Hypotonia-cystinuria syndrome (HCS) is a recessive disorder caused by microdeletions of SLC3A1 and PREPL on chromosome 2p21.  Patients present with generalized hypotonia at birth, failure to thrive, growth retardation and cystinuria type I.  While the initially described HCS families live in small regions in Belgium and France, we have now identified HCS alleles in patients and carriers from the Netherlands, Italy, Canada and United States of America.  Surprisingly, among the nine deletions detected in those patients, only one novel deletion was found.  Furthermore, one previously described deletion was found six times, another twice.  Finally, we have investigated the frequency of both deletions using a random Belgian cohort.  Given the global occurrence, HCS should be considered in the differential diagnosis of neonatal hypotonia

Heulens, Inge

Martens, Kevin

Meulemans, Sandra

Tilstra, David, MD

English

CentraCare Health

CentraCare HealthDigitalCommons@CentraCare HealthArticles Posters and Scholarly Works10-2007Chromosomal Microdeletions and Genes'Functions: A Cluster of ChromosomalMicrodeletions and the Deleted Genes' FunctionsDavid Tilstra MDCentraCare HealthKevin MartensDepartment of Human Genetics, University of Leuven and Flanders Interuniversity Institute for BiotechnologyInge HeulensSandra MeulemansFollow this and additional works at: https://digitalcommons.centracare.com/articlesPart of the Genetic Processes Commons, and the Medical Genetics CommonsThis Article is brought to you for free and open access by the Posters and Scholarly Works at DigitalCommons@CentraCare Health. It has beenaccepted for inclusion in Articles by an authorized administrator of DigitalCommons@CentraCare Health. For more information, please contactschlepers@centracare.com.Recommended CitationTilstra, David MD; Martens, Kevin; Heulens, Inge; and Meulemans, Sandra, "Chromosomal Microdeletions and Genes' Functions: ACluster of Chromosomal Microdeletions and the Deleted Genes' Functions" (2007). Articles. 15.https://digitalcommons.centracare.com/articles/15ARTICLEGlobal distribution of the most prevalent deletionscausing hypotonia–cystinuria syndromeKevin Martens1,2, Inge Heulens1, Sandra Meulemans1, Marco Zaffanello3, David Tilstra4,Frederik J Hes5, Raoul Rooman6, Inge Franc¸ois7, Francis de Zegher7, Jaak Jaeken7,Gert Matthijs2 and John WM Creemers*,11Laboratory for Biochemical Neuro-endocrinology, Department for Human Genetics, University of Leuven and FlandersInteruniversity Institute for Biotechnology, Gent, Belgium; 2Laboratory for Molecular Diagnosis, Department for HumanGenetics, University of Leuven, Leuven, Belgium; 3Department of Mother and Child, Biology-Genetics, Sectionof Paediatrics, University of Verona, Verona, Italy; 4CentraCare Clinic, St Cloud, MN, USA; 5Department of ClinicalGenetics, Leiden University Medical Centre, Leiden, The Netherlands; 6Department of Pediatrics, Antwerp UniversityHospital, Antwerp, Belgium; 7Department of Pediatrics, University Hospitals Leuven, Leuven, BelgiumHypotonia–cystinuria syndrome (HCS) is a recessive disorder caused by microdeletions of SLC3A1 andPREPL on chromosome 2p21. Patients present with generalized hypotonia at birth, failure to thrive, growthretardation and cystinuria type I. While the initially described HCS families live in small regions in Belgiumand France, we have now identified HCS alleles in patients and carriers from the Netherlands, Italy, Canadaand United States of America. Surprisingly, among the nine deletions detected in those patients, only onenovel deletion was found. Furthermore, one previously described deletion was found six times, anothertwice. Finally, we have investigated the frequency of both deletions using a random Belgian cohort. Giventhe global occurrence, HCS should be considered in the differential diagnosis of neonatal hypotonia.European Journal of Human Genetics (2007) 15, 1029–1033; doi:10.1038/sj.ejhg.5201881; published online 20 June 2007Keywords: PREPL; SLC3A1; hypotonia; cystinuria; oligopeptidase; growth retardationIntroductionThe hypotonia–cystinuria syndrome (HCS; MIM 606407)is a recessive congenital disorder characterized by general-ized hypotonia at birth, failure to thrive, growth retarda-tion and cystinuria type I.1,2 Recently, the genetic defectunderlying this syndrome has been characterized in ninefamilies.1 The disease is due to microdeletions that disrupttwo genes on chromosome 2p21, SLC3A1 and PREPL. Asmutations in SLC3A1 cause isolated cystinuria type I andthe flanking genes are normally expressed, the extendedphenotype can be attributed to PREPL.1A larger deletion of 179 kb at this locus has previouslybeen reported,3,4 which causes the recessive 2p21 deletionsyndrome. At least four genes (SLC3A1, PREPL, PPM1B andC2orf34) are deleted, resulting in a much more severephenotype. A detailed comparison between the twosyndromes has been published.5At least three additional deletions involving PREPL havebeen reported in patients with isolated cystinuria type I.However, they were not fully characterized.6 – 8 Finally, onesporadic case with HCS symptoms, accompanied with amitochondrial respiratory chain defect has been reported.Molecular analysis of the SLC3A1/PREPL locus was sug-gested but not performed.9This report focuses on the molecular analysis of four newHCS families, including two previously described cases.Received 22 November 2006; revised 27 April 2007; accepted 22 May2007; published online 20 June 2007*Correspondence: Dr JWM Creemers, Faculty of Medicine, Laboratory forBiochemical Neuroendocrinology, Department for Human Genetics,University of Leuven/V.I.B., Gasthuisberg O/N 6, Box 602, Herestraat49, Leuven B-3000, Belgium.Tel: þ 32-16-346080; Fax: þ 32-16-346073;E-mail: john.creemers@med.kuleuven.beEuropean Journal of Human Genetics (2007) 15, 1029–1033& 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00www.nature.com/ejhgFinally, the frequency of the most prevalent deletionscausing HCS was estimated using a random Belgianpopulation.Materials and methodsPatient materialMaterial from family 10 (University Hospitals Leuven,Belgium), family 11 (University Medical Centre Leiden,The Netherlands) and family 12 (CentraCare Clinic,St Cloud, MN, USA) was sent for molecular diagnosis forthe SLC3A1/PREPL locus. DNA samples for family 13(University of Verona, Italy) and family 14 (McGillUniversity, Montreal, Canada) were obtained upon request.As a random population, we used the CAREGENE10 cohort.Quantitative PCRQuantitative PCR was performed using qPCR Master Mixfor SYBR Green I detection and fluorescein as internalstandard (Eurogentec, Seraing, Belgium) on the MyIQsystem (Bio-Rad, Nazareth-Eke, Belgium), in accordancewith the manufacturer’s guidelines. Primers were developedwith Primer Express software (Applied Biosystems, Nieu-werkerk a/d IJssel, The Netherlands). The relative number ofalleles were calculated using the Livak (2DDCt ) method.11An amplicon within the p53 gene was used as reference.Junction fragment PCR and sequencingPCR products were amplified using 150 ng of genomicDNA, by the use of the Advantage 2 PCR kit (Clontech,Mountain View, CA, USA). Junction fragments werepurified with the Montage PCR Centrifugal Filter Device(Millipore, Billerica, MA, USA) before sequencing (BigdyeTerminator v3.1, Applied Biosystems) on the ABI3130system (Applied Biosystems). Primers used for amplifyingdeletions A and B have been described.1 Primers fordeletion E are 50-TTGCTCCAAAAGTTCCTAACCAA-30 and50-TTCTGTGTTGAGGTTGCACTCC-30.Haplotype analysisGenotypes of microsatellites and single nucleotide poly-morphisms (SNP) were determined as described.1ResultsThe clinical and endocrinological data on the patients aresimilar to those reported earlier.1 Details are listed in Table 1.In summary, the clinical picture is characterized by neonataland infantile hypotonia, and anorexia, often necessitatingnasogastric tube feeding or gastrostomy. Some dysmorphy ispresent, particularly dolichocephaly and ptosis of theeyelids, as well as a striking nasal speech. Anorexia andhypotonia ameliorate with age, but gradually growthvelocity decreases due to IGF-1 insufficiency. Patientsrespond well to growth hormone treatment. Electromyo-graphy and brain magnetic resonance imaging are normal,when performed. In addition, patients have classicalcystinuria type I causing nephrolithiasis at variable ages.The presence or absence of different exons of SLC3A1and PREPL was determined in patients and parents (whenTable 1 Clinical and endocrinological data on the patientsFamily 10 Family 11 Family 12 Family 13Actual age (years) 16 34/12 9 15Sex M M M FBirth weight (g) 1220 3185 3205 3660Birth length (cm) 38 NI 44 50Gestational age (weeks) 31 40 40 40Infantile hypotonia + + + +Infantile anorexia + + + +Nasogastric tube feeding +  + +Gastrostomy   + Dysmorphy Dolichocephaly Deep sunken eyes Dolichocephaly, eyelid ptosisand tented upper lipNasal speech +  + +Unsupported walking from (m) 18 23 14 14Special school  NI + Growth retardation +  + +From age (years) 1–2 NA 7 2.5Growth hormone (basal/peak) 2.4/31 mU/ml NI NI 1.2 /10.2 mU/mlIGF-1 3 SD NI NI 3 SDGrowth hormone treatment Yes NA Yes NoStart of puberty (years) 14 NA  12Age of first symptomaticnephrolithiasis7 years (coral stone) No lithiasis yet No lithiasis yet No lithiasis yetElectromyography Normal  Normal NIBrain CT/MRI Normal MRI  Normal MRI Normal MRINI, no information; NA, not applicableGlobal distribution of HCS deletionsK Martens et al1030European Journal of Human Geneticsavailable) using quantitative PCR. In families 11 and 13,the boundaries for both deleted alleles were located inintron 1 of SLC3A1 and intron 1 of PREPL. As the samefragments were deleted in the previously described dele-tion B,1 we performed a junction fragment PCR specific forthis deletion followed by sequencing of the amplicons(Figure 1). Both patients are homozygous for deletion B,therefore confirming the diagnosis of HCS. The patient infamily 10 is compound heterozygous for two deletions, oneidentical to deletion B, the other extending from exon 10in SLC3A1 to exon 2 in PREPL-like deletion A from Jaekenet al.1 Junction fragment PCR and sequencing confirmedthat the second allele is indeed deletion A (Figure 1). Thepatient in family 12 showed a homozygous loss from exon10 in SLC3A1 to exon 2 in PREPL. Junction fragment PCRand sequencing confirmed that the paternal allele is againdeletion A. The proximal boundary of the deletion on thematernal chromosome is located in intron 1 of SLC3A1,whereas the distal break point is located between 45 and50 kb upstream of PREPL (Figures 1 and 2). Sequencing ofthe junction fragment indicated that the deletion was127 kb in size, with the distal break point located in intron3 of C2orf34, the gene flanking PREPL. This is a novel allele(deletion E) involved in HCS.The patient in family 14 (patient 936 in Saadi et al6) hasisolated cystinuria type I, and has previously beenFigure 1 Genetic analysis of the HCS patients. (A) Quantitative PCR analysis on genomic DNA of HCS patients. Only relevant amplicons are shown.(B) Junction fragments spanning the break point in patients and parents (when available). , negative control; P, patient; M, mother; F, father.(C) Genotype of the patients.Global distribution of HCS deletionsK Martens et al1031European Journal of Human Geneticsdescribed as compound heterozygous for two deletionsinvolving SLC3A1. The deletion on the paternal allele hadbeen shown to extend from exon 1 to exon 7 of SLC3A1,whereas the deletion on the maternal allele starts in intron1 of SLC3A1 and extends beyond exon 10. We have nowdetermined the exact break points on the latter deletion.Quantitative PCR analysis revealed that the proximalboundary was located in intron 1 of SLC3A1, whereas thedistal break point was localized in intron 2 of PREPL. Again,this pointed toward the presence of deletion B (Figure 1).The different deletion alleles were haplotyped usingmicrosatellites and SNPs. Only family 10 showed a partiallyconserved ancestral haplotype for deletion A (Figure 3).SNPs genotyped in families 11–13 identified the samealleles as present in the ancestral haplotype of deletion B;however, the genotypes for families 11 and 13 were notinformative. The SNP data for family 10 could not beobtained due to a lack of parental DNA (data not shown).Because of the high incidence of deletions A and B, wehave screened a random Belgian cohort for the presence ofthese deletions. We found 1 allele with deletion A and 1allele with deletion B in a total of 936 random samples(1872 chromosomes). This leads to an allele frequencyestimate for deletions A or B between 1/333 and 1/7700(binomial distribution; 95% confidence interval).DiscussionHCS was initially described in nine families living inBelgium and France. On 18 independent chromosomes,only four different deletions (deletions A–D) were found.Figure 2 The 2p21 locus. (a) Genomic organization of the 2p21 locus. The flanking microsatellite markers used in the haplotype analysis areindicated with their genomic coordinates. (b) Schematic representation of the deletions detected in the novel HCS families, presented here. Deletions(a and b) have previously been described,1 whereas deletion E represents a new allele. Sequences flanking the break points are shown and arenumbered in accordance with the numbering in the database (Homo sapiens, build 36; http://www.ncbi.nlm.nih.gov/).Figure 3 (A and B) Haplotype analysis for deletion. Completely conserved haplotypes are shaded dark gray, partially conserved haplotypes areshaded light gray. Dashed lines indicate the break point. Note ‘a’ phase unknown.Global distribution of HCS deletionsK Martens et al1032European Journal of Human GeneticsThe frequency of deletions A and B was 4/18 and 11/18,respectively, due to founder effects.1 In the four novel HCSfamilies and the one carrier of a HCS allele described here,the frequency of deletions A and B were 2/9 and 6/9,respectively. The allele frequencies of deletions A or B in arandom Belgian cohort were estimated between 1/333 and1/7700.Deletions A and B are globally distributed, as they werefound in patients from The Netherlands, Italy, Canada andthe United States America. However, the ancestral haplo-types of the microsatellite markers flanking the breakpoints were completely decayed in these families, indicat-ing that the deletions are old. However, it cannot beexcluded that a genetic mechanism is promoting deletionsin this region. If this is the case, however, it is remarkablethat the break points are exactly the same.The patient in family 12 appeared to be compoundheterozygous for deletions A and E, the latter being a noveldeletion involved in HCS. This deletion does not onlydisrupt the coding region of SLC3A1 and PREPL, but also ofC2orf34. C2orf34 has a putative methyltransferase domain,but its function has not yet been described. It is, however,not expected that a loss of one allele is modulating thephenotype in this HCS patient, because the mothershowed no phenotype that might be due to the loss ofone allele of C2orf34.Unexpected results were obtained for family 13. Theproband in this family showed all HCS symptoms,accompanied with a mitochondrial respiratory chaindefect. As a mitochondrial respiratory chain defect wasalso observed in the 2p21 deletion syndrome,3 one mighthave expected that besides SLC3A1 and PREPL, at least oneother gene (PPM1B or C2orf133) was involved. However,this patient is homozygous for deletion B, and thereforeher genotype is identical to some of the previouslypublished patients. She has been the only HCS patientwith a reported respiratory chain defect. This brings up anintriguing question about the cause of the respiratorychain defect in this patient. A possible explanation couldbe the presence of variations in modifier genes, either in cisor in trans, yet to be discovered. Currently, in-depthanalysis of mitochondrial respiratory chain function infibroblast cells from other HCS patients is being performedto determine potential minor defects that might previouslyhave been overlooked.Three deletions involving PREPL have been described inpatients with isolated cystinuria type I. We characterizedthe deletion initially reported in Saadi et al.6 It turns out tobe the most frequent HCS allele, deletion B, only disrupt-ing the coding region of SLC3A1 and PREPL. This leaves thetotal of unique alleles where PREPL is deleted at six: fivedeletions present in HCS and one causing the 2p21deletion syndrome. Possibly, two other alleles exist, butthe break points have not been determined.7,8In conclusion, HCS is a relatively homogenous syn-drome, with common ancestral alleles found in differentpopulations. On the basis of the allele frequencies, thisdisease will be rare (1/1 000 000 for an allele frequency of1/1000); hence, our observations are compatible with afounder effect in the regions from where the HCS patientsoriginate.AcknowledgementsDNA from family 14 was kindly provided by Dr Rozen (McGillUniversity, Montreal, Canada). We thank Dr Vos (Medical CentreHaaglanden, Den Haag, The Netherlands) for assistance with patient11. Grant and scholarship support was provided by the ‘VlaamsInstituut voor de Bevordering van Wetenschappelijk-TechnologischOnderzoek in de Industrie (IWT)’ and the ‘Fonds voor Wetenschappe-lijk Onderzoek Vlaanderen (FWO)’.References1 Jaeken J, Martens K, Francois I et al: Deletion of PREPL, a geneencoding a putative serine oligopeptidase, in patients withhypotonia–cystinuria syndrome. Am J Hum Genet 2006; 78:38–51.2 Clara R, Lowenthal A: Familial aminoaciduria with muscularhypotonia and dwarfism. Bull Acad R Med Belg 1966; 6: 577–611.3 Parvari R, Brodyansky I, Elpeleg O, Moses S, Landau D,Hershkovitz E: A recessive contiguous gene deletion of chromo-some 2p16 associated with cystinuria and a mitochondrialdisease. Am J Hum Genet 2001; 69: 869–875.4 Parvari R, Gonen Y, Alshafee I, Buriakovsky S, Regev K,Hershkovitz E: The 2p21 deletion syndrome: characterization ofthe transcription content. Genomics 2005; 86: 195–211.5 Martens K, Derua R, Meulemans S et al: PREPL: a putative noveloligopeptidase propelled into the limelight. Biol Chem 2006; 387:879–883.6 Saadi I, Chen XZ, Hediger M et al: Molecular genetics ofcystinuria: mutation analysis of SLC3A1 and evidence for anothergene in type I (silent) phenotype. Kidney Int 1998; 54: 48–55.7 Font-Llitjos M, Jimenez-Vidal M, Bisceglia L et al: New insightsinto cystinuria: 40 new mutations, genotype–phenotype correla-tion, and digenic inheritance causing partial phenotype. J MedGenet 2005; 42: 58–68.8 Bisceglia L, Calonge MJ, Dello Strologo L et al: Molecular analysisof the cystinuria disease gene: identification of four newmutations, one large deletion, and one polymorphism. HumGenet 1996; 98: 447–451.9 Zaffanello M, Beghini R, Zamboni G, Fanos V: A sporadic case ofcystinuria, respiratory chain and growth hormone deficiencies.Pediatr Nephrol 2003; 18: 846–847.10 Defoor J, Martens K, Matthijs G et al: The care gene study: muscle-specific creatine kinase gene and aerobic power in coronary arterydisease. Eur J Cardiovasc Prev Rehabil 2005; 12: 415–417.11 Livak KJ, Schmittgen TD: Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-delta delta C(T))method. Methods 2001; 25: 402–408.Global distribution of HCS deletionsK Martens et al1033European Journal of Human Genetics

Chromosomal Microdeletions and Genes\u27 Functions: A Cluster of Chromosomal Microdeletions and the Deleted Genes\u27 Functions

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Chromosomal Microdeletions and Genes\u27 Functions: A Cluster of Chromosomal Microdeletions and the Deleted Genes\u27 Functions

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