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Purpose: Four patients with Saul-Wilson syndrome were reported between 1982 and 1994, but no additional individuals were described until 2018, when the molecular etiology of the disease was elucidated. Hence, the clinical phenotype of the disease remains poorly defined. We address this shortcoming by providing a detailed characterization of its phenotype., Methods: Retrospective chart reviews were performed and primary radiographs assessed for all 14 individuals. Four individuals underwent detailed ophthalmologic examination by the same physician. Two individuals underwent gynecologic evaluation. Z-scores for height, weight, head circumference and body mass index were calculated at different ages., Results: All patients exhibited short stature, with sharp decline from the mean within the first months of life, and a final height Z-score between -4 and -8.5 standard deviations. The facial and radiographic features evolved over time. Intermittent neutropenia was frequently observed. Novel findings included elevation of liver transaminases, skeletal fragility, rod-cone dystrophy, and cystic macular changes., Conclusions: Saul-Wilson syndrome presents a remarkably uniform phenotype, and the comprehensive description of our cohort allows for improved understanding of the long-term morbidity of the condition, establishment of follow-up recommendations for affected individuals, and documentation of the natural history into adulthood for comparison with treated patients, when therapeutics become available.

Short telomeres are frequently identified in patients with idiopathic pulmonary fibrosis (IPF) and its inherited form, familial interstitial pneumonia (FIP). We identified a kindred with FIP with short telomeres who did not carry a mutation in known FIP genes TERT or hTR . We performed targeted sequencing of other telomere-related genes to identify the genetic basis of FIP in this kindred. The proband was a 69 year-old man with dyspnea, restrictive pulmonary function test results, and reticular changes on high-resolution CT scan. An older male sibling had died from IPF. The proband had markedly shortened telomeres in peripheral blood and undetectably short telomeres in alveolar epithelial cells. Polymerase chain reaction-based sequencing of NOP10 , TINF2 , NHP2 , and DKC1 revealed that both affected siblings shared a novel A to G 1213 transition in DKC1 near the hTR binding domain that is predicted to encode a Thr405Ala amino acid substitution. hTR levels were decreased out of proportion to DKC1 expression in the T405A DKC1 proband, suggesting this mutation destabilizes hTR and impairs telomerase function. This DKC1 variant represents the third telomere-related gene identified as a genetic cause of FIP. Further investigation into the mechanism by which dyskerin contributes to the development of lung fibrosis is warranted.

A man with usual interstitial pneumonia (age of onset 58 years) was previously found to have an Ile73Thr (I73T) surfactant protein C (SFTPC) mutation. Genomic DNA from the individual and two daughters (aged 39 and 43 years) was sequenced for the I73T mutation and variations in ATP-binding cassette A3 (ABCA3). All three had the I73T SFTPC mutation. The father and one daughter (aged 39 years) also had a transversion encoding an Asp123Asn (D123N) substitution in ABCA3. The daughters were evaluated by pulmonary function testing and high-resolution CT (HRCT). Neither daughter had evidence of disease, except for focal subpleural septal thickening on HRCT scan in one daughter (aged 39 years). This daughter underwent bronchoscopy with transbronchial biopsies revealing interstitial fibrotic remodeling. These findings demonstrate that subclinical fibrotic changes may be present in family members of patients with SFTPC mutation-associated interstitial lung disease and suggest that ABCA3 variants could affect disease pathogenesis.

Purpose: We hypothesized that functional TGFbeta1 SNPs increase TGFbeta/BMP signaling imbalance in BMPR2 mutation heterozygotes to accelerate the age at diagnosis, increase the penetrance and SMAD2 expression in familial pulmonary arterial hypertension., Methods: Single nucleotide polymorphism genotypes of BMPR2 mutation heterozygotes, age at diagnosis, and penetrance of familial pulmonary arterial hypertension were compared and SMAD2 expression was studied in lung sections., Results: BMPR2 mutation heterozygotes with least active -509 or codon 10 TGFbeta1 SNPs had later mean age at diagnosis of familial pulmonary arterial hypertension (39.5 and 43.2 years) than those with more active genotypes (31.6 and 33.1 years, P = 0.03 and 0.02, respectively). Kaplan-Meier analysis also showed that those with the less active single nucleotide polymorphisms had later age at diagnosis. BMPR2 mutation heterozygotes with nonsense-mediated decay resistant BMPR2 mutations and the least, intermediate and most active -509 TGFbeta1 SNP genotypes had penetrances of 33, 72, and 80%, respectively (P = 0.003), whereas those with 0-1, 2, or 3-4 active single nucleotide polymorphism alleles had penetrances of 33, 72, and 75% (P = 0.005). The relative expression of TGFbeta1 dependent SMAD2 was increased in lung sections of those with familial pulmonary arterial hypertension compared with controls., Conclusions: The TGFbeta1 SNPs studied modulate age at diagnosis and penetrance of familial pulmonary arterial hypertension in BMPR2 mutation heterozygotes, likely by affecting TGFbeta/BMP signaling imbalance. This modulation is an example of Synergistic Heterozygosity.

Objective: To characterize the cause of a sporadic isolated growth hormone deficiency in a single patient., Methods: Genomic DNA was extracted from blood samples of the patient and his family. Exons and exon-intron junctions of the GH-1 gene were amplified by PCR and sequenced. To characterize possible GH-1 deletions we performed Southern blot analysis and PCR-restriction fragment length analyses., Results: An adenine to guanine mutation at the first nucleotide of the initiation codon (Met [ATG](-26)Val [GTG]) of the GH-1 gene was identified in the patient and the mother. A 7.6kb GH-1 deletion was identified in the patient, the brother and the father., Conclusion: The patient was a compound heterozygote for an allele bearing a Met(-26)Val missense mutation inherited from his mother and an allele containing deletion of the entire GH-1 gene inherited from his father. The present missense mutation has not been described previously. Attention should be paid to the heterozygous gene deletion that is difficult to detect by PCR-based genetic analysis. The patient responded to GH replacement therapy fairly well, without developing anti-hGH antibody.

Purpose: Approximately 50% of patients with familial primary pulmonary hypertension (FPPH) have been reported to have mutations within the bone morphogenic protein receptor type 2 (BMPR2) gene. The vast majority of these mutations were identified by PCR amplification and sequencing of individual exons. The aim of our study was to determine if additional BMPR2 mutations not found by exon sequencing alone could account for a significant portion of these negative cases., Methods: We examined DNA samples from 12 families, previously found to be negative for BMPR2 mutations, to identify any large BMPR2 gene rearrangements., Results: Southern blot analysis found large gene rearrangements in four (33%) unrelated kindreds. Further analysis by reverse transcriptase PCR (RT-PCR) of BMPR2 transcripts from two of these kindreds found one to be heterozygous for a exon 10 duplication and the second to be heterozygous for a deletion of exons 4 to 5. Nonhomologous recombination is believed to be the cause of these large insertions/deletions., Conclusion: Our results demonstrate the inherent problems associated with exon-by-exon sequencing and the importance of other screening methods such as Southern blot and RT-PCR in the identification of BMPR2 mutations.

Purpose: Infantile glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by deficiency of acid alpha-glucosidase (GAA). The purpose of this study was to investigate the safety and efficacy of recombinant human GAA (rhGAA) enzyme therapy for this fatal disorder., Methods: The study was designed as a phase I/II, open-label, single-dose study of rhGAA infused intravenously twice weekly in three infants with infantile GSD-II. rhGAA used in this study was purified from genetically engineered Chinese hamster ovary (CHO) cells overproducing GAA. Adverse effects and efficacy of rhGAA upon cardiac, pulmonary, neurologic, and motor functions were evaluated during 1 year of the trial period. The primary end point assessed was heart failure-free survival at 1 year of age. This was based on historical control data that virtually all patients died of cardiac failure by 1 year of age., Results: The results of more than 250 infusions showed that rhGAA was generally well tolerated. Steady decreases in heart size and maintenance of normal cardiac function for more than 1 year were observed in all three infants. These infants have well passed the critical age of 1 year (currently 16, 18, and 22 months old) and continue to have normal cardiac function. Improvements of skeletal muscle functions were also noted; one patient showed marked improvement and currently has normal muscle tone and strength as well as normal neurologic and Denver developmental evaluations. Muscle biopsies confirmed that dramatic reductions in glycogen accumulation had occurred after rhGAA treatment in this patient., Conclusions: This phase I/II first study of recombinant human GAA derived from CHO cells showed that rhGAA is capable of improving cardiac and skeletal muscle functions in infantile GSD-II patients. Further study will be needed to assess the overall potential of this therapy.

A G - C transversion at the fifth nucleotide of intron 3 of GH-I gene was identified in a sporadic case of isolated growth hormone deficiency (IGHD). The mutation was absent in both of the parents, indicating that the mutation occurred de novo. An abnormal hGH mRNA lacking a region encoded by exon 3 was spliced when the mutant GH-I gene was expressed in cultured cells. Since skipping of exon 3 is a common feature for four different mutant GH-I genes identified in patients with autosomal dominantly inherited IGHD, we conclude that the mutation causes IGHD in this case., (Copyright 1999 Harcourt Publishers Ltd.)

Cystinuria is a common inherited aminoaciduria that leads to recurrent cystine nephrolithiasis. Mutations in a gene encoding a renal amino acid transporter (SLC3A1) have been identified in patients with cystinuria establishing one molecular cause for the disease. To facilitate systematic screening of this gene for mutations, we have delineated the complete genomic organization of the SLC3A1 coding region using polymerase chain reaction strategies. The complete coding region of the gene is contained within a single yeast artificial chromosome clone and consists of 10 exons and 9 introns. Oligonucleotide primers capable of amplifying selected exons have been made and used in mutational analysis of DNA from 24 cystinuria probands. We illustrate the usefulness of this approach by identifying two novel SLC3A1 mutations. One novel mutation causes replacement of a highly conserved arginine residue (arginine-452) with tryptophan in the cytoplasmic loop between the putative third and fourth membrane spanning segments. A second previously unreported mutation results in replacement of a highly conserved tyrosine (tyrosine-461) residue with histidine in the same region of the protein. In addition, we detected three previously reported SLC3A1 mutations, R270X, 1500 +1/G to T, and M467T, the latter being present in approximately 20% of cystinuria chromosomes examined. Our findings provide a foundation for the development of more accessible diagnostic screening assays for detecting SLC3A1 mutations using patient genomic DNA, and also contribute to the emerging spectrum of cystinuria genotypes.

Since the renin angiotensin system (RAS) is established as an important factor in renal disease progression, we determined whether RAS alleles that have been linked to variability in outcome in several cardiovascular diseases also affect progression of IgA nephropathy. These genetic variants include: (1) angiotensin I converting enzyme deletion polymorphism in intron 16 (ACE I/D), reported to be associated with increased risk of myocardial infarction as well as left ventricular hypertrophy; (2) a point mutation in the angiotensinogen (Agt) gene resulting in a methionine to threonine substitution at residue 235 (M235T), reported to be associated with hypertension in Caucasians; and (3) an angiotensin receptor type I (ATR) A to C transition at bp 1166 (A1166C) which shows synergy with the deleterious effects of the ACE DD genotype in myocardial infarction. We examined these polymorphisms by PCR amplification of genomic DNA samples from 64 Caucasian patients in the USA (age 6 to 83 years) with biopsy-proven IgA nephropathy whose renal status was followed for an average of almost seven years. Patients who presented with and maintained normal serum creatinine (Cr, < 1.5 mg/dl), had ACE genotype frequencies of II:35%, ID:61%, DD:4%. By contrast, in patients with progression (initially normal Cr increased to a mean of 4.5 +/- 0.86 mg/dl), ACE genotype frequencies were II:22%, ID:44%, DD:33% (P = 0.057 by Fishers's exact test, vs. non-progressors). The association of the DD genotype with progression was even more striking when patients with other risk factors (hypertension and/or heavy proteinuria) were excluded. In this subgroup, the genotype frequencies in patients with stable creatinine versus those with deterioration in renal function was 53%, 47%, and 0% versus 0%, 40%, and 60%, respectively, for II, ID, and DD genotypes (P = 0.009 by Fisher's exact test, progressors vs. non-progressors). Further, sequence analysis of the I gene polymorphism revealed a potential 13 bp silence motif. Neither the Agt 235T nor the ATR A 1166C gene variants, however, was associated with deterioration of renal function. Taken together, these results indicate that, although polymorphism in each of the three genes in the RAS system has been linked to cardiovascular diseases, only the ACE I/D polymorphism is associated with progressive deterioration in renal function in IgA nephropathy. Since previous observations link ACE polymorphism with ACE activity, these findings imply a widespread importance of ACE in modulating destructive processes in different organs.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Administration of a selective angiotensin I type 1 receptor (AT1) antagonist in animals not only nullifies the vasopressor action of angiotensin II, but also induces chloriduria and kaliuria, juxtoglomerular apparatus (JGA) hypertrophy and hyperreninemia, features characteristic of human Bartter's syndrome. We, therefore, explored the possibility that Bartter's syndrome may involve an AT1 abnormality. Using a pair of AT1-specific oligonucleotide primers and two different DNA polymerases (Taq and Pfu), we amplified the approximately 1 kb AT1 coding region of genomic DNA isolated from leukocytes of five patients with Bartter's syndrome by PCR and analyzed the sequence of the product. While the sequence of all clones from four patients were identical to that already reported for the normal human AT1 DNA sequence, 50% of the clones from one patient with Bartter's syndrome were found to have A-->G transition at nucleotide 931 which causes an amino acid substitution (arg-->gly) on the carboxy-terminal cytosolic tail of AT1. This mutation was not found in DNA from 50 normal controls which were screened by restriction enzyme digestion pattern of the PCR products of this region. As PCR-amplified AT1 DNA clones from four other individuals with Bartter's syndrome did not display any abnormality in the coding region, the possibility exists that Bartter's syndrome consists of multiple disease entities, where an AT1 gene abnormality represents a specific subgroup of the syndrome and/or some abnormality includes mutations outside of the coding region.

Molecular biology techniques have begun to shed light on the genetic basis of autosomal dominant central DI, but several very basic questions remain to be answered. The disorder was initially presumed to have a developmental, degenerative, or autoimmune basis based on the autopsy findings in the hypothalamus of a limited number of patients. The molecular cloning of the AVP-NP II gene and the clue from the Brattleboro rat that at least this one form of hereditary DI involved an AVP-NP II gene mutation allowed us to focus on this gene in our study of human hereditary DI. Our initial experiments did not show this gene to have a major structural alteration such as a deletion, insertion, or rearrangement, but the approach was not capable of detecting more suitable defects. The linkage studies provided substantial evidence that one particular OT-NP I haplotype was linked to the disease phenotype in each family, and thus, a mutation in the AVP/OT region of chromosome 20 is responsible for this disease. Ito et al. (1991) then identified a single base change in the AVP-NP II gene in affected members of one Japanese family. This change was not detected in unrelated, unaffected persons and thus is a good candidate for the mutation causing the disease in this family. However, there appears to be diversity in the molecular basis of autosomal dominant central DI as affected members of one of our families did not have this particular base change in either AVP-NP II allele and recently another distinct AVP-NP II gene base change has been associated with this disorder. One interesting question still to be addressed is how a mutation in the NP-II coding region of this gene prevents AVP release from the posterior pituitary in the rat or the human disease. Does the disrupted AVP-NP II coding sequence prevent normal processing of the mRNA so that it can not be properly translated into protein? Does the mutated AVP-NP II glycoprotein precursor protein interfere with normal post-translational processing to prevent release of AVP? Is an altered NP II protein not able to protect the AVP from proteolysis within the magnocellular neuron? An even more puzzling question is how a mutation in the gene encoding a hormone is inherited in an autosomal dominant pattern. The Brattleboro rat model follows the a priori expectation of autosomal recessive inheritance: the animal only exhibits a defect in hormone function if both genes encoding the hormone are defective.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecular defect responsible for moderate and severe hemophilia A has been identified for two unrelated patients with the CRM-positive form of this disorder (factor VIII activity of 0.02 and 0.05 U/mL with factor VIII antigen of 0.87 and 2.20 U/mL). In both cases, the immunopurified dysfunctional factor VIII protein is abnormal, in that the 80 Kd light chain is not cleaved by thrombin at arginine-1689. The basis for this failure was identified by polymerase chain reaction amplification of exon 14 of the variant factor VIII genes and direct sequencing of the amplified products. In both cases, a single base substitution (C to T) was identified that produces an arginine to cysteine substitution at amino acid residue 1689. These data identify the molecular defects of the two identical factor VIII variant proteins. The dysfunctional factor VIII has been designated "Factor VIII-East Hartford," the residence of the patient in whom the defect was first identified.

We have applied gene counting and restriction endonuclease mapping techniques to the study of two American black families in which there were one or more cases of HbH disease. We found deletions of three of the four normal alpha-globin genes in individuals with HbH disease. In two of these individuals, the chromosome containing the single alpha gene could have originated by crossing over between mispaired alpha genes, resulting in a deletion of about 4.2 kilobases (kb).

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.

The alpha-globin genes of five black Americans, two Chinese, and five Filipinos with HbH disease (an alpha-thalassemia state in which there is a single functional alpha gene) were analyzed by restriction endonuclease techniques. All subjects were found to have one chromosome 16, lacking both alpha genes, and another containing a single alpha gene (--/-alpha). Restriction endonuclease patterns of the DNA obtained from all 12 subjects were identical and compatible with unequal crossing-over as the mechanism of origin of the single alpha gene in these individuals.

A DNA polymorphism for an Xbal site in intron 22 of the human factor VIII:C gene extends the utility of DNA methods for carrier detection in families segregating for hemophilia A. While the DNA polymorphism detected by a BclI site in intron 18 of the factor VIII:C gene was informative for 41% of females studied, the BglI/intron 25 polymorphism provided no additional information because of apparent linkage disequilibrium. In contrast, the Xbal intron 22 polymorphism was useful in 53% of women who were uninformative (homozygous) for either the BclI or BglI polymorphisms. Using the BclI/intron 18 and Xbal/intron 22 intragenic polymorphisms, we could provide highly accurate information for 68% of women we studied who were at risk for carriership. The carrier status of the remaining 32% could be determined utilizing the closely linked Taql/St14 DNA polymorphism.