Thirteen year retrospective review of the spectrum of inborn errors of metabolism presenting in a tertiary center in Saudi Arabia

Background Inborn errors of metabolism (IEMs) are individually rare; however, they are collectively common. More than 600 human diseases caused by inborn errors of metabolism are now recognized, and this number is constantly increasing as new concepts and techniques become available for identifying biochemical phenotypes. The aim of this study was to determine the type and distribution of IEMs in patients presenting to a tertiary care center in Saudi Arabia. METHOD: We conducted a retrospective review of children diagnosed with IEMs presenting to the Pediatric Department of King Abdulaziz Medical City in Riyadh, Saudi Arabia over a 13-year period. Results Over the 13- year period of this retrospective cohort, the total number of live births reached 110,601. A total of 187 patients were diagnosed with IEMs, representing a incidence of 169 in 100,000 births (1:591). Of these, 121 patients (64.7 %) were identified to have small molecule diseases and 66 (35.3 %) to have large molecule diseases. Organic acidemias were the most common small molecule IEMs, while lysosomal storage disorders (LSD) were the most common large molecule diseases. Sphingolipidosis were the most common LSD. Conclusion Our study confirms the previous results of the high rate of IEMs in Saudi Arabia and urges the health care strategists in the country to devise a long-term strategic plan, including an IEM national registry and a high school carrier screening program, for the prevention of such disorders. In addition, we identified 43 novel mutations that were not described previously, which will help in the molecular diagnosis of these disorders.


Background
Inborn errors of metabolism (IEMs) are defined as monogenic diseases that result in dysfunctional proteins encoded by different genes, which in many cases lead to loss of activity of the enzymes involved [1]. More than 600 different inborn errors of metabolism have been recognized up to this point, and this number is constantly increasing as new concepts and techniques become available for identifying biochemical phenotypes. IEMs are extremely heterogeneous making their classification a primary challenge. Several informal systems of classification currently exist. IEMs can be classified according to the organs involved, such as neurological or hepatic disorders or according to the organelle involved, such as mitochondrial, peroxisomal or lysosomal disorders. Disorders can also be classified according to the age of presentation ranging from neonatal onset to juvenile and adult onset. Because each of these approaches may depend upon the actual setting, no single classification is universally applied [2]. One common and informative classification system involves the classification of IEMs into small and large molecule disorders [3]. Small molecule IEMs have an acute intoxication presentation with a remitting-relapsing clinical course. These include organic acidemias, vitamin responsive disorders, urea cycle disorders, inborn errors of carbohydrates, haem synthesis defects, cholesterol biosynthesis defects, and amino acids and metal transport defects. Large molecule IEMs have a gradual and insidious progressive presentation. These include glycogen storage disorders, sphingolipidosis, mucopolysaccharidosis, oligosaccharidosis, mitochondrial disorders and congenital disorders of glycosylation [2]. The diagnosis of IEMs is mainly based on biochemical investigations, which include the screening of several metabolites in the blood, urine and cerebrospinal fluid (CSF); analysis of enzymatic activities, and molecular genetics testing.
The incidence of such disorders vary from country to country and from region to region. In one Australian study the incidence was reported to be 15.7 per 100,000 births whereas, in Italy, the reported incidence was 27 per 100,000 births [4,5]. In the West Midlands region in the United Kingdom (UK), the incidence reached up to 1:784 and in British Columbia, Canada, the incidence was reported to be 1:2500 [6,7].
These disorders are usually inherited as autosomal recessive disorders, explaining why IEMs are common in populations with a high rate of consanguineous marriages, such as Saudi Arabia. In Saudi Arabia, the rate of consanguineous marriages reaches up to approximately 60 % [8,9].
Despite the high frequency of IEMs, apart from the eastern region study [10], only a few anecdotal epidemiological studies in Saudi Arabia have discussed the incidence, type and distribution of such devastating disorders. Most of the remaining reports in the literature were limited to case reports and case series. In this study, we report the incidence, type, and distribution of IEMs presenting to King Abdulaziz Medical City (KAMC) in the middle region of Saudi Arabia over 13 years. In addition, we identified 43 novel mutations. The diagnostic algorithm starts by referring patients based on clinical suspicion to genetic/metabolic facility from other departments, and from positive new born screening (NBS) after 2011. All accepted cases undergo phenotype related screening biochemical investigations. Confirmation of diagnosis is then achieved either by measuring enzyme activity and/or by targeted molecular tests. In case of unrevealing confirmatory investigations or vague presentation, whole exome or whole genome sequencing is requested.

Methods
The diagnosis of each IEM was based on clinical and biochemical investigations, including analyses of ammonia, lactic acid, total homocysteine, plasma amino acids, the acylcarnitine profile, urine aminoacids, urine for organic acids, copper, ceruloplasmine levels, very long chain fatty acids, transferrin isoelectrofocusing, carbohydrate-deficient transferrin and urine for polyols.
DNA molecular genetic testing was performed in commercial clinical international labs including CENTO-GENE, GeneDx, Emory Genetics, Cincinnati Children's Hospital Medical Center, Bioscientia and Nijmegen Medical Center. All of the parents of the patients with IEMs were tested for carrier status.
Incidence was calculated by dividing the number of diagnosed cases by the number of total births during the study period and multiplying by 100,000 [11].

Results
Over the 13-year period of this retrospective cohort study, the total number of live births reached 110,601. A total of 187 patients were diagnosed with an IEM, resulting in an incidence of 169 in 100,000 births (1:591). Of these patients, 121 (64.7 %) were identified to have a small molecule disease (Table 1) and 66 (35.3 %) to have a large molecule disease ( Table 2). The overall mean, median and range of age at diagnosis were 3.2 years, 1.2 years and from 1 day to 13 years respectively. Tables 1 and 2 show the type and distribution of IEMs from 2001 to 2014 and illustrate the estimated incidence per 100,000 live births for each group of disorders and the mean, median and range of age at diagnosis for each group. The lysosomal storage disorders (LSDs) were the most common diagnosed group, in general, and were observed in 39/187 patients (20.8 %). Sphingolipidoses represent the largest subgroup of the LSDs (22/39; 56.4 %), and GM1 gangliosidosis (infantile phenotype) was the most prevalent disorder. The second most common category was organic acidemias (34/187; 18.2 %), with propionic acidemia (PA) as the most common disorder in that group. Aminoacidopathies were diagnosed in 30/187 patients (16 %). Fatty acid oxidation defects (FAOD) were diagnosed in 5/187 patients (2.7 %), and the frequency of patients with urea cycle disorders (UCD) was 12/187 (6.4 %). The most common FAOD was very long-chain acyl-CoA dehydrogenase deficiency (VLCAD), while argininosuccinic aciduria was the most common UCD. Mucopolysaccharidoses (MPS) were diagnosed in 15/187 patients (8 %), with MPS VI as the predominant type. Fourty-three novel mutations were identified, and missense mutations were the most common type of mutations (Tables 3 and 4). All the listed novel mutations fit with the clinical features of the disease, biochemical biomarkers support genotype phenotype correlation and they segregate well within the patients and family members.

Discussion
In this report, we describe the incidence of IEMs in a single tertiary center in the middle region of Saudi Arabia over more than a decade. We reported an incidence of 1:591 individuals, which is the highest incidence for IEMs reported to this point. Our study is the second epidemiological report after that of Moammar et al., 2010, who reported a incidence of 1:667 [10]. If we combine the two studies, the cumulative incidence is 1:635 births or 157 per 100,000, which is still one of highest reported incidence rates across the world. KAMC is one of the largest medical institutions in the Kingdom and is also a referral center; therefore to obtain a more accurate estimation of incidence of these disorders we have excluded the 69 patients who were diagnosed with IEM but not born at KAMC. If we combine the referred cases with those of patients born at the hospital, we obtain an incidence of 231.5 in 100,000 births (1:432).
Our study confirms the conclusions drawn by Moammar et al., 2010, LSDs are the most commonly identified group of disorders and organic acidemias are the most prevalent small molecule diseases. Our work also supports the notion that MPS VI is the most common type of mucopolysaccharidosis in Saudi Arabia. The results also support the wide variation in incidence of genetic diseases between Saudi Arabia and other parts of the world [12]. In our study for example the recorded number of PTPS exceeds the classical PKU which is reverse in Caucasian population [13]. This is more evident in PA, in which incidence in Saudi Arabia far exceeds the global incidence [14]. During the course of our study we discovered 9 new PA cases in our center, with incidence of 1 per 12,500 live births.
Our study showed unique phenotypes in comparison with the literature. Our three VLCAD patients for example had early presentation with severe phenotype ended with early death. Their genotype mutations were one novel missense mutation in exon 6, c.494 T > C (p.Phe165Ser) and the other two had previously reported nonsense mutation in exon 2, c.65C > A (p.Ser22*). Although these mutations are not clear null mutations [15], they resulted in severe phenotype. Alternatively, the most common phenotype in VLCAD is the milder late onset with the missense mutation p.Val283Ala being the most prevalent disease causing variant [15]. This reflect the poor genotype-phenotype correlation.
In this study the number of private mutations was almost double that of founder mutations, which support the previous report of Al-Owain et.al (2012) who noted that private mutations outweigh founder mutations in Saudi Arabia [12].
Interestingly, among the diseases discovered by our study, 35 diseases are amenable for treatment. Recent advances in early diagnostic tools like the expanded New Born Screening program list, which can detect 14 of the listed diseases, and the availability of treatment options like enzyme replacement therapy, opened new horizons for these patients and their families.
With the implementation of next generation sequencing (WES and WGS) we were able to solve many obscure cases. Additionally, new diseases and new variants might be discovered more easily. It is possible to see NGS a first line diagnostic tool in the near future.       The markedly high numbers of metabolic diseases in Saudi Arabia in general and in our center in particular, with mostly homogenous genotypes, paves the way for future collaboration with international parties, research centers and drug industry, to help providing treatment for our patients. This environment is ideal for wide spectrum of clinical trials of various phases, to speed up the process of new medications discovery.
The limitations of the presented cohort study are clear. These include the fact that the study is confined to a single center in one particular region and contains a small sample size; therefore, the internal and external validity of the study is threatened. Such a retrospective review increases the risk of selection and information biases. Therefore, the numbers mentioned in this study should be taken with caution until further larger studies confirm or refute such findings. The retrospective nature risks missing cases due to poor documentation. In addition, some patients with metabolic disorders may not be seen at the medical center due to early death prior metabolic intervention. In addition, cases with a relatively mild disease may never have presented to the specialized metabolic center, which also contributes to the bias inherent in this study. The variability in ages at diagnosis is attributed to the delay in referring cases to the metabolic facility from other departments, and these numbers should not reflect the expected age of presentation for the listed diseases.
The incredibly high rate of IEMs in Saudi Arabia compels the health care administration in the country to develop a long-term strategic plan for the prevention of such disorders. First, a national registry should be implemented, and through that registry, a determination of the most common IEM and most common mutations in the population can be made. Second, genetic screening of high school students by DNA molecular testing should be performed to identify carriers for the most common disorders in the Saudi population. Premarital molecular screening can help couples, who carry the same disease causing variants, to take informed decision regarding their marriage and the consequences of their decision. Such a strategy has proven to be effective in another population [16]. Finally, intensive educational campaigns aimed at the community through schools, TV, and web-based social media should be initiated.

Conclusion
In this study, we report the incidence, type, and distribution of IEMs presenting to King Abdulaziz Medical City (KAMC) in the middle region of Saudi Arabia over 13 years. We also identified 43 novel mutations in 37 genes. Our study emphasizes the high incidence of IEMs in the Saudi population and urges the health care administration in the country to develop a long-term strategic plan for the prevention of such disorders, including an IEMs national registry and a high school carrier screening program.