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• W1974746092 abstract "Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development. Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development. Cutis laxa type 2 (ARCL2, [MIM 219200]) is an autosomal-recessive multisystem disorder with prominent connective-tissue features characterized by the appearance of premature aging, particularly wrinkled and lax skin with reduced elasticity. It falls in a family of related disorders including cutis laxa type 1 (MIM 219100), gerodermia osteodysplastica or Walt Disney dwarfism (MIM 231070), and wrinkly skin syndrome (MIM 278250) and is sometimes referred to as cutis laxa with growth and developmental delay, or as cutis laxa with joint laxity and retarded development. Definitive criteria for distinguishing all of these conditions unambiguously is lacking, involving a subjective element to clinical diagnosis. Thus, molecular diagnosis is proving increasingly useful in resolving these phenotypically similar disorders. Mutations have been identified in five genes in patients and families segregating these related clinical presentations, specifically Egf-containing fibulin-like extracellular matrix protein 2 (EFEMP2, alias fibulin 4 [MIM 604633]),1Dasouki M. Markova D. Garola R. Sasaki T. Charbonneau N.L. Sakai L.Y. Chu M.L. Compound heterozygous mutations in fibulin-4 causing neonatal lethal pulmonary artery occlusion, aortic aneurysm, arachnodactyly, and mild cutis laxa.Am. J. Med. Genet. A. 2007; 143A: 2635-2641Crossref PubMed Scopus (88) Google Scholar, 2Hucthagowder V. Sausgruber N. Kim K.H. Angle B. Marmorstein L.Y. Urban Z. Fibulin-4: a novel gene for an autosomal recessive cutis laxa syndrome.Am. J. Hum. Genet. 2006; 78: 1075-1080Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar fibulin 5 (FBLN5 [MIM 604580]),3Claus S. Fischer J. Megarbane H. Megarbane A. Jobard F. Debret R. Peyrol S. Saker S. Devillers M. Sommer P. et al.A p.C217R mutation in fibulin-5 from cutis laxa patients is associated with incomplete extracellular matrix formation in a skin equivalent model.J. Invest. Dermatol. 2008; 128: 1442-1450Crossref PubMed Scopus (35) Google Scholar, 4Markova D. Zou Y. Ringpfeil F. Sasaki T. Kostka G. Timpl R. Uitto J. Chu M.L. Genetic heterogeneity of cutis laxa: a heterozygous tandem duplication within the fibulin-5 (FBLN5) gene.Am. J. Hum. Genet. 2003; 72: 998-1004Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 5Loeys B. Van Maldergem L. Mortier G. Coucke P. Gerniers S. Naeyaert J.M. De Paepe A. Homozygosity for a missense mutation in fibulin-5 (FBLN5) results in a severe form of cutis laxa.Hum. Mol. Genet. 2002; 11: 2113-2118Crossref PubMed Scopus (227) Google Scholar, 6de Schepper S. Loeys B. de Paepe A. Lambert J. Naeyaert J.M. Cutis laxa of the autosomal recessive type in a consanguineous family.Eur. J. Dermatol. 2003; 13: 529-533PubMed Google Scholar ATPase, H+ transporting, V0 subunit a2 (ATP6V0A2 [MIM 611716]),7Kornak U. Reynders E. Dimopoulou A. van Reeuwijk J. Fischer B. Rajab A. Budde B. Nurnberg P. Foulquier F. Lefeber D. et al.Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2.Nat. Genet. 2008; 40: 32-34Crossref PubMed Scopus (260) Google Scholar elastin (ELN [MIM 130160]),8Graul-Neumann L.M. Hausser I. Essayie M. Rauch A. Kraus C. Highly variable cutis laxa resulting from a dominant splicing mutation of the elastin gene.Am. J. Med. Genet. A. 2008; 146A: 977-983Crossref PubMed Scopus (32) Google Scholar, 9Rodriguez-Revenga L. Iranzo P. Badenas C. Puig S. Carrio A. Mila M. A novel elastin gene mutation resulting in an autosomal dominant form of cutis laxa.Arch. Dermatol. 2004; 140: 1135-1139Crossref PubMed Scopus (60) Google Scholar, 10Zhang M.C. He L. Giro M. Yong S.L. Tiller G.E. Davidson J.M. Cutis laxa arising from frameshift mutations in exon 30 of the elastin gene (ELN).J. Biol. Chem. 1999; 274: 981-986Crossref PubMed Scopus (121) Google Scholar, 11Tassabehji M. Metcalfe K. Hurst J. Ashcroft G.S. Kielty C. Wilmot C. Donnai D. Read A.P. Jones C.J. An elastin gene mutation producing abnormal tropoelastin and abnormal elastic fibres in a patient with autosomal dominant cutis laxa.Hum. Mol. Genet. 1998; 7: 1021-1028Crossref PubMed Scopus (128) Google Scholar and SCYL1 binding protein (SCYL1BP1 [MIM 607983]).12Hennies H.C. Kornak U. Zhang H. Egerer J. Zhang X. Seifert W. Kuhnisch J. Budde B. Natebus M. Brancati F. et al.Gerodermia osteodysplastica is caused by mutations in SCYL1BP1, a Rab-6 interacting golgin.Nat. Genet. 2008; 40: 1410-1412Crossref PubMed Scopus (112) Google Scholar Mutations have also been found in a sixth gene, aldehyde dehydrogenase 18 family, member A1 (ALDH18A1, alias pyrroline 5-carboxylate synthase [PYCS] [MIM 138250]), in patients described with a so-called neurocutaneous syndrome closely resembling cutis laxa type 2.13Baumgartner M.R. Hu C.A. Almashanu S. Steel G. Obie C. Aral B. Rabier D. Kamoun P. Saudubray J.M. Valle D. Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding delta(1)-pyrroline-5-carboxylate synthase.Hum. Mol. Genet. 2000; 9: 2853-2858Crossref PubMed Google Scholar, 14Baumgartner M.R. Rabier D. Nassogne M.C. Dufier J.L. Padovani J.P. Kamoun P. Valle D. Saudubray J.M. Delta1-pyrroline-5-carboxylate synthase deficiency: neurodegeneration, cataracts and connective tissue manifestations combined with hyperammonaemia and reduced ornithine, citrulline, arginine and proline.Eur. J. Pediatr. 2005; 164: 31-36Crossref PubMed Scopus (67) Google Scholar, 15Bicknell L.S. Pitt J. Aftimos S. Ramadas R. Maw M.A. Robertson S.P. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.Eur. J. Hum. Genet. 2008; 16: 1176-1186Crossref PubMed Scopus (65) Google Scholar These six genes represent a variety of biochemical processes. The phenotypes also vary among the described patients, both clinically and biochemically, as to whether there is involvement of protein N- or O-glycosylation, found to be defective in assocation with the ATP6V0A2 mutations, although this gene may not explain the symptoms of all such patients.7Kornak U. Reynders E. Dimopoulou A. van Reeuwijk J. Fischer B. Rajab A. Budde B. Nurnberg P. Foulquier F. Lefeber D. et al.Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2.Nat. Genet. 2008; 40: 32-34Crossref PubMed Scopus (260) Google Scholar, 16Wopereis S. Morava E. Grunewald S. Mills P.B. Winchester B.G. Clayton P. Coucke P. Huijben K.M. Wevers R.A. A combined defect in the biosynthesis of N- and O-glycans in patients with cutis laxa and neurological involvement: the biochemical characteristics.Biochim. Biophys. Acta. 2005; 1741: 156-164Crossref PubMed Scopus (33) Google Scholar, 17Morava E. Wopereis S. Coucke P. Gillessen-Kaesbach G. Voit T. Smeitink J. Wevers R. Grunewald S. Defective protein glycosylation in patients with cutis laxa syndrome.Eur. J. Hum. Genet. 2005; 13: 414-421Crossref PubMed Scopus (66) Google Scholar, 18Morava E. Lefeber D.J. Urban Z. de Meirleir L. Meinecke P. Gillessen Kaesbach G. Sykut-Cegielska J. Adamowicz M. Salafsky I. Ranells J. et al.Defining the phenotype in an autosomal recessive cutis laxa syndrome with a combined congenital defect of glycosylation.Eur. J. Hum. Genet. 2008; 16: 28-35Crossref PubMed Scopus (52) Google Scholar Notably, there are numerous literature reports of cases resembling this suite of phenotypes, either unexplained or untested for the known genes.19Imaizumi K. Kurosawa K. Makita Y. Masuno M. Kuroki Y. Male with type II autosomal recessive cutis laxa.Clin. Genet. 1994; 45: 40-43Crossref PubMed Scopus (20) Google Scholar, 20Al-Gazali L.I. Sztriha L. Skaff F. Haas D. Gerodermia osteodysplastica and wrinkly skin syndrome: are they the same?.Am. J. Med. Genet. 2001; 101: 213-220Crossref PubMed Scopus (34) Google Scholar, 21Gupta N. Phadke S.R. Cutis laxa type II and wrinkly skin syndrome: distinct phenotypes.Pediatr. Dermatol. 2006; 23: 225-230Crossref PubMed Scopus (15) Google Scholar, 22Nanda A. Alsaleh Q.A. Al-Sabah H. Marzouk E.E. Salam A.M. Nanda M. Anim J.T. Gerodermia osteodysplastica/wrinkly skin syndrome: report of three patients and brief review of the literature.Pediatr. Dermatol. 2008; 25: 66-71Crossref PubMed Scopus (17) Google Scholar, 23Rajab A. Kornak U. Budde B.S. Hoffmann K. Jaeken J. Nurnberg P. Mundlos S. Geroderma osteodysplasticum hereditaria and wrinkly skin syndrome in 22 patients from Oman.Am. J. Med. Genet. A. 2008; 146A: 965-976Crossref PubMed Scopus (34) Google Scholar, 24Scherrer D.Z. Alexandrino F. Cintra M.L. Sartorato E.L. Steiner C.E. Type II autosomal recessive cutis laxa: report of another patient and molecular studies concerning three candidate genes.Am. J. Med. Genet. A. 2008; 146A: 2740-2745Crossref PubMed Scopus (2) Google Scholar In the course of regular clinical practice, we ascertained two Maritime Canadian pedigrees of French Acadian descent, one with four individuals diagnosed with ARCL2 and one with one such individual (Figures 1A and 1B). All of the patients displayed lax, wrinkled skin with reduced elasticity, lax joints, and mild craniofacial dysmorphic features (Figure 2). The loose skin was most prominent over the dorsum of the hands and feet. Craniofacial dysmorphism included microcephaly, broad and prominent forehead, prominent ears, blue sclerae, and sagging cheeks. The patients looked substantially older than their chronologic ages. Joint laxity was most prominent in the small joints of the hands and feet, and two patients had congenital hip dislocation. All patients had intrauterine growth retardation and at least some degree of postnatal growth deficiency. All five showed developmental delay. Two affected individuals had demonstrated agenesis of the corpus callosum, and one other had been found to have enlarged ventricles. There was no obvious metabolic pathology ascertained by routine blood and urine metabolite analysis. There was no corneal clouding or athetosis, as might be expected for de Barsy syndrome. There was no clinical evidence requiring X-ray or bone mineral density scan; hence, these procedures were not performed. Protein glycosylation and wound healing were normal. See Table 1 for detailed clinical findings. Approval for a research study was obtained from the Isaak Walton Killam (IWK) Hospital Research Ethics Board. All sampled family members or parents provided written informed consent to participate in the study. DNA was obtained from living patients' and relatives' blood samples via routine extraction methods. All procedures were in accordance with ethical and methodological standards for human experimentation.Figure 2Clinical Presentations of Patients with Cutis Laxa Type 2Show full captionShown are (A) hands (11156), (B) face (D05-09864), and (C) feet (11156) of case individuals aged 12, 11, and 12 years, respectively, at the time of photography. Note the appearance of premature aging, particularly wrinkled skin and joint laxity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1CL2 Clinical Details in Maritime Patients and the LiteratureClinical FeatureCL Type 2WSSGONC11807D05-098641155612827No DNAGeneralIUGR+++++++Failure to thrive++++++?Developmental delay+++++++++Feeding problemsreflux+reflux-+DYSMORPHISMMicrocephaly+++++++?+Broad, prominent forehead++++++Long philtrum+-+--Midface hypoplasia+malar+-+Epicanthal folds++---Downslanting palpebral fissures+-+Large, bulbous nose++-Prognathism+-+/−-Retrognathia---Large, protruding ears++++Hypertelorism++/−++-Blue sclera+/?-+-+++Sagging jowls+-++++++Large fontanelle++/NM--+++Pinched nose++++High palate+--Triangular face++-++Deep set eyes-++MusculoskeletalLax joints+++++++++Hip dislocation+++++-?-+Winged scapulaeNM+-----Spinal deformity+---Fractures---Pectus excavatum--++--Diminshed muscle mass+++--Hypotonia++++---Scoliosis++-Long digits++-Clasped thumb+++CutaneousWrinkly skin++++++Prominent veins/translucent skin+++++++++Prominent palmar/plantar creases++-+Lax skin with reduced elasticity+++++++RadiologyOsteoporosis+-Vertebral anomalies+-Wormian bones+-+OtherInguinal hernia+-/NM+1 of 4--+Umbilical hernia+-/NM++ (?)--Congenital heart disease-/NM---+Pulmonary emphysema----Renal defect--/NM---Hydrocephalus-+-+Jaundice+--+CNS involvement++-+Agenesis of the corpus callosum++-Respiratory tract infection?+-?Easy bruising+?+Aortic root aneurysm---Clinical features of Acadian ARCL2 patients (last 5 columns) versus literature review of other descriptions of cutis laxa type 2 with developmental delay, wrinkly skin syndrome (WSS), gerodermia osteodysplastica (GO), and neurocutaneous syndrome (NC).13Baumgartner M.R. Hu C.A. Almashanu S. Steel G. Obie C. Aral B. Rabier D. Kamoun P. Saudubray J.M. Valle D. Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding delta(1)-pyrroline-5-carboxylate synthase.Hum. Mol. Genet. 2000; 9: 2853-2858Crossref PubMed Google Scholar, 14Baumgartner M.R. Rabier D. Nassogne M.C. Dufier J.L. Padovani J.P. Kamoun P. Valle D. Saudubray J.M. Delta1-pyrroline-5-carboxylate synthase deficiency: neurodegeneration, cataracts and connective tissue manifestations combined with hyperammonaemia and reduced ornithine, citrulline, arginine and proline.Eur. J. Pediatr. 2005; 164: 31-36Crossref PubMed Scopus (67) Google Scholar, 15Bicknell L.S. Pitt J. Aftimos S. Ramadas R. Maw M.A. Robertson S.P. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.Eur. J. Hum. Genet. 2008; 16: 1176-1186Crossref PubMed Scopus (65) Google Scholar, 21Gupta N. Phadke S.R. Cutis laxa type II and wrinkly skin syndrome: distinct phenotypes.Pediatr. Dermatol. 2006; 23: 225-230Crossref PubMed Scopus (15) Google Scholar, 22Nanda A. Alsaleh Q.A. Al-Sabah H. Marzouk E.E. Salam A.M. Nanda M. Anim J.T. Gerodermia osteodysplastica/wrinkly skin syndrome: report of three patients and brief review of the literature.Pediatr. Dermatol. 2008; 25: 66-71Crossref PubMed Scopus (17) Google Scholar, 44Lisker R. Hernandez A. Martinez-Lavin M. Mutchinick O. Armas C. Reyes P. Robles-Gil J. Gerodermia osteodysplastica hereditaria: report of three affected brothers and literature review.Am. J. Med. Genet. 1979; 3: 389-395Crossref PubMed Scopus (16) Google Scholar, 45Steiner C.E. Cintra M.L. Marques-de-Faria A.P. Cutis laxa with growth and developmental delay, wrinkly skin syndrome and gerodermia osteodysplastica: A report of two unrelated patients and a literature review.Genet. Mol. Biol. 2005; 28: 181-190Crossref Scopus (9) Google Scholar, 46Mensing H. Krieg T. Meigel W. Braun-Falco O. [Cutis laxa. Classification, clinical aspects and molecular defects.].Hautarzt. 1984; 35: 506-511PubMed Google Scholar Open table in a new tab Shown are (A) hands (11156), (B) face (D05-09864), and (C) feet (11156) of case individuals aged 12, 11, and 12 years, respectively, at the time of photography. Note the appearance of premature aging, particularly wrinkled skin and joint laxity. Clinical features of Acadian ARCL2 patients (last 5 columns) versus literature review of other descriptions of cutis laxa type 2 with developmental delay, wrinkly skin syndrome (WSS), gerodermia osteodysplastica (GO), and neurocutaneous syndrome (NC).13Baumgartner M.R. Hu C.A. Almashanu S. Steel G. Obie C. Aral B. Rabier D. Kamoun P. Saudubray J.M. Valle D. Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding delta(1)-pyrroline-5-carboxylate synthase.Hum. Mol. Genet. 2000; 9: 2853-2858Crossref PubMed Google Scholar, 14Baumgartner M.R. Rabier D. Nassogne M.C. Dufier J.L. Padovani J.P. Kamoun P. Valle D. Saudubray J.M. Delta1-pyrroline-5-carboxylate synthase deficiency: neurodegeneration, cataracts and connective tissue manifestations combined with hyperammonaemia and reduced ornithine, citrulline, arginine and proline.Eur. J. Pediatr. 2005; 164: 31-36Crossref PubMed Scopus (67) Google Scholar, 15Bicknell L.S. Pitt J. Aftimos S. Ramadas R. Maw M.A. Robertson S.P. A missense mutation in ALDH18A1, encoding Delta1-pyrroline-5-carboxylate synthase (P5CS), causes an autosomal recessive neurocutaneous syndrome.Eur. J. Hum. Genet. 2008; 16: 1176-1186Crossref PubMed Scopus (65) Google Scholar, 21Gupta N. Phadke S.R. Cutis laxa type II and wrinkly skin syndrome: distinct phenotypes.Pediatr. Dermatol. 2006; 23: 225-230Crossref PubMed Scopus (15) Google Scholar, 22Nanda A. Alsaleh Q.A. Al-Sabah H. Marzouk E.E. Salam A.M. Nanda M. Anim J.T. Gerodermia osteodysplastica/wrinkly skin syndrome: report of three patients and brief review of the literature.Pediatr. Dermatol. 2008; 25: 66-71Crossref PubMed Scopus (17) Google Scholar, 44Lisker R. Hernandez A. Martinez-Lavin M. Mutchinick O. Armas C. Reyes P. Robles-Gil J. Gerodermia osteodysplastica hereditaria: report of three affected brothers and literature review.Am. J. Med. Genet. 1979; 3: 389-395Crossref PubMed Scopus (16) Google Scholar, 45Steiner C.E. Cintra M.L. Marques-de-Faria A.P. Cutis laxa with growth and developmental delay, wrinkly skin syndrome and gerodermia osteodysplastica: A report of two unrelated patients and a literature review.Genet. Mol. Biol. 2005; 28: 181-190Crossref Scopus (9) Google Scholar, 46Mensing H. Krieg T. Meigel W. Braun-Falco O. [Cutis laxa. Classification, clinical aspects and molecular defects.].Hautarzt. 1984; 35: 506-511PubMed Google Scholar Together, the clinical findings most resemble ARCL2 as described in the literature. In addition to sharing ethnic heritage, the two families reside near each other, consistent with the possibility of a founder effect. To test this, we genotyped a total of 13 DNA samples, including four of the five affected patients (sampled from both families) and nine unaffected relatives from the two pedigrees, using a high-density SNP genotyping array containing approximately 330,000 biallelic markers. Whole-genome SNP scanning was performed at McGill University and the Genome Quebec Centre for Innovation with the use of the Illumina HumanHap300v2_A panel. Homozygosity was assessed by computational inspection for long runs of consecutive homozygous SNPs identical by state in the four Maritime Canadian affected patients, followed by visual inspection of the longest runs for informativeness in unaffected family members.This analysis defined one chromosomal region of approximately 1.4 Mbp, near the tip of chromosome 17 (17q25.3), where all four genotyped affected patients from both families shared 134 consecutive SNPs homozygous with the same genotypic status (Figure 3, Table 2, Figure S1). The next largest region of sharing comprised only 40 consecutive SNPs and fell within the general distribution of miscellaneous short homozygous segments. Formal linkage analysis supported the chromosome 17 locus, although the two-point LOD scores were less than 2 for all hits in the genome (data not shown) as a result of the poorly informative pedigree structure. The region thus defined on chromosome 17 includes all or part of 50 RefSeq annotated genes.Table 2Homozygosity Analysis of CL2nSNPsChrBeginSNPEndSNPBeginEndSize13417rs9894429rs650204377,207,21678,634,3661,427,151401rs4652869rs1207484835,151,52136,357,6641,206,1443320rs6088813rs726353633,438,59534,110,871672,277292rs1455653rs11692344198,186,565198,504,108317,544294rs6531772rs684933933,432,80534,032,949600,145287rs4646450rs696748799,104,25499,440,127335,8742810rs2247247rs71568784,373,25784,561,933188,6772814rs11626364rs210975073,319,56373,741,009421,4472818rs17240415rs143141964,801,86864,889,90988,042255rs4267850rs929375777,683,52077,959,776276,257The ten longest runs of homozygous SNPs shared identically by state (IBS) in the four genotyped affected CL2 patients are shown in decreasing order of number of consistent contiguous SNPs. Headings indicate number of SNPs, chromosome, starting and ending SNPs (meaning first inconsistent heterozygous or non-IBS SNP at each end of each interval, yielding maximal interval extent), and beginning and ending nucleotides of each interval, according to human genome assembly hg36, based on first and last inconsistent SNPs. Interval size is given in megabase pairs (Mbp). Open table in a new tab The ten longest runs of homozygous SNPs shared identically by state (IBS) in the four genotyped affected CL2 patients are shown in decreasing order of number of consistent contiguous SNPs. Headings indicate number of SNPs, chromosome, starting and ending SNPs (meaning first inconsistent heterozygous or non-IBS SNP at each end of each interval, yielding maximal interval extent), and beginning and ending nucleotides of each interval, according to human genome assembly hg36, based on first and last inconsistent SNPs. Interval size is given in megabase pairs (Mbp). For mutation detection, annotated coding exons were amplified from genomic patient DNA by PCR via standard methods and sequenced at McGill University and the Genome Quebec Centre for Innovation, or at Dalhousie University with Sanger fluorescent sequencing and capillary electrophoresis (Table S1). Sequence traces were analyzed with MutationSurveyor (Soft Genetics). One obvious gene candidate, nuclear prelamin A recognition factor (NARF), was screened first because of the association of lamin A with other progeroid genetic disorders. However, NARF did not contain any obvious causal mutations. In total, we sequenced most or all of the coding regions of 11 genes in the interval (NARF [MIM 705349], HGS [MIM 604375], HEXDC, PYCR1 [MIM 179035], RAB40B, ARHGDIA [MIM 601925], DUS1L, MRPL12 [MIM 602375], NPLOC4, RAC3 [MIM 602050], and TBCD [MIM 604649]), until a potentially pathogenic variant, c.797G>A (p.Arg266Gln), was found in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). This was a single-nucleotide change, altering the last amino acid encoded by exon 6 of the gene, R266, and also potentially altering the splice donor site (Figure 4A). The mutation segregated in the family as expected on the basis of SNP genotyping: all four affected individuals were homozygous, all obligatory carriers were heterozygous, and some unaffected siblings were heterozygous for the mutation (data not shown) The mutation was not in dbSNP and was not found among 96 CEPH control DNAs. Among 142 Maritime population control samples, one sample was heterozygous for the mutation; there were no homozygotes. Our local control samples are known to include individuals of Acadian ethnicity, as in our ARCL2 patient families; therefore, it is not unlikely that a heterozygote for a recessively acting causal mutation might be observed among these controls. Our results are consistent with a founder effect of this mutation in the Acadian population; no other identified cases of ARCL2 from the region could be identified through intensive outreach among regional clinicians. The potential pathogenic effect of the mutation was first studied bioinformatically. Residue R266 is completely conserved across vertebrate evolution (Figure S2). The missense mutation R266Q was predicted to have a deleterious effect on protein function by SIFT, PANTHER, and Align-GVGD, but was predicted as benign by PolyPhen (Table S2). The results suggest that the missense mutation R266Q might affect protein function. The potential impact of the mutation on splicing was also analyzed with NetGene2, SplicePort, and SplicePredictor. Genomic sequence NC_000017.9 was used as the input wild-type DNA sequence, and the DNA sequence with one base change, g.2767G>A, was used as the input mutant sequence. The exon 6 splice donor site was correctly predicted in the wild-type sequence by all three programs. The three methods also correctly identified other donor sites of the reference sequence. In the mutant sequence, however, the exon 6 donor site could no longer be identified by any of the three programs. This suggested that the mutation might also interfere with correct splicing of the primary PCYR1 transcript. We tested this directly by examining RNA extracted from patients' blood lymphocytes. We were able to amplify spliced products of PYCR1 by nested RT-PCR from total RNA of blood lymphocytes (although not from salivary RNA). For sequencing of cDNA generated from lymphocyte RNA, buffy coats were prepared from fresh blood draws and approximately 5 × 105cells were extracted with the use of the QIAGEN RNeasy kit, in accordance with standard protocol for cells. Two microliters of total RNA was used as template for two rounds of nested RT-PCR with the Invitrogen M-MLV kit, including the use of Invitrogen RNaseOUT. An unrelated control sample generated the expected size product for mRNA, correctly splicing exons 5, 6, and 7 (Figures 4B and 4C). However, in two homozygous mutation-bearing individuals from our pedigree, a smaller size band was uniquely detected, which upon sequencing represented an aberrant splice product of exon 5 to exon 7, skipping exon 6 (Figures 4B and 4D, Figure S3). Heterozygous mutation carriers contained a mixture of RNA with normal and skipped products (Figures 4B and 4C). Skipping of exon 6 deletes 54 amino acids of the PYCR1 protein, including the conserved reductase functional signature (Figure 5), and also generates an obligatory frameshift in the downstream exon(s), leading to premature termination of the open reading frame, and is reasonably presumed to be pathogenic. The nested RT-PCR protocol is qualitative, although signal strengths were comparable for the correctly spliced and skipped RNA-derived bands in the control and homozygous affected lymphocyte samples treated similarly. In the heterozygous carriers, the skipped RNA signal was weaker than that of the correctly spliced band in the same samples and gel lanes, suggesting some amount of nonsense-mediated decay or other destabilization of the incorrectly spliced product. However, the skipped product was readily detectable in both homozygous affected individuals and heterozygous carriers, so such an effect, if present, appears unrelated to the clinical pene" @default.
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