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“Hyperprolinemia in 22q.11 Deletion Syndrome and its association with Neurological Deficits” Abstract

Type 1 hyperprolinemia is biochemically characterized by a defect of the proline dehydrogenase enzyme involved in the conversion of proline to glutamate (McKusick 239500). The enzyme has been mapped to the PRODH gene on the human chromosome 22 on band q11.2 (Campbell et al. 1997). This gene falls in the region deleted in 22q11 Deletion Syndrome, which includes DiGeorge and Velo-cardio-facial syndromes. Although type 1 hyperprolinemia has been thought to be a benign disorder, severe neurological manifestations have been reported in several affected subjects. In this study, 14 patients with 22q11 deletion syndrome were studied to determine the frequency of hyperprolinemia in these patients and to examine whether hyperprolinemia, when present, was associated with any neurological deficits. Eight subjects had proline levels above normal and seven were within the normal limits (age specific). Furthermore, it was found that three subjects had various degrees of psychomotor deficits, including delayed milestones, global developmental delay and decreased IQ. The data in this study suggest that hyperprolinemia is a common finding in 22q11 deletion syndromes. In addition, it is in agreement with previous studies, which state an association between hyperprolinemia and neurological disorders.

CONTENTS TITLE                                                     PAGE NO:

1.	INTRODUCTION

2.	REVIEW OF LITERATURE

3.	METHODOLOGY

4.	RESULTS

5.	DISCUSSION

6.	SUMMARY

7.	APPENDIX

Chapter 1 INTRODUCTION

1.1	   Background of the Problem 1.2	   Significance of the Study 1.3	   Statement of the problem 1.4	   Aim and Objectives of the Study 1.5	   Methodology in brief 1.6	   Format of the Report

1. INTRODUCTION 1.1  BACKGROUND OF THE PROBLEM: Type I hyperprolinemia (HPI) is a rare, autosomal recessive disorder caused by a deficiency of the enzyme proline dehydrogenase (McKusick 239500) which is encoded by the PRODH (proline dehydrogenase) gene on 22q11 (Campbell et al, 1997; Lin 1998). The PRODH gene falls within the region deleted in 22q11 deletion syndromes, which include DiGeorge and Velocardiofacial syndrome. An elevated level of proline in the blood is referred to as Hyperprolinemia. There are two inherited forms of hyperprolinemia – Type 1 and Type 2. 21 Besides these, hyperprolinemia may also be seen in malnutrition, liver disease and lacticacidemia. Individuals diagnosed with lacticacidemia may have hyperprolinemia because lactic acid inhibits the breakdown of proline. Even though type 1 hyperprolinemia has been considered a benign disorder, neurological manifestation including mental retardation and epilepsy have been reported in a number of affected individuals.5, 6,7,15 HYPERPROLINEMIA An elevated level of proline in the blood is referred to as Hyperprolinemia. There are two inherited forms of hyperprolinemia – Type 1 and Type 2. 21 Besides these, hyperprolinemia may also be seen in malnutrition, liver disease and lacticacidemia. Individuals diagnosed with lacticacidemia may have hyperprolinemia because lactic acid inhibits the breakdown of proline. An elevated plasma proline concentration is a biochemical feature of 22q11deletion syndrome and it has been suggested that individuals with isolated hyperprolinemia should be investigated for deletions at 22q11.2 (Goodman et al.2000) Type 1 Hyperprolinemia: Hyperprolinemia type1 (HP1) is the outcome of the deficiency of the enzyme proline dehydrogenase whose synthesis is regulated by the PRODH gene. This gene is present in the DGCR portion of chromosome 22q and its deletion in 22q11 deletion syndrome leads to a deficit of proline dehydrogenase. The end result is that degradation of the amino acid proline is inhibited and its levels are elevated in plasma. Patients may be asymptomatic even though proline levels may be more than 3 to 10 time the normal level. Seizures, mental retardation or other neurological manifestations may be seen in some individuals.16 Type 2 Hyperprolinemia: Hyperprolinemia type 2 is the fall out of a mutation in the ALDH4A1 gene, which codes for the enzyme pyrroline-5-carboxylate dehydrogenase.26 This enzyme is necessary for the degradation of proline. Proline levels in the blood may be 10 to 15 times higher than normal, and high levels of pyrroline-5-carboxylate may be found. This type of the disorder has signs and symptoms that vary in severity, and is more prone to seizures or mental retardation. Genes related to hyperprolinemia: Mutations in the ALDH4A1 and PRODH genes cause hyperprolinemia. v	Hyperprolinemia type I is caused by a mutation in the PRODH gene. This gene falls in the region deleted in 22q11 deletion syndromes, which includes DiGeorge syndrome. Patients with DiGeorge syndrome should ideally be screened for type 1 hyperprolinemia.8 v	Hyperprolinemia type II is caused by a mutation in the ALDH4A1 gene. CHROMOSOME 22 and 22q11 DELETION SYNDROME The long arm of chromosome 22 (22q) is rich in genes when compared to other chromosomes. Alteration of the gene sequence or dosage on part of 22q is accountable for a number of human congenital disorders including 22q11 Deletion Syndrome. The chromosome also has a schizophrenia susceptibility locus. The following is an ideogram of chromosome 22. The bands describe the location of genes on the chromosome.30 A variation in the number or structure of chromosome 22 can have a range of effects like intellectual disability, delayed development, delayed or absent speech, distinctive facial features, and behavioral problems.7 PRODH GENE: The PRODH gene codes for proline oxidase (proline dehydrogenase), the enzyme responsible for the degradation of the amino acid proline. This gene regulates the synthesis of proline dehydrogenase, which is found principally in the brain, liver and kidney. The PRODH gene falls within the region deleted in the 22q11 deletion syndrome, including DiGeorge syndrome and velocardiofacial syndrome. Mutations in the PRODH gene have been linked to the following disorders: 1.	Type 1 Hyperprolinemia: Mutations in the PRODH gene reduce the activity of the enzyme proline dehydrogenase. This leads to the buildup of proline in the body and in severe cases of hyperprolinemia can cause seizures, intellectual disability, or other neurological or psychiatric problems. 2.	Schizophrenia: Several studies have shown an association between variations in the PRODH gene and neurological disorders like schizophrenia. Mutations in the gene lead to reduced activity of the enzyme, thereby leading to an accumulation of proline. Research has shown that elevated proline levels affect the activity of neurotransmitters, resulting in an increased propensity towards neurological disorders.10, 16

1.2  SIGNIFICANCE OF THE STUDY Type 1 hyperprolinemia, previously thought to be a benign disorder is now being re-examined in view of the various studies linking it to neurological disorders including schizophrenia.25 Several studies have been carried out worldwide providing substantial evidence that elevated levels of proline in the plasma can significantly affect the cognitive and learning abilities of individuals in the affected state. 7    In type 1 hyperprolinemia, the defective activity of the enzyme proline dehydrogenase results in the accumulation of proline and inhibits further metabolism. Consequently, synthesis of glutamate is affected, as proline is the precursor for glutamate, especially in the glutamatergic synapses. Another fallout of this defect is that the synthesis of GABA, which is the chief inhibitory neurotransmitter in the mammalian nervous system, is also threatened.2 Elevated levels of proline adversely affect its normal functions in the glutamatergic synapses. This, coupled with the reduced synthesis of GABA, has been cited as the potential link between hyperprolinemia and neurological disorders.2, 12 The defective activity of proline dehydrogenase in type 1 hyperprolinemia is due to mutations in the PRODH gene, located on chromosome 22q.11. This gene falls in the region that is deleted in 22q.11 Deletion Syndromes. The PRODH gene, whose mutation results in the above disorder, has been cited as a susceptibility gene for schizophrenia.39 These findings form the basis for this study. Previous research has shown that growth is improved in hyperprolinemic children with 22q11 deletion syndrome on reduction of plasma proline levels.5, 7 An individual affected with 22q11 deletion syndrome does not have the choice of a complete cure. But, if the quality of life can be improved through reduction of plasma proline levels, then that must be given emphasis in the management of such patients. 1.3 STATEMENT OF THE PROBLEM: The 22q11 deletion syndrome has an incidence of 1:4000.47 Several studies have been carried out world wide to describe the disorder. The lack of existing data with regard to hyperprolinemia in 22q11 deletion syndrome in India prompted this study to be undertaken. 1.4 AIMS AND OBJECTIVES OF THE STUDY: 1.4.1 Aim: This study aims to describe the frequency of type1 hyperprolinemia in 22q11deletion syndromes. 1.4.2 Objectives: ·	To study the frequency of type 1 hyperprolinemia in patients diagnosed with 22q11deletion syndromes. ·	To examine the association between plasma proline levels and clinical features (neurological deficits) of the above patients.

1.5 METHODOLOGY IN BRIEF: In this study, plasma proline levels were assayed in 14 children affected with 22q11 deletion syndrome. The proline levels were estimated using high performance liquid chromatography (HPLC). The neurological and development statuses of the hyperprolinemic subjects were studied.

1.6 FORMAT OF THE REPORT: The report has been divided into 6 chapters. Chapter 1            : Introduction – It includes background of the    study, significance of the study, statement of the problem, aims and objectives, methodology in brief and format of the report. Chapter 2                 : Review of Literature - It includes the review of literature under the headings of structure and function of proline, metabolism of proline, hyperprolinemia, PRODH gene, 22q11 deletion syndrome and neuropsychiatric problems in 22q11 deletion syndrome. Chapter 3                : Methodology – It includes study design, study setting, study population, sample, duration and time of study, materials and tools, data collection process, data processing, data analysis and statistical tests used. Chapter 4                  :  Results Chapter 5                  : Discussion

Chapter 6              : Summary – It includes summary, limitations, and suggestions.

Chapter 2 REVIEW OF LITERATURE 2.1   PROLINE: STRUCTURE AND FUNCTION 2.2   METABOLISM OF PROLINE 2.3   HYPERPROLINEMIA 2.3   PRODH GENE 2.5   22q11 DELETION SYNDROME (22q11 DS) 2.6   NEUROPSYCHITRIC PROBLEMS IN 22q 11 DS 	                              2. LITERATURE REVIEW

Type 1 hyperprolinemia, a rare metabolic disorder, is the result of a mutation in the PRODH gene, which codes for the enzyme proline dehydrogenase.20 This enzyme initiates the conversion from proline to glutamate and reduced activity leads to elevated plasma proline levels. Since the PRODH gene falls in the region that is deleted in 22q11 deletion syndromes (22q11DS), hyperprolinemia is a biochemical feature in this syndrome.8 An excessive amount of proline affects normal functioning of brain neurotransmitters and it has been suggested as one of the causes of neurological problems seen in 22q11deletion syndromes.2 A number of studies have been undertaken worldwide to assess the incidence and effects of hyperprolinemia in 22q11deletion syndromes, but very little research has been done in this arena in India. With the introduction of newer techniques for the early detection and identification of individuals with 22q11DS, it is imperative that maximum steps be taken to improve the quality of life in these patients. 5, 7      This study aims to look at the prevalence of hyperprolinemia in patients diagnosed with 22q11deletion syndrome. Furthermore this study intends to examine the association between hyperprolinemia and neurological disorders in 22q11DS patients.2

2.1   PROLINE- Structure and Function Proline is unique as it is an iminoacid (it contains an NH2 rather than an NH3 group). As a result proline is unable to occupy many of the main chain conformations that are easily adopted by other aminoacids. Fig.2: Structure of Proline Proline, despite being aliphatic and hydrophobic is usually found on the protein surface due to its preference for turn structure. The concentration of proline is relatively high near the transmembrane helices which has led to the suggestion that proline plays a role in signal transduction.9 Another key function of proline is molecular recognition, particularly intracellular signaling. Proline containing peptides are therefore vital parts of many signaling cascades.9 Proline acts as an osmolyte in certain cells. It is also part of a redox shuttle. Its action as a modulator of synaptic pathways, 24 added to the fact that it is the precursor for glutamate in glutamergic synapses is a potential pathophysiologic link between proline and neuropsychiatric disorders in humans.2, 12 Vorstman, Turetsky, Sijmens-marcus, de Sain, Dorland, Sprong, Rappaport, Beemer, Emanuel, Kahn, van Engeland, and Kemner (2009) conducted a study on 56 22q11DS to evaluate the relationship between 22q11DS and abnormal brain function. The children were assessed to determine pre-pulse inhibition (PPI) of startle, P50 auditory sensory gating and smooth pursuit eye movements (SPEM). Since the genes PRODH and COMT lie in the region deleted in 22q11DS, their responses were compared to their plasma proline levels and COMT genotype. They established that SPEM and PPI were abnormal in the patient sample when compared to normal controls. Children with elevated proline levels and low activity COMT allele were found to have poor SPEM. This study was consistent with a pattern in which accumulation of proline has a negative effect on brain function due to the increase of dopamine in the pre-frontal cortex. 6

2.2   METABOLISM OF PROLINE Before we examine the causes and features of hyperprolinemia, an idea of the synthesis, metabolism and degradation of proline is essential. As shown in fig.3, note that both the synthesis and degradation of proline proceed through the Δ-pyrroline-5-carboxylate intermediate.

Fig.3: Proline – Synthesis and Degradation ·	 PRODH – proline dehydrogenase ·	PC5R – pyrroline-5-carboxylate reduce ·	OAT – ornithine aminotransferase ·	P5CDH – pyrroline- 5 carboxylate dehydrogenase ·	GPR – gamma glutamyl phosphate reductase ·	GK – gamma glutamyl kinase ·	GSA – Glutamate  semialdehyde ·	ORN – ornithine Proline dehydrogenase (PRODH) is responsible for the first step in the catabolism of proline; whereas the enzyme pyrroline-5-carboxylate reductase (P5CR) is necessary for the synthesis of proline. A deficiency of either proline oxidase or pyrroline-5-carboxylate dehydrogenase results in a buildup of proline in the body known as hyperprolinemia. Proline is a precursor of glutamate especially in the glutamatergic synapses.2 Three steps are involved in the conversion of proline to glutamate. Fig.4: Conversion of proline to glutamate 1. Proline Dehydrogenase 2. pyrroline- 5 carboxylate dehydrogenase 3. Non-enzymatic As can be seen, any defect in the metabolism will affect the synthesis of glutamate. The domino effect is that the synthesis of GABA will be affected. GABA is the chief inhibitory neurotransmitter in the mammalian nervous system and its deficiency will eventually lead to neurological disorders.12 Abnormalities in proline metabolism have been linked to several diseases: six monogenic inborn errors linking metabolism and/or transfer of proline and its immediate metabolites have been described.11 Impaired proline metabolism has also been linked to schizophrenia.1, 2 Mitsubuchi, Nakamura, Matsumoto, and Endo (2008) describes type 1 hyperprolinemia, an inborn error of metabolism resulting from a paucity in the proline dehydrogenase enzyme coded by the PRODH gene which falls in the region deleted in22q11deletion syndrome. Moreover they suggested that the above gene locus is related to an inclination to schizophrenia.1

Nadler, Wang, and Hakim (1988) found that excessive administration of L-proline destroyed pyramidal and granule cells. They suggested that proline should be included in the list of brain excitotoxins and furthermore stated that the excitotoxic action of proline just might be the root of neurological and cognitive deficits seen in hyperprolinemia.3

In a study conducted by Kishor, Sangam, Amrita, Sri Laxmi, Naidu, Rao, Sreenath Rao, Reddy, Theriappan, and Sreenivasalu (2005) on transgenic plants, they noted that accumulation of proline lead to a reduction in growth. Another finding was that proline levels and growth were inversely correlated in cells grown under normal osmotic settings.4

2.3   HYPERPROLINEMIA Excess of proline in the blood is known as hyperprolinemia. It can be classified as Type 1 and type 2 based on the cause.17 Type 1 hyperprolinemia (#239500) arises due to the decreased activity of the enzyme proline dehydrogenase (proline oxidase). 14 The gene PRODH that is located on the long arm of the 22nd chromosome encodes this enzyme. To be more precise, this gene falls in the DGCR region i.e. the portion that is deleted in 22q11 deletion syndromes. Consequentially, hyperprolinemia is associated with the above deletion syndrome. 7, 8 Earlier it was though that type 1 hyperprolinemia was a benign disorder but it is now of considerable interest that seizures, mental retardation and other neurological symptoms have been linked to this disorder. 13, 15, 16     Hyperprolinemia type 2 is caused by a mutation in the ALDH4A1 gene, which provides instructions for producing the enzyme pyrroline-5-carboxylate dehydrogenase. This enzyme helps to break down the pyrroline-5-carboxylate, converting it to the amino acid glutamate.26 A study conducted by Raux, Bumsel, Hecketsweiler, et al (2007) gave emphasis to the fact that there is an inverse relationship between plasma proline levels and IQ. Microdeletions in the 22q11 region are associated with an increased tendency towards psychosis and mental retardation. They assessed 8 children with hyperprolinemia and compared their phenotypes, plasma proline levels and PRODH genotypes. They found that mental retardation was a common characteristic; usually appearing in early childhood and that most of the children had psychomotor delay, thus establishing the fact that type 1 hyperprolinemia is undeniably associated with neurological features in children. Furthermore they evaluated 92 VCFS patients with plasma proline levels ranging from138 to 1275 µmole/l. 34 of the subjects were found to be hyperprolinemic and possessed a phenotype that was very different from that of the other VCFS patients. Another finding was that increased plasma proline levels inversely affect IQ. The study proved that 0-30% residual activity of proline dehydrogenase results in type 1 hyperprolinemia, whereas activity in the range 30-50%is associated with either normal plasma proline levels or otherwise mild-to-moderate hyperprolinemia. Their findings have important therapeutic implications with regard to improving the quality of life in 22q11DS patients by reducing the intake of proline either by adopting a low proline diet or by pharmacologic means.7

Researchers Goodman, Rutberg, Lin, Pulver and Thomas (2000) after extensive research concluded that hyperprolinemia is a rather frequent finding in patients with 22q11 deletion syndromes. Their results were based on a study in which plasma proline levels were assayed in 15 individuals with 22q11DS. Eight of the subjects had proline levels in the range278-849 µmoles/L (normal laboratory range: 51-271 µmoles/L). The study suggested that determination of plasma proline levels should be a mandatory biochemical test in the diagnosis of 22q11 deletion syndrome.8

Oyanagi, Tsuchiyama, Itakura, Tamura, Nakao, Fujita, and Shiono (1987) studied the clinical, biochemical and enzymatic features in a 11-month old infant with type 1 hyperprolinemia and chromosomal abnormality. At the time this was the first case of type 1 hyperprolinemia associated with chromosomal abnormality. The infant presented with short status, delayed milestones, convulsions and dysmorphic facies. They found that the child suffered from mental retardation and growth defects. On adoption of a low proline diet it was noted that the growth of the child improved, though there was no change in the mental retardation. Initial level of plasma proline was 830 µmole /L, and this was reduced on adopting a low-proline diet (maintained in the range 174-348 µmoles /L). In the above study the researchers stated that the cause of type 1 hyperprolinemia was the deficit of the enzyme proline dehydrogenase, but they were not aware as to which chromosome contained the gene that coded for the said enzyme. 5     Thus we see that in recent years it has been proven that type 1 hyperprolinemia is caused by a mutation in the PRODH gene and this inherited metabolic disorder is associated with certain neurological symptoms.6, 7, 15, 16 In a study in 1996, Jaeken et al described a case of type 1 hyperprpolinemia in whom a 22q11 microdeletion was demonstrated by FISH analysis. Besides, the patient showed symptoms of DiGeorge syndroem and had a deficiency of heparin cofactor 2 which maps to 22q11.On further examination of the parents and sibling it was seen that they had normal levels of proline and no deletions at 22q11 were noted.17 In recent years it has been proven that type 1 hyperprolinemia is caused by a mutation in the PRODH gene and this inherited metabolic disorder is associated with certain neurological symptoms.6, 7, 15, 16

2.4  PRODH GENE The PRODH gene falls in the DGCR (DiGeorge Chromosomal Region) region, which is deleted in 22q11deletion syndrome, including Digeorge syndrome and velocardiofacial syndrome. This gene codes for proline dehydrogenase, 23 which is the first enzyme essential for the degradation of proline. As discussed earlier, a defect in the PRODH gene is the cause for Type 1 Hyperprolinemia.15, 16 The PRODH gene spans 23.8 kb with 15 exons and is localized to 22q11.2 at 17.3Mb. It is located on the long arm (q) of chromosome 22 at position 11.2. 10                                  Fig.  5: PRODH gene- Location on Chromosome 22

Beside hyperprolinemia, variations in the PRODH gene locus have been linked to psychiatric disorders like schizophrenia. Elevated levels of proline affect the activity of neurotransmitters and this may perhaps be the reason for associated psychological problems. 16     Jacquet et al. (2003), examined a case of a 4-year old child with psychomotor delay, hyperactivity, sleep disturbance and epilepsy. Hyperprolinemia and hyperprolinuria were seen. The disorder was identified as type 1 hyperprolinemia and a complete homozygous deletion of the PRODH gene on the chromosome 22q11 was detected. The study was concluded on the note that severe forms of type 1 hyperprolinemia associated with neurological deficits were related to homozygous inactivating alterations in the PRODH gene.15 A study conducted by Afenjar, A., Moutard, M. L., Doummar, & D., Guet, A. et al. (2007) identified biallelic abnormalities in 8 patients as a result of which the activity of proline dehydrogenase was greatly reduced. Missense and non-sense mutations, deletions of PRODH, a microdeletion at 22q11and biallelic PRODH mutations were detected. The characteristic feature noted in the subjects was early psychomotor development delay with predominant cognitive failings, autistic features and epilepsy. The study showed that early onset and severe neurological features was associated with biallelic mutations of PRODH. 28      Prasad, Howley & Murphy (2008), emphasized the presence of an association between 22q11 deletion syndrome and neurological disorders like schizophrenia. Linkage studies of schizophrenics have alluded to the presence of susceptibility genes three of which lie in the 22q11.2 region– PRODH, COMT and Gnb1L.27 Zinstok, Schmitz, van Amelsvoort, Moeton, Baas, & Linszen (2008) studied the link between polymorphisms in COMT and PRODH with brain anatomy in young patients with schizophrenia. Regional grey matter and white matter density differences and volume differences between genotype groups were studied. It was seen that patients with one or two mutations in the PRODH allele had an increased white matter density in the left inferior frontal lobe. The study concluded that genetic variation in COMT and PRODH has notable effects on brain areas affected in schizophrenia.29 Vorstman, Turetsky, Sijmens-marcus, de Sain, Dorland, Sprong, Rappaport, Beemer, Emanuel, Kahn, van Engeland, and Kemner (2009) conducted a study on 56 22q11DS to evaluate the relationship between 22q11DS and abnormal brain function. The children were assessed to determine pre-pulse inhibition (PPI) of startle, P50 auditory sensory gating and smooth pursuit eye movements (SPEM). Since the genes PRODH and COMT lie in the region deleted in 22q11DS, their responses were compared to their plasma proline levels and COMT genotype. They established that SPEM and PPI were abnormal in the patient sample when compared to normal controls. Children with elevated proline levels and low activity COMT allele were found to have poor SPEM.6

2.5  22q11 DELETION SYNDROMES: The 22q11 deletion syndrome (22q11 DS) is fallout of a meiotic deletion of genetic material at the q11.2 on chromosome 22. This kind of deletion is the most frequent known interstitial deletion in man with an incidence of 1 in 4000 live births.38 The end result is that 30 genes are deleted including two that encode the specific neurotransmitter modulators catechol O-methyltransferase (COMT) and proline dehydrogenase (PRODH). Loss of these alleles leads to a dysregulation of dopaminergic, glutamatergic and GABA-ergic transmission. Congenital anomalies may be seen in some children and they include: v	heart defects v	 immunologic deficits v	 craniofacial dysmorphologies v	 velopharyngeal defects such as overt or sub mucous cleft palate v	 Inflammation-related pain syndromes. Fig. 6: Child with DiGeorge Syndrome A number of syndromes like the "conotruncal anomaly face syndrome" (heart defect with facial dysmorphologies), "velocardiofacial syndrome" (velopharyngeal, heart, and facial anomalies), and "DiGeorge syndrome" (immunologic insufficiency) are grouped under the banner of 22q11 deletion syndromes. A consistent neuropsychological profile is seen in children with this syndrome35: v	Delays in gross motor, fine motor and expressive language skills in the early years. v	Learning disabilities and academic failures, attention impairment, and behavioral anomalies in an estimated 90%–100% of the school-age children. v	 Adult studies have suggested that approximately 25% of the children with 22q11 deletion syndrome go on to develop schizophrenia in late adolescence or early adulthood. 35     The above profile clearly brings to light a strong association between 22q11deletion syndromes and schizophrenia-like illness. Judicious assessment of the neurocognitive status during the presymptomatic childhood years is vital and could eventually play a decisive role in providing targeted strategies for early intervention. 2.6  NEUROPSYCHIATRIC PROBLEMS IN 22q11 DS       Several studies have reported an increased incidence of neurological problems in children and adults affected with 22q11 DS. 32, 33, 34 Linkage and association studies have been undertaken to determine susceptibility genes for schizophrenia. Karayiorgou et al (1995) conducted a study on schizophrenic patients to determine the presence of a 22q11 deletion In the second phase of the study they examined whether there was any correlation between the length of the deletion and the schizophrenic phenotype. They obtained results consistent with linkage studies, which implicate the 22q11 region in containing susceptibility genes predisposing to schizophrenia.36 Debbane, Glaser, David, Feinstein & Eliez (2006) studied forty-three children and adolescents with 22q 11 deletion syndrome and found psychotic symptoms in 28% of the total sample. Furthermore, it was accompanied by reduced verbal IQ performance and decreased adaptive social skills. 40     A study conducted by Raux, Bumsel, Hecketsweiler, et al (2007) gave emphasis to the fact that there is an inverse relationship between plasma proline levels and IQ. Microdeletions in the 22q11 region are associated with an increased tendency towards psychosis and mental retardation. They assessed 8 children with hyperprolinemia and compared their phenotypes, plasma proline levels and PRODH genotypes. They found that mental retardation was a common characteristic; usually appearing in early childhood and that most of the children had psychomotor delay. Another finding was that increased plasma proline levels inversely affect IQ.7 In a study carried out by Bassett, Chow, Husted, Weksberg, Caluseriu, Webb and Gatzoulis (2005), 78 adults with 22q11 deletion syndrome were examined and 43 distinct features were present in more than 5% of the patients.The features included intellectual disabilities (92.3), hypocal;cemia (64%), palatal abnormalities (42%), and cardiovascular abnormalities (25.8%). A significant finding in their study was that schizophrenia was present in 22.6% of patients. 41  Researchers Bartsch, Nemeckova, Kocarek, Wagner, Puchmajerova, Poppe, Ounap & Goetz (2003), studied the position of the deletion in 58 subjects identified with DiGeorge/velocardiofacial syndrome and suggested that intellectual and or behavioural outcome may be better with the proximal versus the distal (common) deletion.42 DiRosa, Pustorini, Spano, Campion, Calabro, Aquennouz, Caccamo et al. (2008) studied 4 Italian children with neurological manifestations and clinical geatures including early motor and cognitive developmental delay, speech delay, autistic features, seizures etc.They found that all patients had elevated plasma and urine proline levels and biallelic mutations on the PRODH gene. They concluded that type 1 hyperprolinemia may be associated with neuropsychiatric disorders, which may ultimately correlate with PRODH genotype.43

Chapter 3 METHODOLOGY 3.1	  STUDY METHOD 3.2	  STUDY SETTING 3.3	  STUDY POPULATION 3.4	  SAMPLE 3.5	  DURATION AND TIME OF STUDY 3.6	  MATERIALS AND TOOLS 3.7	   DATA PROCESSING 3.8	  DATA ANALYSIS AND STATISTICAL TESTS USED 3.1 STUDY METHOD: Hospital based prospective study.

3.2 STUDY SETTING: The study was carried out in the department of Human Cytogenetics, Amrita Institute of Medical Sciences, Kochi, Kerala, INDIA.

3.3	STUDY POPULATION: Individuals affected with 22q11 deletion syndrome being managed at Amrita Institute of Medical Sciences, Kochi.

3.4	SAMPLE: Since this was the first study carried out in relation to hyperprolinemia in 22q11 deletion syndrome in India, there were no references for the sample size to be assessed. 3.4.1  Age – Group: Less than 10 years 3.4.2   Sex: 11 Male and 3 Female 3. 4.3  Subjects: Fourteen children identified with 22q11 deletion syndrome were subjects in this study. Plasma proline levels were assessed in the subjects. In addition, clinical features and plasma amino acid levels of the subjects were examined.

3.5  DURATION AND TIME OF STUDY: The study was undertaken for a period of 6 months from December 2008 – May 2009. The initial month of the study was taken up in review of literature and setting up of the study design.

3.6	MATERIALS AND TOOLS: 3.6.1	Determination of Plasma Proline levels: Heparinised blood samples were used to estimate plasma proline levels. On receipt, all samples were centrifuged and the plasma separated via centrifugation for further analysis. All samples were analysed in the same laboratory using high-pressure liquid chromatography (HPLC) on a SHIMADZU LC instrument.

3.6.2  Amino acid analysis by HPLC: Principle: HPLC is an analytical process classified designed to separate, quantify and analyse components of a solution. The basic components of an HPLC system include 1.	solvent reservoir 2.	sample injector 3.	pump(s) 4.	analytical column 5.	detector(s) 6.	data recorder Other important elements are an inlet solvent filter, post-pump inline filter, sample filter or precolumn filter, guard column, back-pressure regulator and/or solvent sparging system. Samples are introduced to a solvent flow path via an injector; carried through a column for component separation; and the data is obtained through the combination of a detection mechanism coupled with a data recording system. The different compounds in the mixture or solution pass through the column at different rates based on their partitioning behaviour between the mobile liquid phase and the stationary phase. This enables their separation and consequent detection as they are eluted from the column. 3.6.3  Analytical conditions: Column: LUNAC-18 (250 x 4.6 mm) Mobile phase: Gradient elution Flow rate: 1ml/min Temperature: 40o C Detection:  Diode array detection (254nm) A gradient HPLC system with ODS (octa-decyl-silane) Hypersil 5μm × 4.6 mm × 25 cm C18 column with guard column (filter) was used. The column is housed in an incubator oven, which was set at a constant temperature of 40oC. The mobile phase was composed of phosphate buffer and acetonitrile. A photodiode array detector was used. Separation was effected by gradient elution using phosphate buffer and acetonitrile mixture. Flow rate was set at 1ml/minute. The samples for analysis were deproteinised with 10% 5-sulphosalicylic acid and derivatised using phenyl-iso-thiocyanate (PITC)and triethylamine (TEA) reagents following which they were run through the HPLC system. Prior to the test run a set of aminoacid standards was run through the column.

3.6.4 Reagents: 3.	100mM phenyl-iso-thio-cyanate(PITC) in acetonitrile 4.	1M triethylamine in acetonitrile 5.	10% 5-sulpho-salicylic acid 6.	Amino acid standard: The amino acid standard kit contains 10 ampoules of 1mL each. Each 1mL contains 17 amino acids and ammonium. The concentration of each amino acid is 2.5μmol/mL, except for cysteine whose concentration is half that of the other amino acids.

3.6.5  Standard pre-treatment: v	100μL of 100mM phenyl-iso-thio-cyanate and 100μL 1M tri-ethyl-amine were added to 200 µL of the standard solution. v	The mixture was kept at room temperature for 2.5 hours. v	400μL of hexane (HPLC grade) was added to the solution and vortexed. v	The hexane layer was separated and discarded. v	20μL from the bottom layer was taken in a Hamilton syringe and used for analysis.

3.6.6  Sample pre-treatment: ·	400μL of the sample was added to 200μL of 10% 5-sulpho-salicylic acid and kept at 4oC for half an hour. ·	The mixture was centrifuged and 100μL of 100mM phenyl-iso-thio-cyanate and 100μL of 1M tri-ethyl-amine were added to 200μL of the supernatant. ·	The treated solution was allowed to stand at room temperature for 2 hours. ·	400μL of hexane (HPLC grade) was added to the solution and vortexed. ·	The hexane layer was separated and discarded. ·	20μL of the bottom layer was then used for analysis. ·	Pre-treated samples can be stored at -20oC for 8 to 10 days. . 3.6.7  Sample injection: 1.	20μL of the pre-treated sample was taken in a Hamilton syringe and introduced into the injector. It was ensured that no air bubbles were introduced during the process. 2.	After injecting the sample the injection loop was brought back to the injection position. 3.	The syringe was rinsed in Milli Q water (HPLC grade water) after each injection. 4.	 After the run concentrations of the standards and the test samples were calculated.

3.7	DATA PROCESSING: 3.7.1 Quantification of plasma proline levels: Resolution of amino acid derivatives was monitored through a diode-array detector with absorbance at 254nm. The chromatograph of the standards gave a series of peaks in correlation with the concentration of the standard injected. The area under each peak was calculated and the data used to develop a calibration curve. Concentration of aminoacids in the test sample (samples obtained from the subjects in the study) was then determined by comparison with the known standards.

3.8 DATA ANALYSIS AND STATISTICAL TESTS USED: 3.8.1 Plasma proline levels: Reports were generated using the SHIMADZU LC solution analysis software. 3.8.2 Statistical tools: SPSS.11 was used to calculate the mean values of plasma proline levels, its standard deviation and the frequency of hyperprolinemia.

Chapter 4 RESULTS 4.1	 PLASMA PROLINE LEVELS IN THE STUDY SUBJECT 4.2	 DESCRIPTION OF NEUROLOGICAL STATUS OF THE SUBJECTS 4. RESULTS

4.1   PLASMA PROLINE LEVELS IN THE STUDY SUBJECTS: Plasma proline levels were assayed in 14 patients, ranging in age from newborn to 6 years. ID:	         AGE 	     SEX	Plasma Proline (μmol/L) 1	       4y	      M	497 2	       6y	      M	         227 3	       9m	      M	         311 4	       9m	      M	         274 5	       1y	      M	         326 6	       6y	      M	         198 7	       1y	      M	         200 8	       1m	      F	         166 9	       1y	      M	         266 10	       1m	      M	         181 11	       4m	      F	217 12	       1m	      F	216 13	 1m	M	236 14	4m	M	190

Table 1: Plasma Proline levels in the subjects.

Reference Values: 44 1st week after Birth:  120-344μmol/L 1 week – 1 month:  65-457μmol/L 1 - 4 months: 98-254μmol/L 4month – 2 years:  53-201μmol/L 2 – 10 years:  93-221μmol/L 10 – 18 years:  75-307μmol/L Based on the reference range for plasma proline, the 14 children were grouped into three categories for easier processing of data. PLASMA PROLINE LEVELS AGE GROUP	MINIMUM	MAXIMUM	MEAN	STANDARD DEVIATION 1 to 4 months	166	236	197.8	28.03 5 to 24 months	200	326	265.67	49.89 25 to 120 months	198	497	307.33	164.89

Table 2: Mean plasma proline levels There were 5 children in the first category, 6 in the second and 3 in the third category. All subjects in the age group 1 – 4 months gave plasma proline values within the normal reference range. There were 6 subjects in the second group and only one child had normal proline levels. All the others had elevated proline values. There were 3 children in the third category and two of them had high levels of plasma proline. The plasma proline levels of the 14 subjects ranged from 166 - 497μmol/L with a mean value of 250.36μmol/L (standard deviation = 85.952).

Chart 1: Mean Plasma Proline levels 7/14 subjects show plasma proline levels above the normal reference range.

4.2  DESCRIPTION OF NEUROLOGICAL STATUS OF THE SUBJECTS: The subjects were assessed for neurological status and it was seen that 3/7 subjects with hyperprolinemia had neurological deficits. Two patients had psychomotor delay since birth. One patient showed global developmental delay including delayed speech (at 3 years) and walking at the age of 1.5 years. One subject had poor scholastic performance and on assessment of IQ levels gave results below normal (IQ test performed by the concerned clinicians).

Chapter 5 DISCUSSION

5.1	  HYPERPROLINEMIA IN 22Q11 DELETION SYNDROME 5.2	  ASSOCIATION BETWEEN HYPERPROLINEMIA AND      NEUROLOGICAL DISORDERS

5. DISCUSSION

5.1 HYPERPROLINEMIA IN 22Q11 DELETION SYNDROME: Fourteen subjects were tested for plasma proline levels and values above normal were seen in seven cases. It was noted that even though the remaining subjects did not show elevated values, their proline levels had a tendency towards the upper limit of normal. In a study conducted by Raux et al. (2007), the question of reproducibility of proline values was assessed and it was seen that there was a 12% variation between independent measures and the mean variation was 8%. 7      7/14 subjects in this study were found to have elevated proline levels (Table 1). In table 2, all subjects in the age group 1- 4 months had normal proline levels. But in the second group, 5/6 children gave elevated proline levels. 2/3 in the third group had hyperprolinemia. In chart 1, it was seen that plasma proline levels had an increasing trend with increase in age. Goodman et al. (2000) conducted a study on fifteen patients with 22q11 deletions and detected hyperprolinemia in eight patients. In a previous study they found elevated plasma proline levels in nine out of twenty-four subjects.8 Goodman et al. concluded that elevated plasma proline levels are a frequent finding in individuals with 22q11 deletion syndrome.8 Goodman et al. suggested that the hyperprolinemia is caused by the hemizygous deletion of the proline dehydrogenase (PRODH) gene that maps to the region, which is commonly deleted in 22q11 deletion syndrome. They concluded that elevated plasma proline levels should be considered as a biochemical feature of 22q11 deletion syndrome. The results obtained in this study are in agreement with previous studies conducted (as compared to international research work, as no previous study has been carried out in India).

5.1	ASSOCIATION BETWEEN HYPERPROLINEMIA AND NEUROLOGICAL    DISORDERS: Type 1 hyperprolinemia was previously thought to be a benign disorder. But several studies in the recent past have shown this disorder to be associated with delayed psychomotor development, mental retardation and decreased IQ.6, 7 In this study the small sample size makes it difficult to properly evaluate the association between hyperprolinemia and neurological disorders. However, it was seen that two patients had psychomotor delay since birth. One patient showed global developmental delay, which comprised delayed speech (at 3 years) and walking (at 1.5 years). One subject had poor scholastic performance and on assessment of IQ levels gave results below normal (IQ test performed by the concerned clinicians). The clinical department assessed the neurological and developmental statuses.

5.2.1  Subject ID: 1 The child had a plasma proline concentration of 497μmol/L and was found to have global developmental delay. The subject had delayed psychomotor development since birth. He was able to walk only at the age of 1.5 years. Speech was delayed, beginning only at three years of age. This subject did not have any cardiac abnormality. 5.2.2  Subject ID: 2 This child had mild developmental delay since birth. At the time of the study the subject was known to have poor scholastic performance. IQ measurement, assessment of social functioning and developmental screening tests were performed by the clinical department and it was seen that the child had an IQ that was below normal, a mental age of four years, and a social development of three years. These findings are consistent with previous studies in which patients with 22q11 deletion syndrome were found to have decreased IQ and deficits in cognitive, sensorimotor and learning abilities.37, 46 5.2.3 Subject ID: 9 This child has gross developmental delay since birth with delayed milestones. Note: In this study 7/14 subjects were found to have hyperprolinemia. Neurological deficits were recorded in 3/7 children with elevated plasma proline levels. They were 4, 6 and 1 year(s) of age respectively i.e. they were older than majority of the subjects in this study. Ten subjects were in the age range of 1 month – 12 months. Assessment of neurological status, cognitive functions and IQ was not possible in these ten children due to their young age. Another finding in this study was that 4 of the subjects had poor weight gain in the first years of life. This finding has been previously elucidated in a study carried out on 73 patients with 22q11 deletion (Digilio, 2001). Their study showed that patients with 22q11 deletion syndrome had a characteristic pattern of weight deficiency in the first years of life.45 Shivananda, Christopher and Kumar (2000) reported a family with Type 1 hyperprolinemia who was investigated for recurrent uncontrolled seizures. Their work in hyperprolinemia showed that seizures are a neurological feature seen in type 1 hyperprolinemia. This study was significant because hyperprolinemia was previously thought to be a benign disorder.25 This is one study that has been conducted in India, with regard to type 1 hypreprolinemia, but the subjects were not affected with 22q11 deletion syndrome.

Chapter 6 SUMMARY 6.1	SUMMARY 6.2	LIMITATIONS 6.3	SUGGESTIONS

6. SUMMARY 6.1 SUMMARY Type 1 hyperprolinemia is fallout of a defect in the activity of the enzyme proline dehydrogenase coded by the PRODH gene which maps to the 22q11.2 region. Since genes in this region are deleted in individuals affected with 22q11 deletion syndrome, hyperprolinemia is a common finding in this disorder. In this study 14 patients with 22q11 deletion syndrome were assessed for plasma proline levels. 7/14 showed elevated proline levels. 3/7 hyperprolinemic subjects showed varied neurological status and abnormal development patterns. In concordance with previous studies, this study finds that hyperprolinemia is a common finding in 22q11 deletion syndrome. Evaluation of association between hyperprolinemia and neurological status of the subjects was difficult due to the small sample size and the young age of majority of the subjects. 6.2	LIMITATIONS This study had few limitations. First and foremost, the dearth of previous reference studies made it difficult to asses the sample size and study design. Moreover, the short period of study resulted in a small sample size. Further more, the young age of the majority of the subjects made it impossible to form a clear association between hyperprolinemia and neurological abnormalities in 22q11 deletion syndrome. 6.3	SUGGESTIONS: Type 1 hyperprolinemia has not found a cure to date. But, it has been found that decreasing the plasma proline levels does improve the growth and development of the affected child, though mental retardation cannot be reversed. This is an important factor in improving the quality of life in those with 22q11 deletion syndrome. The subjects in this study were below 10 years of age. It is suggested that future studies in an older population will give a better association between hyperprolinemia and neurological disorders in 22q11 deletion syndrome.