Diagnosis of cystathionine beta-synthase deficiency by genetic analysis

Journal of the Neurological Sciences 347 (2014) 305–309

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Diagnosis of cystathionine beta-synthase deficiency by genetic analysis Fatemeh Suri a, Mehrnaz Narooie-Nejad b, Iman Safari a, Hamidreza Moazzeni c, Mohammad-Reza Rohani d, Ali Khajeh e, Brandy Klotzle f, Jian-Bing Fan f, Elahe Elahi a,g,⁎ a

School of Biology, College of Science, University of Tehran, Tehran, Iran Genetics of Non-communicable Disease Research Center, Zahedan University of Medical Science, Zahedan, Iran Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran d Division of Ophthalmology, Zahedan University of Medical Sciences, Zahedan, Iran e Children and Adolescent Health Research Center, Zahedan University of Medical Sciences, Zahedan, Iran f Illumina, San Diego, CA, USA g Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran b c

a r t i c l e

i n f o

Article history: Received 8 September 2014 Received in revised form 14 October 2014 Accepted 16 October 2014 Available online 22 October 2014 Keywords: Whole genome SNP homozygosity mapping Intellectual disability Homocystinuria Diagnosis Cystathionine beta-synthase (CBS) p.Gly116Arg

a b s t r a c t Intellectual disability like other common diseases is often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. We present diagnosis of cystathionine beta-synthase (CBS) deficiency in a multiply affected Iranian family with obvious intellectual disability based on whole genome SNP homozygosity mapping. Diagnosis based on clinical presentations had not been made because of unavailability of appropriate medical services. Genetic analysis led to identification of homozygous c.346GNA in CBS that causes p.Gly116Arg in the encoded protein, cystathionine beta-synthase. CBS is the most common causative gene of homocystinurea. Later, the same mutation was found in three other apparently unrelated Iranian homocystinuria patients. p.Gly116Arg was reported once before in a Turkish patient, suggesting it may be a common CBS deficiency causing mutation in the Middle East. Clinical features of the patients are reported that evidence to variable presentations caused by the same mutation. Finally, observations in heterozygous carriers of the mutation suggest data that a single allele of the p.Gly116Arg causing mutation may have phenotypic consequences, including cardiac related phenotypes. Our study attests to the powers of genetic analysis for diagnosis especially for some forms of intellectual disability, with known genetic causing agents. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Homocystinuria (OMIM #236200), the classical form of which is often known as cystathionine beta-synthase (CBS) (Enzyme Commission (EC) number; EC 4.2.1.22) deficiency, is an inherited disorder of methionine and sulfur metabolism whose inheritance is autosomal recessive [1,2]. Its clinical features were first described in 1962 after studying patients affected with mental retardation and analysis of their urinary amino acid profiles [3,4]. CBS is a homotetramer that catalyzes a reaction in the trans-sulfuration pathway of the methionine cycle during which serine and homocysteine are conjugated to form cystathionine. Homocysteine can also undergo remethylation to form methionine. Dysfunction of the CBS enzyme, therefore, may result in accumulation of both homocysteine and methionine. Mutations in CBS on chromosome 21q22 that encodes cystathionine beta-synthase are the major cause of homocystinuria [5]. Worldwide, at ⁎ Corresponding author at: College of Science, University of Tehran, Enghelab Ave., Tehran 1417614411, Iran. Tel.: +98 9122181251; fax: +98 2166405141. E-mail addresses: [email protected], [email protected] (E. Elahi).

http://dx.doi.org/10.1016/j.jns.2014.10.031 0022-510X/© 2014 Elsevier B.V. All rights reserved.

least 181 mutations in CBS have been reported (Human Genome Mutation Database; http://www.hgmd.cf.ac.uk/ac/index.php). In addition to CBS, mutations in other genes involved in methionine metabolism have sometimes been reported as cause of homocystinuria (http://omim.org/entry/236200). Dietary recommendations and treatment with vitamins B6 (pyridoxal 5-phosphate), B9 (folic acid), and B12 (cobalamin) can be very advantageous in preventing clinical presentations of homocystinuria, particularly if administered early [1]. Treatment even after presentation of symptoms can be beneficial and is recommended. The vitamins are involved in the functions of the various enzymes associated with homocystinuria. The most common clinical manifestations of CBS deficiency include anomalies in nervous, skeletal, ocular, and vascular systems [1]. The earliest manifestations usually occur in the first or second decade of life. Ectopia lentis is the most consistent presentation of the disorder. Other ocular manifestations include high myopia, glaucoma, cataract, and retinal detachment [6]. Skeletal abnormalities include dolichostenomelia which results in a Marfan syndrome-like appearance [7]. Ectopia lentis and skeletal anomalies may in part be due to disruptions of proteins with high concentrations of cysteine such as fibrillin-1 which is involved in the etiology of both conditions [8]. The disruptions

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are expected to be caused by malfunction of sulfur metabolism. Intellectual disability of varying severity is the most frequent abnormality of the nervous system and usually the earliest manifestation of CBS deficiency [9]. Vascular occlusions can result in thromboembolism and are the most frequent cause of death [10]. Additionally, homocystinuria may affect the liver and skin; hypopigmentation of the skin is common [9]. Although the total clinical presentation among various patients is heterogeneous, a “full-blown” presentation is likely to progressively develop if the condition is left untreated [11]. Biochemical testing of homocystinuria is performed by urinary and preferably blood amino acid profiling [1]. The hallmark of CBS deficiency is increased levels of homocysteine and methionine. Based on biochemical screening of more than 200,000 newborns in various countries, a detection rate of 1 in 344,000 was reported [9]. The actual prevalence is thought to be higher, particular in certain populations such as those of Ireland (1 in 65,000), Norway (1 in 6400), and Qatar (1 in 1800) [12-14]. Its incidence in Iran is unknown. A controversial issue with regard to CBS deficiency is potential pathological consequences of being a heterozygous carrier of a CBS mutation [15-17]. This is important because an association between increased plasma homocysteine and cardiovascular disease has been reported [18-21]. To the best of our knowledge, we here report for the first time clinical findings and genetic analysis of Iranian patients affected with CBS deficiency. Correct diagnosis was based on results of genetic analysis.

2. Subjects and methods 2.1. Subjects The CBS deficiency family (HCU-220) studied here included three affected siblings (HCU-220-4, HCU-220-5, and HCU-220-6) (Fig. 1). Blood samples of the patients and family members were sent to us for genetic analysis. The patients were originally reported to be affected with intellectual disability (ID). With the objective of finding the causative gene of the condition, we proceeded to perform whole genome linkage analysis. Recently, genetic analysis on a large cohort of Iranian ID patients had resulted in identification of 73 causative genes [22]. Autosomal recessive inheritance of ID in the family was inferred on the basis of multiple affected children being born to consanguineous unaffected parents. The family was of Sistani ethnicity, and lived in a remote village in the province of Sistan and Baluchestan. The results, as described below, along with biochemical testings and clinical examinations, led to correct diagnosis. After completion of studies on family HCU-220, three other CBS deficiency families were identified and genetic analyses on these were done as well.

Fig. 1. Pedigree of cystathionine beta-synthase deficiency pedigree HCU-220. ■, ●: affected; □, ○: not affected. *, Individuals on whom genome-wide SNP genotyping was performed.

2.2. Genome-wide genotyping and homozygosity mapping DNA was isolated according to standard phenol-chloroform methods. Genome-wide single-nucleotide polymorphism (SNP) genotyping was carried out using HumanCytoSNP-12v1-0_D BeadChips and the iScan reader (Illumin; www.illumin.com). Nine individuals, including seven siblings and their parents were genotyped (Fig. 1). SNPs that had not been genotyped in one or more individual and SNPs that exhibited Mendelian error were removed from the analysis. MERLIN was used for linkage analysis under an autosomal recessive model [23]. Additionally, homozygous regions common to the three affected siblings with a minimum length of 1 Mb and absent in non-affected individuals were sought using the Homozygosity Detector Tool within the GenomeStudio V2010.3 program (Illumina). The SNP chip data output was subsequently exported to Microsoft Excel software and homozygous regions were confirmed within the EXCLUDEAR spreadsheet. Genomic regions are reported with reference to Human Genome Build 37.3. 2.3. Mutation screening The 15 coding exons and flanking intronic sequences of CBS in all three affected children were amplified by polymerase chain reaction (PCR) and then sequenced using the dye terminator chemistry (Big Dye kit and the Prism 3700 sequencer; Applied Biosystems, Foster City, CA, USA). Sequences were analyzed with the Sequencher 4.8 software (Gene Codes, Ann Arbor, MI, USA). Sequence variations were identified by comparison with reference sequences available at the National Center for Biotechnology Information: NC_000021.9, NM_000071.2, and NP_000062.1. Having identified the putative disease associated sequence variation in CBS, the mutation status was assessed in the remaining members of the family by direct sequencing. The mutation was also screened in CBS deficiency patients of three additional families from the same province who report to be unrelated. Sequences of all primers used are available upon request. 3. Results 3.1. Genetic analysis Linkage analysis using MERLIN showed that the highest logarithm of odds (LOD) score (2.8) was associated with a region on chromosome 21q22 (Fig. 2A). A score higher than 1.6 was not obtained anywhere else on the genome. The GenomeStudio Homozygosity Detector tool also only identified the same region on 21q22 to be homozygous in the three affected siblings and not homozygous in the unaffected parents and siblings (Fig. 2B). The homozygous region common to the three affected siblings expanded ~14 Mb and was bound by proximal and distal markers, respectively, rs2018308 (21:33898917 bp) and rs7278087 (21:48098824 bp). The locus includes 296 annotated protein coding genes, one of which is CBS. CBS was considered a candidate causative gene because homocystinuria is accompanied with ID. Sequencing of the gene revealed c.346GNA that causes p.Gly116Arg in the homozygous state in the DNAs of the three affected siblings (Fig. 2C). It was the only variation observed in CBS, and this variation was either absent or present in the heterozygous state in the remaining members of the family (Table 1). C.346GNA was previously reported as a CBS causative mutation in a Turkish patient who harbored it in the compound heterozygous state [16]. P.Gly116 is positioned close to p.Lys119, which is the putative binding site of phosphopyridoxal phosphate [24]. The PolyPhen (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http:// sift.jcvi.org/) bioinformatics tools both predict that p.Gly116Arg is damaging. The results of the genetic analysis suggested that the sequence variation that causes p.Gly116Arg in CBS is the cause of disease in family HCU-220, and that the disease of the affected individuals is homocystinuria due to cystathionine beta-synthase deficiency. To obtain confirmatory evidence for this diagnosis and for gaining a more

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definitive clinical profile, all family members were transported to the provincial center for best available medical examination. 3.2. Clinical findings The clinical features of HCU-220 family members are presented in Table 1. The parents report that the earliest sign of a disorder in the three affected siblings was delayed cognitive responses which were apparent as early as when they were two years old; they were unable to attend school and are considered mentally retarded. Their optical presentation is ectopia lentis. Additionally, they all show increased length of long bones, genu varum, and hypopigmentation of skin and eyes. Notably, glaucoma and mitral valve prolapse were observed in HCU-220-3, who was a heterozygous carrier of the p.Gly116Arg mutation. Glaucoma and mitral valve prolapse are occasional presentations of CBS deficiency [1,25]. Mild mitral regurgitation was observed in the three remaining heterozygous carriers (HCU-220-1, HCU-220-2 and HCU-220-7); these individuals had no other notable presentation. We were able to obtain biochemical data on only three individuals, HCU220-5 who is homozygous for the mutated allele, HCU-220-3 who is a hetrozygote, and HCU-220-9 who is homozygous for the wild type allele. Only HCU-220-5 had elevated levels of plasma homocysteine, urinary cysteine, and plasma methionine. The plasma methionine level (820 μm/l) was extremely high. HCU-220-6 died during a surgery that was performed in the course of this study. Anesthesia-related precautions recommended for CBS deficiency patients were not followed during the surgery [26]. 3.3. Other CBS deficiency patients in province of Sistan and Baluchestan in Iran After having identified the genetic cause of the disorder in family HCU-220, an attempt was made to identify other individuals in the province of Sistan and Baluchestan potentially affected with CBS deficiency through consultation with metabolic disorder specialists and review of hospital records. Four candidate patients based on clinical presentations were identified, and these were from three apparently unrelated families (Table 2). Direct sequencing of CBS coding exon 3 in the DNA of the four individuals showed that they were all homozygous for the same c.346GNA that causes p.Gly116Arg, and they were, therefore, diagnosed with CBS deficiency. As no variation other than c.346GNA was observed in the CBS amplicons of family HCU-220, and as only one amplicon was sequenced in the newly identified patients, it cannot definitively be determined whether the various mutated alleles are identical by decent. 4. Discussion

Fig. 2. Identification of c.346GNA in CBS that causes p.Gly116Arg as cause of disease in family HCU-220. A. LOD plot for chromosome 21 under autosomal-recessive model of inheritance that shows linkage to locus on 21q. B. Homozygosity mapping of HCU-220 family members. Bookmarks indicate homozygous regions. The bookmarks on 21q22 (large bookmarks in third, fourth, and fifth plots from top) that include CBS were present in the three affected individuals and absent in all unaffected members of the family. C. DNA sequence chromatograms showing the c.346GNA in CBS that causes p.Gly116Arg. Chromatograms obtained using reverse primers are shown. Top: homozygous mutated; middle: heterozygous genotype; bottom: homozygous wild type.

The research presented is an example of disease diagnosis based on genetic analysis. Genetic diagnosis by whole genome exome sequencing as compared to whole genome linkage analysis is more common [27-29]. Delayed diagnosis of CBS deficiency is particularly unfortunate, because timely diagnosis allows inexpensive and simple treatments that result in prevention or significant amelioration of the drastic manifestations of the disorder if left untreated [30]. Even delayed treatment that includes prescriptions of vitamins and restricted methionine diets may prevent or slow progression of symptoms [31]. Treatment has been initiated for all the patients included in this study. Clearly, had acceptable medical services been available to the family, early diagnosis of homocystinuria could have been established by obtainment of plasma amino acid profiles and measurement of total homocysteine levels in the blood. This would have been pursued with very early initiation of appropriate dietary-based treatments and concomitant increased likelihood of prevention of the devastating presentations. In some countries, including the United States, some European countries, Australia, and Japan, routine screening of homocystinuria based on measurement

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Table 1 Clinical data on CBS deficiency family HCU-220. CBS deficiency diagnosis

CBS genotype*

HCU-220-1 HCU-220-2 HCU-220-3 HCU-220-4 HCU-220-5 HCU-220n6 HCU-220-7 HCU-220-8 HCU-220-9

− − − + + + − − −

wt/mut wt/mut wt/mut mut/mut mut/mut mut/mut wt/mut wt/wt wt/wt

Present age**

31 24 17 22

yrs yrs yrs yrsΔ

21 yrs

Mental retardation

Ectopia lentis

Skeletal abnormalityα

Genu varum

Hypopigmentation

Cardiac disorder

Glaucoma

− − − + + + − − −

− − − + + + − − −

− − − + + + − − −

− − − + + + − − −

− − − + + + − − −

+β +β +γ

− − + − − − − − −

+β − −

Plasma homocysteine (μm/l)δ

Urinary cysteine

Plasma methionine (μm/l)ε

−

7.7 305

++

820

−

4.7

*wt, wild type allele; mut; p.Gly116Arg; ** and age of clinical examination and biochemical measurements; Δ, died during course of study; α, elongated long bones; β, mitral regurgitation; γ, mitral valve prolapse; δ, upper normal limit: 15 μm/l; ε, normal range: 6–40 μm/l. Data not available are left blank.

Table 2 Clinical data on CBS deficiency patients homozygous for p.Gly116Arg and apparently unrelated to family HCU-220.

**

Patient ID

Present age** Mental retardation Ectopia lentis Skeletal abnormalityα Genu varum Hypopigmentation

Cardiac disorder Glaucoma Plasma homocysteine (μm/l)δ Urinary cysteine Plasma methionine (μm/l)ε

HCU-1013 HCU-1023 HCU-1033 HCU-1034 HCU-220*

10 yrs.

+

+

+

+

−

8 yrs.

−

+

+

−

−

−

3 yrs

+

−

−

−

−

−

640

++

2 yrs

+

−

−

−

−

−

379

++

888

17–24 yrs.

+

+

+

+

+

−

305

++

820

+β

−

330

++

142

++

108

and age of clinical examination and biochemical measurements; α, elongated long bones; β, mitral valve prolapse; δ, upper normal limit: 15 μm/l; ε, normal range: 6–40 μm/l ; *, original HCU pedigree (HCU-220) studied with three affected members. Data not available are left blank.

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Individual ID

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of blood methionine levels is performed on all newborns [32-35]. Newborn screening should seriously be considered for newborns in Iran. This having been said, the exact manifestations of homocystinuria in the patients of the different families studied were variable despite the fact that all harbored the same mutation in the same gene (Table 2). Even mental retardation, ectopia lentis, and Marfanoid skeletal features, that are the cardinal signs of CBS deficiency, were each absent in at least one patient. Compared to the Turkish patient who carried the p.Gly116Arg in the compound heterozygous state, the plasma concentration of homocysteine (305–640 vs. 90 μm/l) in the Iranian patients with the mutation in the homozygous state was much higher (Table 2). The methionine concentration in the Iranian patients was more variable (108–888 μm/l), but still higher than in the Turkish patient (66 μm/l). Although intra-family manifestations in our two families that included more than one affected individual (HCU-220 and HCU-103) were quite similar, variable expression even within families with the p.Gly307Ser mutation has been reported [36]. The variable expressions signify that CBS deficiency should be considered even in patients who present only some of the features commonly associated with CBS deficiency. The phenotypic consequences of being a heterozygous carrier of a single mutated CBS allele, particularly with respect to cardiovascular disorders, are controversial [15]. All four heterozygotes in family HCU220 showed features that are sometimes associated with CBS deficiency. The cardiac disorders observed among the various homozygous or heterozygous carriers of the p.Gly116Arg mutation were considered mild. Although we were unable to perform post-methionine load plasma homocysteine measurements, this measurement was elevated in the heterozygous mother of the Turkish patient with the p.Gly116Arg mutation [16]. Taken together, the data suggest that a single allele of the p.Gly116Arg causing mutation may have phenotypic consequences. Finally, it was notable that all four unrelated CBS deficiency patients from the province of Sistan and Baluchestan were homozygous for the same p.Gly116Arg mutation in CBS and that this same mutation was previously reported only in a Turkish patient [16]. It may be appropriate to specifically screen for this mutation in patients of Middle East origin. We are in the process of recruiting additional homocystinuria patients in Iran. The common CBS mutation in the Arab population of Qatar is p.Arg336Cys (c.1006CNT) [37]. Common CBS mutations have been reported in patients of various populations, including p.Ile278Tyr in Holland; p.Gly307Ser and p.Ile278Tyr in Australia; p.Thr353Met in the United States, p.Thr191Met in Spain and South America, and p.Gly307Ser in patients of Celtic origin (http://omim. org/entry/236200). This distribution of various prevalent mutations facilitates screening protocols in the respective populations, and may reflect founder effects. Acknowledgments We thank the patients and their family members for participating in this study. We acknowledge the Iran National Science Foundation and the Ophthalmic Research Center of Shahid Beheshti University of Medical Sciences for funding this research. References [1] Yap S. Homocystinuria due to cystathionine β-synthase deficiency. In: Saudubray J-M, editor. Orphanet; 2005. [2] Finkelstein JD, Mudd SH, Irreverre F, Laster L. Homocystinuria Due to Cystathionine Synthetase Deficiency: the Mode of Inheritance. Science (New York, NY 1964,146(3645):785–787 [3] Carson NA, Neill DW. Metabolic abnormalities detected in a survey of mentally backward individuals in Northern Ireland. Arch Dis Child 1962;37:505–13. [4] Gerritsen T, Vaughn JG, Waisman HA. The identification of homocystine in the urine. Biochem Biophys Res Commun 1962;9:493–6. [5] Kraus JP, Janosik M, Kozich V, Mandell R, Shih V, Sperandeo MP, et al. Cystathionine beta-synthase mutations in homocystinuria. Hum Mutat 1999;13(5):362–75. [6] Burke JP, O'Keefe M, Bowell R, Naughten ER. Ocular complications in homocystinuria —early and late treated. Br J Ophthalmol 1989;73(6):427–31.

309

[7] Brenton DP, Dow CJ, James JI, Hay RL, Wynne-Davies R. Homocystinuria and Marfan's syndrome A comparison. J Bone Joint Surg 1972;54(2):277–98. [8] Hubmacher D, Tiedemann K, Bartels R, Brinckmann J, Vollbrandt T, Batge B, et al. Modification of the structure and function of fibrillin-1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria. J Biol Chem 2005;280(41): 34946–55. [9] Mudd SH, Levy HL, Skovby F. Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill; 1995. p. 1279–327. [10] Carson NA. Homocystinuria: clinical and biochemical heterogeneity. In: Cockburn F, Gitzelmann R, editors. Inborn errors of metabolism in humans. Lancaster, England: MTP Press Limited; 1982. p. 53–67. [11] Andria G, Sebastio G. Homocystinuria due to cystathionine b-synthase deficiency and related disorders. In: Fernandes J, Saudubray J-M, Van Den Berghe G, editors. Inborn metabolic diseases diagnosis and treatment. 2nd ed. New York: SpringerVerlag Berlin Heidelberg; 1996. p. 177–82. [12] Gan-Schreier H, Kebbewar M, Fang-Hoffmann J, Wilrich J, Abdoh G, Ben-Omran T, et al. Newborn population screening for classic homocystinuria by determination of total homocysteine from Guthrie cards. J Pediatr 2010;156(3):427–32. [13] Naughten ER, Yap S, Mayne PD. Newborn screening for homocystinuria: Irish and world experience. Eur J Pediatr 1998;157(Suppl. 2):S84–7. [14] Refsum H, Fredriksen A, Meyer K, Ueland PM, Kase BF. Birth prevalence of homocystinuria. J Pediatr 2004;144(6):830–2. [15] Guttormsen AB, Ueland PM, Kruger WD, Kim CE, Ose L, Folling I, et al. Disposition of homocysteine in subjects heterozygous for homocystinuria due to cystathionine beta-synthase deficiency: relationship between genotype and phenotype. Am J Med Genet 2001;100(3):204–13. [16] Sperandeo MP, Candito M, Sebastio G, Rolland MO, Turc-Carel C, Giudicelli H, et al. Homocysteine response to methionine challenge in four obligate heterozygotes for homocystinuria and relationship with cystathionine beta-synthase mutations. J Inherit Metab Dis 1996;19(3):351–6. [17] Guilland JC, Favier A, Potier de Courcy G, Galan P, Hercberg S. [Hyperhomocysteinemia: an independent risk factor or a simple marker of vascular disease? 1. Basic data]. Pathol Biol 2003;51(2):101–10. [18] Chao CL, Tsai HH, Lee CM, Hsu SM, Kao JT, Chien KL, et al. The graded effect of hyperhomocysteinemia on the severity and extent of coronary atherosclerosis. Atherosclerosis 1999;147(2):379–86. [19] Wilcken DE, Reddy SG, Gupta VJ. Homocysteinemia, ischemic heart disease, and the carrier state for homocystinuria. Metabolism 1983;32(4):363–70. [20] Linnebank M, Junker R, Nabavi DG, Linnebank A, Koch HG. Isolated thrombosis due to the cystathionine beta-synthase mutation c.833TNC (1278 T). J Inherit Metab Dis 2003;26(5):509–11. [21] Lussana F, Betti S, D'Angelo A, De Stefano V, Fedi S, Ferrazzi P, et al. Evaluation of the prevalence of severe hyperhomocysteinemia in adult patients with thrombosis who underwent screening for thrombophilia. Thromb Res 2013;132(6):681–4. [22] Najmabadi H, Hu H, Garshasbi M, Zemojtel T, Abedini SS, Chen W, et al. Deep sequencing reveals 50 novel genes for recessive cognitive disorders. Nature 2011; 478(7367):57–63. [23] Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 2002;30(1):97–101. [24] Kery V, Bukovska G, Kraus JP. Transsulfuration depends on heme in addition to pyridoxal 5′-phosphate cystathionine beta-synthase is a heme protein. J Biol Chem 1994;269(41):25283–8. [25] Profitlich LE, Kirmse B, Wasserstein MP, Diaz GA, Srivastava S. High prevalence of structural heart disease in children with cblC-type methylmalonic aciduria and homocystinuria. Mol Genet Metab 2009;98(4):344–8. [26] Zahedi H, Nikooseresht M, Seifarabie MA, Sheibani K. Anesthetic management of a child with type homocystinuria. World J Med Surg Case Rep 2013;2:1–4. [27] Jaberi E, Chitsazian F, Ali Shahidi G, Rohani M, Sina F, Safari I, et al. The novel mutation p.Asp251Asn in the beta-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine. J Hum Genet 2013;58(8):526–30. [28] Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, et al. Exome sequencing identifies the cause of a Mendelian disorder. Nat Genet 2010;42(1):30–5. [29] 29.Alavi A, Nafissi S, Shamshiri H, Nejad MM, Elahi E. Identification of mutation in NPC2 by exome sequencing results in diagnosis of Niemann-Pick disease type C. Molecular genetics and metabolism,110(1–2):139–144 [30] Champion MP, Turner C, Bird S, Dalton RN. Delay in diagnosis of homocystinuria. Neonatal screening avoids complications of delayed treatment. BMJ 1997; 314(7077):369–70. [31] Grobe H. Homocystinuria (cystathionine synthase deficiency) results of treatment in late-diagnosed patients. Eur J Pediatr 1980;135(2):199–203. [32] Aoki K. Newborn screening in Japan. Southeast Asian J Trop Med Public Health 2003;34(Suppl. 3):80. [33] Bodamer OA, Hoffmann GF, Lindner M. Expanded newborn screening in Europe 2007. J Inherit Metab Dis 2007;30(4):439–44. [34] Kaye CI, Committee on G, Accurso F, La Franchi S, Lane PA, Hope N, Sonya P, S GB, Michele AL. Newborn screening fact sheets. Pediatrics 2006;118(3):e934–63. [35] Wilcken B, Haas M, Joy P, Wiley V, Bowling F, Carpenter K, et al. Expanded newborn screening: outcome in screened and unscreened patients at age 6 years. Pediatrics 2009;124(2):e241–8. [36] Kraus JP. Komrower lecture molecular basis of phenotype expression in homocystinuria. J Inherit Metab Dis 1994;17(4):383–90. [37] Zschocke J, Kebbewar M, Gan-Schreier H, Fischer C, Fang-Hoffmann J, Wilrich J, et al. Molecular neonatal screening for homocystinuria in the Qatari population. Hum Mutat 2009;30(6):1021–2.