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3-Methylglutaconic Aciduria Research

A distinct type of 3-methylglutaconic aciduria due to a mutation in the Translocase of Inner Mitochondrial Membrane 50 (TIMM50) gene
Abstract
BACKGROUND: 3-methylglutaconic aciduria biochemically characterized by increased urinary excretion of 3-methylglutaconic acid result from defective leucine metabolism and disorders affecting mitochondrial function though in many cases the cause remains unknown. Recently mutations in mitochondrial TIMM50 gene has been reported in four patients from two unrelated families. We report additional mutations in TIMM50 gene in 6 individuals from two unrelated consanguineous families with a distinctive type of 3-methylglutaconic aciduria.
METHODS: We report on three patients of South Asian ancestry with intractable epilepsy, microcephaly, developmental delay, visual deficit spastic quadriplegia and three Caucasian patients of eastern European origin with intellectual disability with or without seizure. Metabolic testing revealed mild lactic acidosis and excretion of large amount of 3-methylglutaconic acid in urine in all patients. Full exome sequencing was performed using genomic DNA isolated from one surviving patient, two healthy siblings and both parents of South Asian family. Exome sequencing was also performed for Caucasian patients of eastern European origin.
RESULTS: Exome sequencing identified two homozygous mutation Gly372Ser and Iso392Thr mutations in the gene TIMM50. There were no other candidate alterations in exome that could explain the phenotype in the proband. The mutations are located in the conserved C-terminal domain of the Tim50 protein that interacts with the N-terminal domain of the Tim23 protein in the intermembrane space and regulates mitochondrial protein import of presequence-containing polypeptides Both parents are heterozygous.
CONCLUSION: Given the phenotypic similarilty of the patients from two unrelated families and an earlier report of mutations in additional family, we conclude that TIMM50 gene mutation results in a novel mitochondrial disorder with 3-methyl glutaconic aciduria.
INTRODUCTION 3-methylglutaconic aciduria (MGCA), an increase in urinary 3-methylglutaconic acid or 3-methylglutaric acid, can be a nonspecific finding in mitochondrial disorders, organic aciduria, urea cycles disease, neuromuscular disorders. but is a consistent abnormality of 3-methylglutaconyl-CoA hydratase deficiency and patients with mutations in TAZ, SERAC1, OPA3, DNAJC19 and TMEM70 gene1. These genes all encode mitochondrial membrane or membrane related proteins. In 3-methylglutaconyl-CoA hydratase deficiency due to mutation in AUH gene , 3-methylglutaconic acid derives from 3-methylglutaconyl CoA (3MG CoA), an intermediate in leucine catabolism1. It has been proposed that in other disorders, 3-methylglutaconic acid derives from aberrant isoprenoid shunting from cytosol to mitochondria via mevalonate pathway or redirection of mitochondrial acetyl CoA toward production of 3MGA due to an increase in the intra-mitochondrial NADH/NAD ratio resulting from mutation induced impairment in electron transport chain or Kreb cycle function 2.
Examples of mitochondrial include Barth syndrome, a cardioskeletal myopathy with neutropenia, abnormal mitochondria and MGCA. Barth syndrome is caused by X-linked recessive mutations in the TAZ gene which encodes the mitochondrial membrane localized transacylase involved in the maturation of cardiolipin.
Autosomal recessive mutations in the OPA3 gene (OMIM: 606580), the mouse ortholog of which encodes a mitochondrial inner membrane protein of unknown function, cause MGCA3 (OMIM: 258501), a neuroopthalmologic syndrome characterized by early-onset bilateral optic atrophy and later-onset spasticity, extrapyramidal dysfunction and cognitive deficit.
MGCA5 (OMIM: 610198) is yet another form of MGCA caused by autosomal recessive mutations in the DNAJC19 gene (OMIM: 608977) and in addition to increased urinary excretion of 3-methylglutaconic acid, patients present with infancy or childhood onset dilated cardiomyopathy, microcytic anemia, mild muscle weakness and ataxia. Many patients die of cardiac failure. The DNAJC19 gene encodes the human homolog of the yeast Tim14 which is a part of the Tim23 mitochondrial protein import machinery and hasbeen shown to interact with the mtHsp70 in an ATP-dependent manner to regulate Tim23 function (Davey, 2006).
WE report a distinct type of 3-methylglutaconic aciduria resulting from a mutation in mitochondrial TIMM50 gene in 3 sibs from a consanguineous family. We initially reported these xases in abstract form. Recently two different mutations in mitochondrial TIMM50 gene have been reported in four patients with 3 methylglutaconic aciduria, epilepsy, severe intellectual disability and lactic acidosis.
Subjects Family 1
Family 1 has three affected sibs of South Asian ancestry with intractable epilepsy, microcephaly, developmental delay, visual deficit spastic quadriplegia. Two affected sibs died unexpectedly when they were visiting families in a remote area of a South Asian country. Metabolic testing had revealed large amount of 3-methylglutaconic acid in urine in all three affected sibs. The patients have a healthy brother and a healthy sister. Mother and father are first cousins. Detailed clinical history, imaging, EEG and metabolic testing were obtained for all affected persons. Full exome sequencing was performed using genomic DNA isolated from one surviving patient, two healthy siblings and both parents.
Patient IV-1. Patient IV-1 was the first born child of the parents and was born at 36 weeks gestation after a normal pregnancy and delivery. Her weight at birth was 1.99 kg. Her weight, height and head circumference were always below 5th centile. She also had asthma and frequent episodes of pneumonia presumably due to aspiration, but the family refused G-tube placement. She was severely delayed. She never sat, stand or spoke. She has poor head control, truncal hypotonia but very brisk tendon jerks and sustained clonus. Funduscopy revealed bilateral optic atrophy. She developed seizures at 1 year of age. EEG revealed multifocal spikes arising from both hemispheres. She was treated with phenobarbital and Zonegran but family was noncompliant with medications. She continued to have daily myoclonic jerks. MRI at 2.5 and 5 years of age showed increased T2 signal in basal ganglia and periventricular white matter, brain atrophy, prominent ventricle, increased extraxial fluid. Normal liver enzymes and blood count, normal blood and CSF glucose and a serum ammonia of 21. Several serum lactate levels were mildly elevated. Lactate 2.8, 4.5 (Pyruvate 0.23), 5.4 (normal 0.7 to 2.1) Lactate to pyruvate ratio 20:1. Urine organic acid analysis revealed very high lactic acid, 3-methylglutaconic acid, and 3-methylglutaric acid. Muscle biopsy revealed only scattered atrophic muscle fibers on electron microscopy. Respiratory chain enzyme activities were within normal limits. She died at 7.5 years of age apparently due to complications from an infection while she was visiting families in a remote area of a South Asian country.
Patient IV-4 was twin A born at 36 weeks gestation after an uncomplicated twin pregnancy. Her weight, height and head circumference were always below 5th centile. She was severely delayed. She never sat, stand or spoke. She has poor head control, truncal hypotonia but very increased reflexes and spasticity in the limbs. At nine-month-of age, she started to experience several episodes of eye fluttering and body jerking. Her EEG reved slow background, poor sleep architecture and frequent multifocal spike and sharp wave activities coming from both the left and right hemispheres. Her seizures were treated with Zonegran and was poorly controlled but parents refused more aggressive treatment of seizures. Metabolic testing revealed mild elevation of lactate and moderate increase of 3 methylglutaconic, 3 methylglutaric acids in urine. A brain MRI at 11-month-of age revealed diffuse volume loss supratentorially with prominent sulci and extraaxial fluid spaces, mild enlargement of the ventricles and patchy signal abnormalities in the basal ganglia bilaterally, especially involving the caudate nuclei and putamen. On spectroscopy with voxel placed in the right basal ganglia with short and long TE, there was a lactate peak which inverted on long TE spectrum. Also, the NAA peak was low with NAA to creatinine being 1.15 on short echo and 1.29 on long echo spectrum. Also, the choline was elevated with choline/creatine ratio being 1.00 on short echo and 1.41 on long echo images. She died at 1.5 years of age apparently due to complications of an infection while she was visiting families in a remote area of a South Asian country.
Patient IV-5 is a 13 year old female of South Asian ancestry, with 3-methylglutaconic aciduria intractable epilepsy, microcephaly, developmental delay, visual deficit and spastic quadriplegia. She was born at 36 weeks gestation after an uncomplicated twin pregnancy. She was twin B and stayed in NICU for 18 days for feeding issues. Her weight was 1.4 kg and she was not intubated. Patient first presented with seizures at 3 months of age with eyelid fluter and jerking of extremities. Her initial EEG revealed multifoal spikes. Initial biochemical evaluation revealed normal serum and CSF glucose, normal ammonia and liver enzymes. Serum lactate and CSF lactate 4.24 mmol were mildly elevated . Lactate was 2.7. Ammonia 25. Serum amino increased alanine 43.6 micromol/dl (9.9-34.5). Csf lactate 4.24 mmol. CSF alanine 7 micromol/dl (0.6 -4.7). There were also mild elevations of serum and CSF valine, leucine, isoleucine and alanine and lysine. Urine organic analysis revealed moderate increase of 3 methylglutaconic, 3 methylglutaric, glutaric, adipic, suberic, and sebacic acids. MRI of brain at 11 months of age revealed severe atrophic changes involving gray and white matter, predominantly of the cerebrum. Grossly abnormal signal is seen in the basal ganglia, particularly the caudate nucleus and the putamen with relative sparing of the globus pallidus and thalamus. A recent MRI (at age 13 years) reveals severe but stable atrophic changes of the gray and white matter of the supra and infratentorial brain, stable white matter changes of the putamen, caudate nucleus and periventricular white matter, Scattered diffusion restriction in the retrotrigonal white matter, compatible with active demyelination and atrophic changes of the optic nerves. Her seizures were treated with with Keppra, Lamictal, Zonegran and Onfi. She also receives carnitine. She continues to have brief episodes of whole body stiffening each week, but the family was also not very compliant with medications. Her current EEG shows slow background for age, poorly formed sleep spindles indicatvie of diffuse neuronal dysfunction, frequent multifocal interictal spike and wave suggests increased risk of seizures arising from multiple foci and hypsarrhythmia in sleep . She has failure to thrive despite G-tube feeding. At 12 years of age, G-tube was placed due to history of aspirations. Height, weight and head circumference below 5th centile. She is severely delayed. She is nonverbal and never learned to sit independently, stand or walk. She recognizes family members, responds to their voice and looks and smiles at them. Her fundoscopy shows mild optic atrophy. She has bilateral esotropia and dysconjugate gaze. She has poor head control and truncal hypotonia, but her limbs are spastic and her tendon reflexes are very brisk.
Family 2
Patient V:1 was the first son of Caucasian consanguineous parents (IV:4 and IV:5) of Eastern European origin. Within the context of an organic acid and amino acid study in young and adult subjects with non-syndromic developmental delay and intellectual disability, he was investigated at the age of 17 years and presented with a developmental language disorder (involving semantic, syntactic, and pragmatic components of the linguistic system), emotional and communicative problems (fearful, aggressive, and loner), and hyperactivity. On neuropsychological testing he showed a short attention span. The child was born at term after an uneventful pregnancy and his birth weight was 2.9 kg. At 4 months of age he was affected by myoclonic jerks that were controlled by administration of valproic acid and lamotrigine. Developmental delay was observed starting from the middle of the first year of life, accompanied by decreased muscle tone. He could walk without support only at 6 years. At last medical exam, the patient showed a reduced muscle mass (height 148 cm, Z-score 3.43; weight 38 kg, Z-score 4.21; BMI 17.1 kg/m2, Z-score 2.02) and a head circumference of 51 cm (Z-score 2.76). Due to refusal of parents, no brain imaging studies could be performed. Fundoscopic examination was normal. Laboratory tests, including creatine phosphokinase (CPK), liver enzymes and plasma amino acids, were normal. The profile of urinary organic acids showed a large peak of 3-methylglutaconic acid (113 mmol/mol creatine) and a slightly increased level of 3-methylglutaric acid (17 mmol/mol creatinine).
Patient V:3 was the younger brother of V:1, the third child of IV:4 and IV:5. He was investigated at the age of 11 years and presented with a clinical phenotype (developmental delay and intellectual and behavioral disorder) similar to that of his brother. The pregnancy and early postnatal course was unremarkable and birth weight was 3.1 kg. At 3 months he received valproic acid and lamotrigine to control tonic seizures with sudden stiffening movements of arms and legs. The boy walked independently at 4 years. When he was 9 years, his growing parameters were: height 119 cm (Z-score 2.47), weight 22 kg (Z-score 1.91), BMI 15.5 kg/m2 (Z-score 0.38), and head circumference 48 cm (Z-score 3.52). Neuropsychological exam revealed mental retardation and impaired communicative skills, including poor language abilities (few repetitive words with no sentences). Occasionally, the patient is aggressive. Ophthalmologic examination revealed left esotropia. High levels of 3-methylglutaconic acid (155 mmol/mol creatine) were identified in urine, together with smaller amounts of 3-methylglutaric acid (22 mmol/mol creatinine).
Patient V:5 was the second son of consanguineous parents (IV:9 and IV:10) related to those of patients V:1 and V:3. The girl was delivered by cesarean section because of growth arrest at 37 week. The neonate showed no external malformations. Birth weight was 2.1 kg. In the following years, the clinical phenotype was characterized by delayed developmental milestones, nocturnal enuresis, severe cognitive impairment, speech retardation, and lack of communicative skills. Results of the electroencephalogram were normal. No brain imaging data are available. On a few occasions, levels of ammonia and lactic acid were found to be slightly elevated, but these results could not be confirmed by repeated blood analyses. Plasma levels of amino acids are within normal range. Fundoscopic examination was normal up to 7 years, but since then there is evidence of mild bilateral optic atrophy. Urine levels of of 3-methylglutaconic acid and 3-methylglutaric acid were 176 mmol/mol creatine and 29 mmol/mol creatinine, respectively.
DISCUSSION Deleterious Nature of the TIMM50 gene alteration:
TIMM50 NM_001001563 c.1114G>A p.G372S
The p.G372S variant (also known as c.1114G>A), located in coding exon 9 of the TIMM50 gene, results from a G to A substitution at nucleotide position 1114. The glycine at codon 372 is replaced by serine, an amino acid with somewhat similar properties. The alteration is not observed in healthy cohorts: Based on data from the NHLBI Exome Sequencing Project (ESP), the TIMM50 c.1114G>A alteration was not observed among 6,503 individuals tested. Allele frequency data for this nucleotide position are not currently available from the 1000 Genomes Project and the alteration is not currently listed in the Database of Single Nucleotide Polymorphisms (dbSNP). Though some variants may appear to be rare due to database-specific ethnic underrepresentation, rare missense alleles commonly exhibit a deleterious effect on protein function (Kryukov, 2007; Tennessen, 2012). The altered amino acid is conserved throughout evolution: The G372 amino acid position is completely conserved in eukaryotes all the way from the yeast Saccharomyces cerevisiae to humans (Mokranjac, 2003). The alteration is predicted deleterious by in silico models: The p.G372S alteration is predicted to be probably damaging and deleterious by PolyPhen and SIFT in silico analyses, respectively. The amino acid is located in a functionally important protein domain: The p.G372S alteration is located in the conserved C-terminal domain of the Tim50 protein that interacts with the N-terminal domain of the Tim23 protein in the inter membrane space and regulates mitochondrial protein import of presequence-containing polypeptides (Geissler, 2002; Yamamoto, 2002; Guo, 2004). The alteration cosegregated with disease in the family herein: Co-segregation analysis revealed that this alteration is present in a heterozygous form in the mother, father and brother, and absent in the sister.
Based on the available evidence, the TIMM50 c.1114G>A (p.G372S) alteration is classified as a likely pathogenic mutation.
The TIMM50 gene is not currently known to underlie Mendelian disease (aka “clinically novel”). The TIMM50 gene function is consistent with the proband’s clinical presentation:
The Translocase of Inner Mitochondrial Membrane 50 (TIMM50) gene (OMIM: 607381) is located on human chromosome 19q13.2 and consists of 11 exons. It encodes the Tim50 protein, a 353 amino acid 40 kDA homolog of the yeast Tim50 protein that functions as an integral part of the mitochondrial Tim23 protein import machinery by linking protein translocation across the outer and inner mitochondrial membranes. This interaction was confirmed by the coprecipitation of Tim50 with an antibody against Tim23 (Geissler, 2002; Yamamoto, 2002; Guo, 2004). The authors further confirmed that the C-terminal domain of Tim50 is located in the inter-membrane space (IMS) where it stably binds to the segment of Tim23 that spans the IMS and regulates its function.
Nuclear encoded mitochondrial proteins are synthesized in the cytosol and subsequently imported into the mitochondria through the function of translocators, the TOM complex of the outer mitochondrial membrane (OMM), and the Tim23 and Tim22 complexes of the inner mitochondrial membrane (IMM) (Jensen, 2002). While the Tim22 complex is involved in the transport and insertion of proteins lacking the presequence into the inner membrane, the Tim23 complex is required to process and insert presequence-containing precursor proteins. The IMM generates a proton motive force that is critical for cellular energy synthesis (Stock, 2000) and the permeability barrier of the IMM needs to be maintained during the transport of proteins through the pore-forming Tim23 protein associated with other IMM proteins such as Tim14 (human DNAJC19), Tim17, Tim21, Tim44 and Tim50. Using various yeast IMM protein mutants, Meinecke et al. (2006) demonstrated that tim17 and tim21 mutant mitochondria displayed membrane potential values that were comparable to wild type mitochondria, whereas tim50 mutant mitochondria showed a drastic reduction of the membrane potential. Further functional studies revealed that the Tim23 channel is tightly regulated by Tim50 in its inactive state to maintain the IMM permeability barrier and is opened only when presequence-containing polypeptide chains need to be translocated into the mitochondrial matrix or the inter membrane space (IMS). Loss of Tim50 function in yeast led to cellular growth arrest and reduced cell viability (Mokranjac, 2003). Knockdown to Tim50 expression in cultured human cells using RNA mediated interference resulted in an increase in the release of cytochrome c and apoptosis in response to cell death stimuli (Guo, 2004).
A 50 kDa isoform of the human mitochondrial TIM50, TIM50a, consisting of 456 amino acids has been found to localize in nuclear speckles, specifically in the Cajal bodies, and interact with small nuclear ribonuclear proteins (snRNPs), the coilin protein and the Survival of Motor Neurons (SMN) protein (Xu, 2005) which has been implicated in Spinal Muscular Atrophy (SMA). The protein sequences of the mitochondrial TIM50 and the nuclear TIM50a are identical with the exception of additional 103 amino acids at the N-terminal of TIM50a that are the result of an alternative translational start sequence. This additional N-terminal sequence in TIM50a is thought to contain a putative nuclear localization sequence that allows the Tim50a isoform to display a nucleus specific localization. Based on their results, Xu et al. hypothesized that Tim50a might be involved in the regulation of snRNP biogenesis and possibly the function of the nuclear SMN protein encoded by the SMN1 gene. One of our patients had mulsle biopsy. Although there were atrophic changes, no neuropthic pattern was seen.
Reference List (1) Wortmann SB, Kluijtmans LA, Rodenburg RJ et al. 3-Methylglutaconic aciduria–lessons from 50 genes and 977 patients. J Inherit Metab Dis 2013;36:913-921.
(2) Ikon N, Ryan RO. On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism. J Inherit Metab Dis 2016;39:749-756.
Legends
Legend to Figure 1
Five-generations pedigree of the family with mild 3-methylglutaconic aciduria in which the TIMM50 p.(Ile293Thr) was identified. Subjects V:1, V:3, and V:5 (filled symbols) are patients suffering from intellectual disability and increased urinary excretion of 3-methylglutaconic acid. They are born to consanguineous parents and homozygous for the TIMM50 c.1011T>C mutation predicting the replacement of isoleucine 293 with threonine in the encoded protein. The mutation was inherited by a common ancestor (either I:1 or I:2) and has been identified in the heterozygous state in the clinically and biochemically unaffected subjects III:3, III:4, III:9, IV:2, IV:4; IV:5; IV:9, IV:10, and V:2.

Parasitic Plants and Their Hosts: Types and Characteristics

Survival of one life form is dependent not only on its ability to search and utilize available life sustaining resources but also upon its healthy competition for various such requisites with other life forms belonging to either the same or different species. To facilitate this, different life forms have evolved with special assets of abilities to exist, work and exhibit a tendency to proliferate so as to flag their dominance on ecological niche. Scientific communities at times are amazed by the kinds of interaction in which different life forms co-exist with each other and also with the surroundings. In plants, interaction science has emerged as a major area of research which deals with investigating and studying such phenomena in detail, their mechanism, reasoning for interactions of plant with plant, animals, and/or micro-organism. Evolution of a new species from an existing ancestral mob assures adaptation to new and more efficient modes of existence, wherein lesser inputs are invested for more possible benefits. One such example is the parasitic plants or epiphytes.
Parasitic plants comprise 19 families in angiosperms and constitute to about 4,100 species (Nickrent. and Musselman, 2004). Among these with various parasitic habits, mistletoes are well known as perrenial, polyphyletic group of angiospermic aerial plant parasites (Devkota, 2005) infecting generally host stem and/or branches, and belong to families, Viscaceae and Loranthaceae.
Mistletoes are considered as an important component of biodiversity (Watson, 2001; Shaw et al., 2004), and have been appraised not only due to their unique parasitic relationship with their respective hosts, their growth habits, mechanism of seed dispersal but also because of their miraculous medicinal values which includes their use as sympathetic medicine to take down abnormal tissue growth under cancer (Kunwar et al.2005).
They are predominantly known to occur on fruit trees. Mistletoes are characterised by the development of a root like absorptive organ called haustorium which forms the host parasite interface and draws nutrients from the host conductive tissues to the parasite (Devkota, 2005).
Dendrophthoe falcata is a hemi-parasitic plant belonging to the Loranthaceae mistletoes and is considered as the commonest of all other mistletoes widely occurring in India (Parker acnd Westwood, 2009). The genus Dendrophthoe comprises of about 31 species spread across tropical Africa, Asia, and Australia (Flora of China, 2003) among which 7 species are found in India. The sp. falcata has its hold in India since ancient times and has been in use as traditional folklore medication for various ailments. Though a parasite incurring huge losses to commercial fruit and timer production, it is considered and welcomed as a sacred sign of prosperity in few areas of rural India. Well globally, much of the advanced scientific investigations have centered on other mistletoes such as Orobanche, Viscum etc. These studies have aimed towards understanding the parasite behaviour, sap flow dynamics at the host parasite interface, chemical compositions of floral organs, designing successful control strategies, evaluating medicinal potentials etc.
The present review will focus on the existing literature earlier attempts aiming study of various scientific aspects associated with D.falcata. The review considerably outlines the variety of studies performed revolving around the host-parasite relationship, morphology and geographical distribution, and studies aiming towards in vitro establishment and attempts towards scientific justification of the ethno-medicinal properties, and various existing and possible control strategies for this mistletoe family member.
Morphology and biogeography:
Evolutionarily, mistletoes belonging to Loranthaceae are considered older than those of Viscaceae and ever since their origin from dense Gondwanan forests; they have successfully encompassed tropics, subtropics and arid portions on the globe through their nutritional competition-driven conquests (Devkota et al, 2005). Notes on floral morphology and embryology in D.falcata have been provided by Singh (1952).
D.falcata bears grey barks, thick coriaceous leaves variable in shape with stout flowers (Wealth of India. 2002). The flowering inflorescences in D.falcata was previously referred to as axillary or as developing on the scars of fallen leaves, but Y.P.S Pundir (1996) verified it to be of strictly cauliflorous nature and also notified that it shares fundamental similarity to that of Ficus glomerata, F. pomifera and F. hispida.
Two of its varieties are widespread in India namely, var. falcata (Honey Suckled Mistletoe) and var. Coccinea (Red Honey Suckled Mistletoe) distinguished by occurrence of white and red flowering, respectively (Flowers of India, a World Wide Web resource). A comprehensive list of almost all the species within the genus Dendrophthoe can be availed (from web source at http://www.naturekind.org/taxpage/0/binomial/Dendrophthoe_falcata).
Haustorial polymorphism, leaf mimicry, explosive flowering, fruit adhesive pulp etc.
Singh, B., 1952. A contribution to the floral morphology and embryology of Dendrophthoe falcata (L. f.) Ettingsh. Jour. Linn. Soc., Lond., 53, 449.
Dendrophthoe homoplastica (mistle- toe) mimic those of its common host, Eucalyptus shirleyi. (Robert L. Mathiasen, David C. Shaw, Daniel L. Nickrent, David M. Watson,2008 Plant Disease / Vol. 92 No. 7)
Hosts and host-parasite relationships:
Mistletoes occur mostly on forest, fruit and ornamental host trees preferentially harboring zones rich in biodiversity and thus are found excessively on mountain ridges provided with favorably optimum light intensities and in few in slopes and plains (Devkota, 2005 and references therein). Mistletoes do not follow a uniform pattern of distribution which is affected by local environments and effected by habits of seed dispersing avian visitors.
Host range:
Earliest, B.Singh (1962) reported that the leafy mistletoe D.falcata is parasitic on over 300 hosts in India, Pakistan, Southeast Asia, and northern Australia. India�s rich plant biodiversity in 1977 could report exclusive host range numbering 345 plants susceptible to infection from this mistletoe (Siva Sarma and Vijayalakshmi 1977) which is a significant number as it contributes to its comparatively recent global record for 401 hosts (Shaw 1993). Till date, D.falcata represents the only known mistletoe with the largest global host range (Calvin and Wilson, 2009) which is continuously and rapidly widening.
Haustoria:
Mistleoes are known to form haustorial structures at the point of attachment to the host.
Among angiosperms, parasitic relationship through the formation of haustorial linkages is known to be widespread (Wilson and Calvin, 2006). In general, haustorial connections among 72 (of the 75) ariel parasitic genera may belong to either of the four types viz., epicortical roots (ERs), clasping unions, wood roses, and bark strands (Calvin and Wilson, 1998). ERs may run along the host branches in either direction forming haustorial structures at variable intervals while �unions� occur as single points of attachment of individual parasites hence pronounced as solitary. In D,falcata on different hosts two of the haustorial kinds have been observed viz., solitary unions as on Sugar apple (Annona squamosa), and epicortical roots as on sugar apple (Achras sapota), guava (Psidium guajava), pomegranate (Punica granatum) have been observed. It is unknown about what factors decide formation of different haustorial types by the leafy mistletoe on different hosts.
The host branches infected with D.falcata show a gradual reduction in growth and diameter as compared to other healthy uninfected branches (Karunaichamy et al, 1999).
It has also been reported that the development of mistletoe plants on the host tree is a dynamic process which necessarily leads to the death of the host tree and that the whole process may last for about a decade (Karunaichamy et al, 1999). It is known that mistletoes have higher nutrient titer than their host (Lamont, 1983; Karunaichamy et al, 1993) and this could probably be attributed to a competitively higher water (including dissolved nutrient and growth promoting metabolite) uptake rate by the parasite at its haustorium at a point in the host branch in comparison to that in the protruding lateral ends of the branch (that extends after the haustorium). This might render key nutrients be unavailable to the host branches that could repress growth post-infection (Stewart and Press, 1990). But in work by Karunaichamy et al (1999) one D.falcata seed per stem of Azadirachta indica seedling has been tested which has shown the life threatening effect on whole plant which again itself is at an immature seedling stage (so death of the whole plant could be expected). In a broad sense, physiological and metabolic perturbations induced by the parasite in the whole host plant outlined at the seedling stage surmised by the reduced host leaf area, leaf number, growth performance upon infection cannot be correlated to that in a fully mature host.
(statement in bold italics) could be justified by that there may be an increment in the probability of multiple mistletoe seed dispersals or extended reach of haustorial structures to other branches of the same tree (as we saw in Guava) that might possibly lead to a gradual depletion of key metabolites (thereby lowering an essential threshold titer for the same) required for growth of the host plant altogether. Alternatively though unknown, it might also be possible or still unknown if the hemi-parasites dissipate any unknown metabolite in to the host xylem at the point of anchor formation that may inhibit growth and metabolism in the host�s axillary meristems and the whole host plant life may only be at stake if such inhibitory effects are perceived by all plant parts. This again is contradictory hypothesis as evident from fact that the haustorial connections of the parasite with the plant are devoid of any retranslocation system (Smith and Stewart, 1990, also see later).
Once more, a report by Sridhar and Rama Rao (1978) unraveled the infection of D.falcata surprisingly hosting itself directly on the fruit (Table 1) of Acharas sapota (Sapota). Almost all the hemi-parasitic members of the Loranthaceae tap the xylem vessels of their host to avail water and minerals but to a considerable extent produce their own supply of assimilates (Kuijt, 1969). D.falcata does not have an indigenous rooting system and is dependent on the host for water and minerals. Nutrient dynamics have shown that a higher titre of N, P , K, Mg and Na in the leaves of mistletoe than the leaves of uninfected and infected hosts which may be due to differential translocation of elements within the host phloem (Prakash et al, 1967; Karunaichamy et al, 1999). Nitrogen loss is well pronounced in mistletoe infected hosts and a higher potassium levels in mistletoe is an indicative of higher transpiration rates and a lack of re-translocation system (Karunaichamy et al, 1999 and references therein). Though outlined above that the total phosphorous in mistletoe leaves is present in excess as compared to that the host leaves, fractionation studies have elucidated that percentage acid-insoluble phosphate is comparatively lesser in the parasite and also that further fractionation again infers a lower percentage of phospholipid, RNA, DNA and phosphoproteins. The DNA content of the infected host leaves however demonstrate a dramatic increase compared to the healthy uninfected leaves (Prakash et al, 1967).
Mistletoes are believed to mimic their hosts in floral structures. At some instance it was supported by a hypothesis which explains that such behaviour imparts protection to the mistletoe from the herbivores. Later, the hypothesis was field tested, rejected and replaced by a belief that a host parasite resemblance might help to safeguard hosts� individual fitness by signaling the birds to expel mistletoe seeds on other uninfected hosts (Atsatt, 1979). Moreover, the hosts and parasite follow individual developmental scheduling of events under which the parasite might experience an influence from a firm epigenetic hormonal control on leaf size and shape thus evolving the mimicry or resemblance to the host().
List of hosts:
Loranthaceous parasites were known in India since 1885 and until then only 35 species were known. Specifically for D.falcata from among the earliest of the reports, 268 host species have been known to be under the clutch of the deadly hemiparasite (Ravindranath and Narsimha Rao, 1959), and shortly later B.Singh (1962) reported its parasitic relation with over 300 hosts reported in India, Pakistan, Southeast Asia, and northern Australia. Until 1993, a report by Shaw III depicts a global increase of host range to 401. In Indian tropics 37 species of tree have been reported as the favourite hosts for D.falcata (Ghosh et al., 2002). Due to the ever increasing host range (Sridhar and Rama Rao, 1978) currently it is difficult to draw a margin between the more and less common host genera.
Vectors for seed dispersal and pollination:
Seed dispersal as well as pollination is usually mediated by the birds that thrive on fruits from the parasite and/or host. Particularly in southern India, Tickel�s Flowerpecker which is also named as Pale-billed Flowerpecker (Dicaeum erythrohynchos, Latham) is reported to facilitate seed dispersal of D. falcata among Neem (Azadirachta indica A. Juss. belonging to Meliaceae) through fecal excretions or regurgitations (Karunaichamy et al, 1999; Hambali, 1977 and references therein). The mistletoe bird also eats insects and therefore has a grinding gizzard. When eating the mistletoe berry the bird is able to close the gizzard and the toxic seeds are usually swallowed as a whole and are shunt through their gut in about 3-4 minute (Murphy et al., 1993) and because the seed has a sticky coating, to get rid of the seed the bird applies its vent to the edges of the host tree branches and may turn around whereby the seed then sticks onto the branch where it may subsequently germinate (Ali, 1931; Ali 1932; Davidar, 1985). These birds also act as vectors for pollination in the hemi-parasite whose flowers bear a mechanism that causes pollen to explosively spray on the plumage of the visiting flowerpeckers (Karunaichamy et al., 1999; Vidal-Russell and Nickrent, 2008). Studies conducted in the higher altitudes of Western Ghats (where both the mistletoes and the flowerpeckers occur predominantly), which parallel the western coast of India infer that the flowerpecker pollinated mistletoes have particularly developed feature specialized to attract a unique vector both to facilitate pollination and seed dispersal: the fruit and flowers have similar resemblance and more significantly, the fruiting time overlap with the next flowering season (Davidar, 1983). Hair-crested Drongo or Spangled Drongo (Dicrurus hottentottus) is an Asian bird of the family Dicruridae and sunbirds (family=Nectariniidae) also known to feed on the nectar from the D.falcata flowers also adds to the list of pollinators to this mistletoe (Kunwar et al, 2005 and references therein).
Phytochemistry:
Results for the preliminary tests in phytochemical screening analyses using ethanolic extract of the whole plant reports the presence of carbohydrates, glycosides, steroids, tannins

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