype–phenotype correlations

Enzyme activity levels of G6PD deficient neonates estimated (see sample recruitment section above) were correlated with their respective mutation profiles in males (hemizygous) and females with homozygous or heterozygous mutations (table 2; figure 2). G6PD activity determined by either of the two methods used (Victor 2D or GSP) was lowest for the Mediterranean variant. A few cases with deviation from the expected enzyme activity levels were seen in patients with Mediterranean, Orissa and Kerala-Kalyan mutation types (figure 2).

Table 2 Mutation status and corresponding mean enzyme activity measured in G6PD deficient males (hemizygous) and females with homozygous or heterozygous mutations females.
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Figure 2
figure 2

Mutation-wise distribution of G6PD activity in G6PD deficient neonates (n=570) measured by (a) Victor 2D and (b) GSP.

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Other clinical complications

Documenting chronic complications such as nonspherocytic haemolytic anaemia might help us in understanding the actual burden of the disease but that was possible to be evaluated in only a subset of the samples. Of the 163 intramural neonates who were followed up during the first week of life, 68 neonates (41.7%) developed clinical jaundice. The mean age for the development of jaundice was 2.6 days (±1.7), mean serum bilirubin was 24.4 ng/dL (±7.8) with a direct fraction of 1.04 ng/dL. Of these, 23 (33.8%) required phototherapy and only two required exchange transfusion. The mutation distribution in these samples is provided in table 3.

Table 3 Clinical status and corresponding mutation distribution in G6PD deficient neonates.
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Discussion

With well-established clinical and allelic heterogeneity, prevalence of G6PD deficiency is ~4.9% worldwide (Nkhoma et al. 2009), but with varying frequency across different populations based on geographical locations as well as caste and ethnic groups suggestive of ethnic disparity (Nair 2009). A high frequency of enzyme-deficient individuals is reported from the Middle East, tropical Africa, tropical and subtropical Asia, Papua New Guinea and some areas of the Mediterranean. It is also widespread across malaria-endemic regions with the frequency being highest among the population of sub-Saharan Africa and less common across Americas (Howes et al. 2012). In India, malaria is a national health problem and the adverse drug reaction to antimalarials in G6PD deficient individuals also makes G6PD screening an important public health initiative. Furthermore, with 390,000 annual births with G6PD deficiency in India (Nair 2009), it certainly emerges as the most predictable and preventable inborn metabolic error. Of note, north and west India are generally reported to have a comparatively higher frequency of G6PD deficiency than south India (Reddy and Tripathy 2007; Verma et al. 2020). In addition, mutation spectrum in deficient subjects has been reasonably reported from central, western and southern parts of the Indian subcontinent (Kapoor and Thelma 2018) but most of these have been retrospective studies with small sample sizes. Of note, though prevalence data from the northern part of the country is available (Verma et al. 2020), mutation spectrum in deficient subjects and genotype–phenotype correlation (G×P) is negligible. Previous studies, mostly small scale, from different states of India and in caste groups such as Muslims, Jats, Parsis, Brahmins, Rajputs and schedule castes have revealed that its prevalence is endogenous and community-specific and ranges from 0 to 27% and is higher in tribal communities than caste groups (Bhasin 2006; Joshi et al. 2001; Reddy and Tripathy 2007; Moinuddin et al. 2017). However, a recent meta-analysis that included a total of 72 Indian studies confirmed the overall magnitude of G6PD deficiency to be ~8.5% in the Indian population (Kumar et al. 2016). Mediterranean mutation with the lowest enzyme activity and most severe phenotype (figures 1 and 2) is in conformity with a previous report in Indian population (60.4%) (Sukumar et al. 2004) as well as other populations namely from Jordan (76.2%) (Al-Sweedan and Awwad 2012), Baghdad (74.3%) (Al-Musawi et al. 2012), Italy (84%) (Cappellini et al. 1996) and Iran (56%) (Zahedpasha et al. 2013).

This study was to establish the mutation spectrum and G×P correlations in a moderate-sized subset (n=570) of G6PD deficient neonates (from among a large cohort of ~200,000 neonates screened in the only prospective study programme to date) from north India. As is well documented for an X-linked condition, of the 570 enzyme-deficient neonates, 473 (~83%) were males and 97 were females (table 1). Of note, all these deficient neonates were accounted for either one of the several known mutations in G6PD (figure 1). Further, enzyme activity in deficient samples in this study (figure 2; table 2) was mostly in accordance with previously documented levels for the different mutations (Cunningham et al. 2017). G6PD, is an X-linked disorder and these mutations are known to lead to loss of function. Accordingly, in males (hemizygous for all X-linked genes) with a mutation there would be G6PD deficiency; and ideally in female neonates with a heterozygous mutation, one would expect a higher enzyme activity than those with homozygous mutations. However, in females with heterozygous mutations, the phenomenon of Lyonization (random inactivation of one of the two X chromosomes early in embryonic life) (Davidson et al. 1963; Domingo et al. 2019), may result in a mixed population of normal and enzyme-deficient cells which explains the deviation of enzyme activity levels observed in a few cases (figure 2).

Mediterranean mutation (c.653C>T; p.Ser218Phe) is the most common in populations of southern Europe, the Middle East and the Indian subcontinent and is characterized by a very low enzyme activity (<10% of normal) (Kurdi-Haidar et al. 1990). In a recent study that screened newborns from different regions of India, Orissa mutation (56.5%) was found to be the most predominant variant followed by Mediterranean (23.6%) (Devendra et al. 2020). However, over 50% of the positive samples were from Maharashtra and Madhya Pradesh, thus the mutation spectrum presented in the article may not be a representation of the whole India. In another study constituting only male patients from 14 different population groups of India, Mediterranean was found to be the commonest (60.4%) deficient variant followed by Kerala-Kalyan (24.5%) and Orissa (13.3%) (Sukumar et al. 2004). Other studies has been done regionally and with very small sample size, and shows varied predominance level based on the population studied, for example, central India and east India show a higher prevalence of Orissa mutation (Devendra et al. 2017; Kumar et al. 2020), Namoru followed by Kerala-Kalyan most predominantly found among the Dravidian speaking tribes of Nilgiri district, Tamil Nadu (Chalvam et al. 2008; Mukherjee et al. 2015).

It is noteworthy that we were able to obtain the mutation spectrum in G6PD in this prospective study but the limitation remains that the long-term follow-up of these neonates to document chronic complications such as non-spherocytic haemolytic anaemia which could have helped us in understanding the actual burden of the disease was not possible for all the samples, due to poor response of parents of deficient neonates to recall for a non-lethal condition, such as G6PD deficiency. Jaundice was observed in neonates harbouring all mutations except Jammu in our study. But no correlation between jaundice and enzyme activity levels could be tested in our study due to the small sample size. Similar findings were however reported from the Apulia region in Italy for 54 G6PD deficient, unrelated male neonates (Pietrapertosa et al. 2001), and another study which reported low haemoglobin and haemocrit levels and severe jaundice, warranting higher intervention compared to jaundiced neonates with normal G6PD activity levels (Çelik et al. 2013).

In conclusion, knowing G6PD activity/mutation status may enable triaging those neonates with jaundice whose closer surveillance may help in preventing kernicterus and its long term sequelae. It may also be mentioned that frequency of G6PD deficient alleles have been reported to be very high in subtropical regions and females can be homozygous or compound heterozygous and can present with the same clinical manifestations as hemizygous males (van den Broek et al. 2016; de la Caridad Oliva Venereo and Viñas Martínez 2018). The benefits of such screening would therefore extend well beyond the neonatal period, especially in malaria-endemic zones where anti-malarial drugs are frequently prescribed. In addition, mutational analysis in deficient subjects besides contributing to prediction of disease severity and appropriate timely management may also help in the identification of heterozygous females who otherwise would be missed by routine screening for G6PD.