Separation of SNP profiles from DNA mixtures with two contributors via massively parallel sequencing technology

ABSTRACT

Massively parallel sequencing technology offers the opportunity to analyse forensically challenging samples, such as degraded samples and mixtures. In the current study, we developed a perl-based pipeline to separate the DNA mixture into its components and to predict the most probable single nucleotide polymorphism (SNP) genotypes of each contributor to the mixed profile. We examined the usefulness of this method by detecting both artificially constructed DNA mixtures and mixtures from crime cases using the Precision ID Identity Panel on the Ion PGM platform. The separated genotypes of mixtures were validated by genotypes of each of the donors detected independently. The results indicate that the method performed well in identifications of both the artificially constructed mixtures and case-type mixtures, even when the two contributors are immediate relatives (mother and son), which demonstrated the practical usefulness of this method in forensic casework. Our research presents an efficient and different strategy for identification and paternity testing of DNA mixtures in forensic genetics.     

Forensic application of a novel MPS-based panel (90 STRs and 100 SNPs) in a non-exclusion parentage case with three autosomal STRs incompatibilities

In kinship tests, the investigating of the forensic STRs usually provides decisive information to resolve relationship cases. We describe a parentage case with 3 genetic incompatibilities (D6S1043, D18S51 and D2S1338) between the child and alleged parent. With 90 STR loci and 100 SNP loci, the massively parallel sequencing (MPS)-based genotyping results support the certainty of parentage, and the mismatched alleles were considered to be mutations. MPS can provide additional allele sequence structures that can be used to infer the origins of the mutations. SNPs as supplementary markers can provide effective information to give an unequivocal statement of the parentage.     

Caprine mucopolysaccharidosis IIID

Mucopolysaccharidosis IIID (MPS IIID) is a lysosomal storage disease associated with deficient activity of the enzyme N-acetylglucosamine 6-sulfatase (EC 3.1.6.14), a lysosomal hydrolase in the heparan sulfate glycosaminoglycan (HS-GAG) degradation pathway. In caprine MPS IIID, enzyme replacement therapy reversed early postnatal systemic but not primary or secondary central nervous system (CNS) substrate accumulations. The caprine MPS IIID large animal model system was used in this investigation to define the developmental profile of morphological and biochemical perturbations to estimate a time frame for therapeutic intervention. Light and electron microscopy were used to compare the CNS, liver, and kidney of normal +/+, MPS IIID carrier +/-, and MPS IIID-affected -/- goat kids (kids), at 60, 113–114, 128–129, and 135 d gestation (dg) of a 150-d gestational period, at birth, and at 59–64 d of postnatal (d-pn) age. In the CNS of -/- kids, morphological correlations of HS-GAG and glycolipid accumulations were evident in early differentiating neurons at 60 dg. CNS and systemic developmental, regional, and cellular differences in -/-kids at all time points included more prominent and earlier accumulation of lucent, putative HS-GAG substrates in lysosomes of meningeal and perivascular macrophages and hepatic sinusoidal cells than in CNS, hepatic, or renal parenchymal cells. The amounts and compositions of HS-GAG substrates in the brain and liver of +/+, +/-, and -/- kids were determined at 60, 65, 113–114, and 128–135 dg, at birth, and 53–78 d-pn. In the CNS of -/- kids, HS-GAG concentrations were variable and exceeded those of age-matched control tissue samples in the third but not the second trimester. In contrast, hepatic HS-GAG levels in -/- kids exceeded control values at all time points evaluated and paralleled the progressive morphological alterations. CNS and hepatic HS-GAG compositions in -/- kids were similar to each other and were more complex at all pre- and postnatal ages than those from control kids. Based on the time frame of development of CNS lesions and biochemical perturbations, prenatal therapeutic intervention in caprine MPS IIID is likely to be necessary to prevent or ameliorate substantive CNS and systemic lesions.     

Radiological findings in mild forms of Morquio's disease (MPS 1V A)

Clinical and radiological features of seven patients with Morquio's disease Type A (MPS 1V A) are presented. The diagnosis was established by two-dimensional electrophoresis of the urinary glycosaminoglycans and enzyme assay. The clinical manifestations were mild or moderate in all patients and the radiological changes were milder than those described in classical Morquio's disease. Offprint requests to: P.S. Thomas     

Sanfilippo B syndrome: molecular defects in Greek patients

Sanfilippo syndrome type B [mucopolysaccharidosis IIIB (MPS IIIB] is the most prevalent type of MPS III in Greece, accounting for 81% of all MPS III cases diagnosed at the Institute of Child Health (Athens) over the last 20 years. The majority of the patients originated from East Central/Central Greece, Thessaly, and Macedonia. We present the results of mutation analysis in 21 Greek patients from 18 different families, all of whom had the severe form of the disorder. Patients were initially screened for five previously known mutations by restriction enzyme digestion of polymerase chain reaction products. Unknown mutations were identified by single-strand conformation polymorphism analysis and DNA sequencing and were confirmed by restriction enzyme analysis. Seven previously described mutations (Y140C, R626X, 503-512del, H414R, G292R, 334del25, and E452K) and four novel mutations (P516L, L242P, E446K, and R482Q) were identified. Expression of the latter and H414R showed that they were all null activity mutations. Considerable genetic heterogeneity has been described in MPS IIIB patients of different origins. In our population, Y140C, H414R, and R626X account for approximately 70% of the studied alleles. Our findings, especially in combination with the origin of individual patients, can improve carrier detection and genetic counseling in affected families.     

Enzyme replacement therapy for mucopolysaccharidosis I: a randomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase)

OBJECTIVE: To confirm the efficacy and safety of recombinant human alpha-L-iduronidase (laronidase) in patients with mucopolysaccharidosis I (MPS I). STUDY DESIGN: This was a randomized, double-blinded, multinational study of 45 patients with MPS I administered 100 U/kg (0.58 mg/kg) laronidase, or placebo intravenously weekly for 26 weeks. The coprimary efficacy end points compared the median change from baseline to week 26 between groups in percentage of predicted normal forced vital capacity (FVC) and in 6-minute walk test (6MWT) distance through the use of the Wilcoxon rank sum test. RESULTS: The laronidase (n=22) and placebo (n=23) groups had similar baseline characteristics. After 26 weeks, patients receiving laronidase compared with placebo showed mean improvements of 5.6 percentage points in percent of predicted normal FVC (median, 3.0; P=.009) and 38.1 meters in 6MWT distance (median, 38.5; P=.066; P=.039, analysis of covariance). Laronidase also significantly reduced hepatomegaly and urinary glycosaminoglycans, and, in more severely affected patients, improved sleep apnea/hypopnea and shoulder flexion. Laronidase was well-tolerated. Nearly all patients receiving enzyme had development of IgG antibodies, without apparent clinical effects. CONCLUSIONS: In patients with MPS I, laronidase significantly improves respiratory function and physical capacity, reduces glycosaminoglycan storage, and has a favorable safety profile.     

Clinical findings in 12 patients with MPS IV A (Morquio''s disease)

Clinical heterogeneity in MPS IV A (Mucopolysaccharidosis IV A, Morquio Disease Type A)has become more clearly identified in recent years. The clinical findings in 12 cases of MPS IV A are described. Clinical presentation was variable, and some cases were only mildly affected. All showed deficiency of N-acetylgalactosamine-6-sulphate sulphatase in fibroblasts, but the patient with the mildest clinical presentation showed a high residual enzyme activity. The urinary glycosaminoglycans (GAGs) were examined on all patients by a two-dimensional electrophoresis technique which proved to be highly reliable and efficient. In particular, no false negative results were obtained, a problem often encountered with routine screening methods. These cases support the division of MPS IV A into three subgroups: the severe "classical" type, an intermediate type and a mild type, all caused by N-acetylgalactosamine-6-sulphate sulphatase deficiency. Residual enzyme activity may be an important prognostic indicator.     

Mucopolysaccharidosis IVA within Tunisian patients: Confirmation of the two novel GALNS gene mutations

Mucopolysaccharidosis type IVA or Morquio A disease is an autosomal recessive disease resulting from a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate-sulfatase, which hydrolyses N-acetylgalactosamine-6-sulfate and galactose-6-sulfate in glycosaminoglycans. Phenotypes in Morquio A disease vary from the classical form with severe bone dysplasia, heart valve involvement, corneal opacity, short trunk dwarfism and a life span of 20 to 30 years, to attenuated forms with normal life span, mild bone involvement and mild visceral organ involvement. Unlike the other forms of mucopolysaccharidoses, Morquio A disease is characterized by normal intelligence.