Analysis of Laboratory Repeat Critical Values at a Large Tertiary Teaching Hospital in China

Context

As a patient safety measure, laboratories are required to have a critical values policy by regulatory agencies. Unfortunately, little information is available on repeat critical values for the same analyte(s) on the same patient.

Objective

To investigate the occurrence and distribution of repeat critical values and the relationship between the frequency of such values and patient outcome to provide information for hospitals on improving reporting policies.

Methods

Eleven laboratory critical value lists, including chemistry and hematology analytes, were selected from a tertiary hospital in China in the year 2010. The distribution and interval time for each repeat critical value were calculated. Serum potassium and platelet count were used as examples to illustrate the relationship between the frequency of the repeat critical values and patient outcome.

Results

All test items on the critical value list were prone to the occurrence of repeat critical values. On average, each patient who experienced critical values had 2.10 occurrences. The median interval time for each repeat critical value varied, with most being longer than 8 hours. For those patients who had repeat critical values of serum potassium and platelet count, along with the increased frequency, the patients had a longer hospital stay and a generally worse outcome.

Conclusions

Patient can have a number of repeat critical values and the frequency of these values is closely related to patient outcome. A careful evaluation is warranted if a laboratory chooses to adopt a policy of not reporting each repeat critical value.     

Patients with biallelic GGC repeat expansions in NOTCH2NLC exhibiting a typical neuronal intranuclear inclusion disease phenotype

We report two patients with autosomal dominant neuronal intranuclear inclusion disease (NIID) harboring the biallelic GGC repeat expansion in NOTCH2NLC to uncover the impact of repeat expansion zygosity on the clinical phenotype. The zygosity of the entire NOTCH2NLC GGC repeat expansion and DNA methylation were comprehensively evaluated using fluorescent amplicon length PCR (AL-PCR), Southern blotting and targeted long-read sequencing, and detailed genetic/epigenetic and clinical features were described. In AL-PCR, we could not recognize the wild-type allele in both patients. Targeted long-read sequencing revealed that one patient harbored a homozygous repeat expansion. The other patient harbored compound heterozygous repeat expansions. The GGC repeats and the nearest CpG island were hypomethylated in all expanded alleles in both patients. Both patients harboring the biallelic GGC repeat expansion showed a typical dementia-dominant NIID phenotype. In conclusion, the biallelic GGC repeat expansion in two typical NIID patients indicated that NOTCH2NLC-related diseases could be completely dominant.     

De novo facioscapulohumeral muscular dystrophy: frequent somatic mosaicism, sex-dependent phenotype, and the role of mitotic transchromosomal repeat interaction between chromosomes 4 and 10

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is caused by deletion of most copies of the 3.3-kb subtelomeric D4Z4 repeat array on chromosome 4q. The molecular mechanisms behind the deletion and the high proportion of new mutations have remained elusive. We surveyed 35 de novo FSHD families and found somatic mosaicism in 40% of cases, in either the patient or an asymptomatic parent. Mosaic males were typically affected; mosaic females were more often the unaffected parent of a nonmosaic de novo patient. A genotypic-severity score, composed of the residual repeat size and the degree of somatic mosaicism, yields a consistent relationship with severity and age at onset of disease. Mosaic females had a higher proportion of somatic mosaicism than did mosaic males. The repeat deletion is significantly enhanced by supernumerary homologous repeat arrays. In 10% of normal chromosomes, 4-type repeat arrays are present on chromosome 10. In mosaic individuals, 4-type repeats on chromosome 10 are almost five times more frequent. The reverse configuration, also 10% in normal chromosomes, was not found, indicating that mutations may arise from transchromosomal interaction, to which the increase in 4-type repeat clusters is a predisposing factor. The somatic mosaicism suggests a mainly mitotic origin; mitotic interchromosomal gene conversion or translocation between fully homologous 4-type repeat arrays may be a major mechanism for FSHD mutations.     

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10–40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients. Received: 10 March 1997 / Accepted: 20 May 1997     

Patient mutations in doublecortin define a repeated tubulin-binding domain

Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. Here we show that the two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro and in vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a beta-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif.     

Determination of the origin of nondisjunction in a 49,XXXXY male using hypervariable dinucleotide repeat sequences

Summary We present a patient with a 49,XXXXY chromosome constitution in whom the origin of the extra X chromosomes was determined by analysis of five polymorphic CA (or GT) dinucleotide repeat sequences. This class of DNA marker has recently been demonstrated to be hypervariable with heterozygosity values up to 80%. By polymerase chain reaction (PCR) analysis of the dinucleotide repeat length polymorphisms, we have shown that all four X chromosomes were of maternal origin.     

Analysis of a CGG sequence at the FMR-1 locus in fragile X families and in the general population.

In this study, we have characterized a CGG repeat at the FMR-1 locus in more than 100 families (more than 500 individuals) presenting for fragile X testing and in 247 individuals from the general population. Both Southern blot and PCR-based assays were evaluated for their ability to detect premutations, full mutations, and variability in normal allele sizes. Among the Southern blot assays, the probes Ox1.9 or StB12.3 with a double restriction-enzyme digest were the most sensitive in detecting both small and large amplifications and, in addition, provided information on methylation of an adjacent CpG island. In the PCR-based assays, analysis of PCR products on denaturing DNA sequencing gels allowed the most accurate determination of CGG repeat number up to approximately 130 repeats. A combination of a Southern blot assay with a double digest and the PCR-sequencing-gel assay detected the spectrum of amplification-type mutations at the FMR-1 locus. In the patient population, a CGG repeat of 51 was the largest to be stably inherited, and a repeat of 57 was the smallest size of premutation to be unstably inherited. When premutations were transmitted by females, the size of repeat correlated with risk of expansion to a full mutation in the next generation. Full mutations (large repeats typically associated with an abnormal methylation pattern and mitotic instability) were associated with clinical and cytogenetic manifestations in males but not necessarily in females. In the control population, the CGG repeat ranged from 13 to 61, but 94% of alleles had fewer than 40 repeats. The most frequent allele (34%) was a repeat of 30. One female had an allele (61 repeats) within a range consistent with fragile X premutations, while two other individuals each had a repeat of 52. This suggests that the frequency of unstable alleles in the general population may be approximately 1%.     

SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients,including those with SCA6

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85-399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as alpha(1A)-voltage-dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients.     

GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer.     

Repair of DNA double-strand breaks within the (GAA*TTC)n sequence results in frequent deletion of the triplet-repeat sequence

Friedreich ataxia is caused by an expanded (GAA•TTC)_n sequence, which is unstable during intergenerational transmission and in most patient tissues, where it frequently undergoes large deletions. We investigated the effect of DSB repair on instability of the (GAA•TTC)_n sequence. Linear plasmids were transformed into Escherichia coli so that each colony represented an individual DSB repair event. Repair of a DSB within the repeat resulted in a dramatic increase in deletions compared with circular templates, but DSB repair outside the repeat tract did not affect instability. Repair-mediated deletions were independent of the orientation and length of the repeat, the location of the break within the repeat or the RecA status of the strain. Repair at the center of the repeat resulted in deletion of approximately half of the repeat tract, and repair at an off-center location produced deletions that were equivalent in length to the shorter of the two repeats flanking the DSB. This is consistent with a single-strand annealing mechanism of DSB repair, and implicates erroneous DSB repair as a mechanism for genetic instability of the (GAA•TTC)_n sequence. Our data contrast significantly with DSB repair within (CTG•CAG)_n repeats, indicating that repair-mediated instability is dependent on the sequence of the triplet repeat.     

A variable tandem repeat locus mapped to chromosome band 10q26 is amplified and rearranged in leukocyte DNAs of two cancer patients.

A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation.     

Heart Failure Re-Admission: Measuring the Ever Shortening Gap between Repeat Heart Failure Hospitalizations

Many quality-of-care and risk prediction metrics rely on time to first rehospitalization even though heart failure (HF) patients may undergo several repeat hospitalizations. The aim of this study is to compare repeat hospitalization models. Using a population-based cohort of 40,667 patients, we examined both HF and all cause re-hospitalizations using up to five years of follow-up. Two models were examined: the gap-time model which estimates the adjusted time between hospitalizations and a multistate model which considered patients to be in one of four states; community-dwelling, in hospital for HF, in hospital for any reason, or dead. The transition probabilities and times were then modeled using patient characteristics and number of repeat hospitalizations. We found that during the five years of follow-up roughly half of the patients returned for a subsequent hospitalization for each repeat hospitalization. Additionally, we noted that the unadjusted time between hospitalizations was reduced ∼40% between each successive hospitalization. After adjustment each additional hospitalization was associated with a 28 day (95% CI: 22-35) reduction in time spent out of hospital. A similar pattern was seen when considering the four state model. A large proportion of patients had multiple repeat hospitalizations. Extending the gap between hospitalizations should be an important goal of treatment evaluation.     

Base Excision Repair of Chemotherapeutically-Induced Alkylated DNA Damage Predominantly Causes Contractions of Expanded GAA Repeats Associated with Friedreich's Ataxia

Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich''s ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)₂₀ repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5''-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5''-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.     

Linking SNPs to CAG repeat length in Huntington's disease patients

Allele-specific silencing using small interfering RNAs targeting heterozygous single-nucleotide polymorphisms (SNPs) is a promising therapy for human trinucleotide repeat diseases such as Huntington's disease. Linking SNP identities to the two HTT alleles, normal and disease-causing, is a prerequisite for allele-specific RNA interference. Here we describe a method, SNP linkage by circularization (SLiC), to identify linkage between CAG repeat length and nucleotide identity of heterozygous SNPs using Huntington's disease patient peripheral blood samples.     

ECHOCARDIOGRAPHIC ABNORMALITIES OF THE MITRAL VALVE ASSOCIATED WITH A LARGE PERICARDIAL EFFUSION

Features of mitral stenosis and systolic anterior motion of the mitral valve were observed on the echocardiogram of a patient with a large posterior pericardial effusion. The abnormal findings disappeared with resolution of the fluid. Because pericardial effusion may result in the echocardiographic alteration of cardiac structures, repeat study is necessary after the effusion has cleared.     

Genetic confirmation of facioscapulohumeral muscular dystrophy in a case with complex D4Z4 rearrangments

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat on chromosome 4q. Genetic confirmation of the clinical diagnosis of FSHD is complicated by the presence of a homologous repeat on chromosome 10q and the frequent repeat exchanges between both chromosomes. Here, we describe the genetic evaluation of an FSHD patient with a complex D4Z4 allele constitution in which the potentially pathogenic allele seemingly resides on chromosome 10, despite FSHD being exclusively linked to chromosome 4. Complementary allele typing and segregation analysis confirmed the clinical diagnosis of FSHD by revealing the chromosome 4 origin of the pathogenic allele in the presence of two exchanged repeat arrays, one on chromosome 4 and one on chromosome 10, an allele constitution that cannot be identified by conventional DNA diagnosis.