Computed Tomography-Measured Psoas Muscle Density as a Predictive Factor for Hypophosphatemia Associated With Refeeding.

BACKGROUND: Initiation of parenteral nutrition (PN) after a period of starvation can be complicated by refeeding syndrome (RFS). RFS is associated with electrolyte abnormalities including hypomagnesemia, hyponatremia, and hypophosphatemia. Risk factors include recent weight loss, low body mass index, and electrolyte deficiencies; however, these associations are not strong. We hypothesized that a validated measure of nutrition risk, computed tomography (CT)-measured psoas muscle density, can be used to predict the development of hypophosphatemia associated with RFS. METHODS: A retrospective analysis of surgical patients initiated on PN with an abdominal CT scan within the past 3 months was conducted. CT-measured psoas muscle density was assessed as a predictive variable for the development of electrolyte abnormalities. Daily electrolyte and clinical outcome measures were recorded. RESULTS: One hundred nine patients were stratified based on Hounsfield unit average calculation (HUAC). The lowest 25th percentile of patients had HUAC <25. Low HUAC was associated with a significant percent decrease in phosphate levels from baseline to PN day 3 (P < .01) and significant difference in serum phosphate value on PN day 3 (P < .01). The low muscle density quartile also experienced longer days on the mechanical ventilator (P = .01) compared with patients with a higher psoas muscle density. CONCLUSION: Psoas muscle density predicted the development of hypophosphatemia in patients initiated on PN. This measurement may aid in identifying patients at highest risk of experiencing RFS. A mean psoas HU <25 may prompt additional precautions, including additional phosphate replacement and slower initiation of PN.

The "Negative" Impact of a Subgaleal Drain: Post-cranioplasty Negative Pressure Subgaleal Drain-induced Ascending Transtentorial Herniation.

One commonly practiced procedural step to reduce the risk of postoperative hematoma accumulation when performing cranioplasties is to place a closed negative-pressure subgaleal drain. We present a patient with sinking skin flap syndrome that underwent such a procedure and subsequently experienced immediate postoperative ascending transtentorial herniation and intracranial hemorrhage remote from the surgical site. On determining that the subgaleal drain was the responsible cause, it was immediate removed, and the patient had neurological recovery. Fewer than 30 cases of life-threatening subgaleal drain-related complications have been documented, and this is the first reported case of ascending herniation occurring after cranioplasty. This report illustrates the potential risks of subgaleal drainage, the importance of early recognition of this rare phenomenon and that intervention can be potentially life-saving.

Computed Tomography-Measured Psoas Density Predicts Outcomes After Enterocutaneous Fistula Repair.

BACKGROUND: Low muscle mass and quality are associated with poor surgical outcomes. We evaluated computed tomography (CT)-measured psoas muscle density as a marker of muscle quality and physiologic reserve and hypothesized that it predicts poor outcomes after enterocutaneous fistula repair (ECF). METHODS: We conducted a retrospective cohort study of patients 18-90 years old with ECF who failed nonoperative management, requiring elective operative repair at The Ohio State University (2005-2016), and who received preoperative abdomen/pelvis CT scan with intravenous contrast within 3 months of the operation. Psoas Hounsfield unit average calculations were measured at the L3 level. One-year leak rate, mortality (90 days, 1 years, and 3 years), complication risk, length of stay, dependent discharge, and 30-day readmission were compared with Hounsfield unit average calculation (HUAC). RESULTS: One hundred patients met inclusion criteria. Patients were stratified into interquartile ranges based on HUAC. The lowest HUAC interquartile was our low muscle quality (LMQ) cutoff, which was associated with 1-year leak (relative risk [RR] = 2.10, P < .005), 1-year mortality (RR = 2.22, P < .04) and 3-year mortality (RR = 2.13, P < .007), complication risk (RR = 1.54, P < .001), and dependent discharge (RR = 2.50, P < .004) compared to patients without LMQ. CONCLUSIONS: Psoas muscle density is a significant predictor of poor outcomes in ECF repair. This readily available measure of physiologic reserve can identify patients with ECF who have increased risk and may benefit from additional interventions and recovery time before operative repair.

Computed tomography measured psoas density predicts outcomes in trauma.

BACKGROUND: Age-related loss of muscle mass and function (sarcopenia) is linked to poor outcomes after operation and trauma. Here we evaluate computed tomography measured psoas muscle density and area using quick and simple tools available to the bedside clinician. We hypothesize these measures will predict poor outcomes after blunt traumatic injury. METHODS: We conducted a retrospective cohort study of patients ages ≥45 years in the Ohio State University Trauma Registry in 2008 that received a computed tomographic scan of the abdomen and/or pelvis with intravenous contrast. Psoas Index and Hounsfield unit average calculation were measured at the L3 level. In the study, 90-day mortality, complication, duration of stay ≥7 days, and dependent discharge were compared with Psoas Index and Hounsfield unit average calculation. RESULTS: In the study, 151 patients met the inclusion criteria. Patients were stratified into interquartile ranges based either on Psoas Index or Hounsfield unit average calculation values. After adjustment with sex-specific cutoffs, the lowest interquartile range of Psoas Index was associated with 90-day mortality (relative risk [RR] 5.95, P < .008), but did not reach significance in other outcomes. The lowest interquartile range of Hounsfield unit average calculation was associated with 90-day mortality (RR 5.95, P < .008), duration of stay ≥ 7 days (RR 1.63, P = .048), complication risk (RR 2.30, P = .002), and dependent discharge 2.14, P = .015). CONCLUSION: Psoas muscle density is a significant predictor of poor outcomes after traumatic injury. This objective, quick, and readily available measure of sarcopenia can identify patients requiring aggressive nutritional and physical therapy to improve prognosis, prevent recurrent traumatic injury, and aid in discharge planning.

pH-Sensitive and Thermosensitive Hydrogels as Stem-Cell Carriers for Cardiac Therapy.

Stem-cell therapy has the potential to regenerate damaged heart tissue after a heart attack. Injectable hydrogels may be used as stem-cell carriers to improve cell retention in the heart tissue. However, current hydrogels are not ideal to serve as cell carriers because most of them block blood vessels after solidification. In addition, these hydrogels have a relatively slow gelation rate (typically >60 s), which does not allow them to quickly solidify upon injection, so as to efficiently hold cells in the heart tissue. As a result, the hydrogels and cells are squeezed out of the tissue, leading to low cell retention. To address these issues, we have developed hydrogels that can quickly solidify at the pH of an infarcted heart (6-7) at 37 °C but cannot solidify at the pH of blood (7.4) at 37 °C. These hydrogels are also clinically attractive because they can be injected through catheters commonly used for minimally invasive surgeries. The hydrogels were synthesized by free-radical polymerization of N-isopropylacrylamide, propylacrylic acid, hydroxyethyl methacrylate-co-oligo(trimethylene carbonate), and methacrylate poly(ethylene oxide) methoxy ester. Hydrogel solutions were injectable through 0.2-mm-diameter catheters at pH 8.0 at 37 °C, and they can quickly form solid gels under pH 6.5 at 37 °C. All of the hydrogels showed pH-dependent degradation and mechanical properties with less mass loss and greater complex shear modulus at pH 6.5 than at pH 7.4. When cardiosphere-derived cells (CDCs) were encapsulated in the hydrogels, the cells were able to survive during a 7-day culture period. The surviving cells were differentiated into cardiac cells, as evidenced by the expression of cardiac markers at both the gene and protein levels, such as cardiac troponin T, myosin heavy chain α, calcium channel CACNA1c, cardiac troponin I, and connexin 43. The gel integrity was found to largely affect CDC cardiac differentiation. These results suggest that the developed dual-sensitive hydrogels may be promising carriers for cardiac cell therapy.

Modest agreement in ECG interpretation limits the application of ECG screening in young athletes.

BACKGROUND: Athlete ECG screening has been recommended by several international sporting bodies; however, a number of controversies remain regarding the accuracy of ECG screening. An important component that has not been assessed is the reproducibility of ECG interpretation. OBJECTIVE: The purpose of this study was to assess the variability of ECG interpretation among experienced physicians when screening a large number of athletes. METHODS: A sports cardiologist, a sports medicine physician, and an electrophysiologist analyzed 440 consecutive screening ECGs from asymptomatic athletes and were asked to classify the ECGs according to the 2010 European Society of Cardiology criteria as normal (or demonstrating training related ECG changes) or abnormal. When an abnormal ECG was identified, they were asked to outline what follow-up investigations they would recommend. RESULTS: The reported prevalence of abnormal ECGs ranged from 13.4% to 17.5%. Agreement on which ECGs were abnormal ranged from poor (κ = 0.297) to moderate (κ = 0.543) between observers. Suggested follow-up investigations were varied, and follow-up costs ranged from an additional A$30-A$129 per screening episode. Neither of the 2 subjects (0.45%) in the cohort with significant pathology diagnosed as a result of screening were identified correctly by all 3 physicians. CONCLUSION: Even when experienced physicians interpret athletes' ECGs according to current standards, there is significant interobserver variability that results in false-positive and false-negative results, thus reducing the effectiveness and increasing the social and economic cost of screening.

Cardiac differentiation of cardiosphere-derived cells in scaffolds mimicking morphology of the cardiac extracellular matrix.

Stem cell therapy has the potential to regenerate heart tissue after myocardial infarction (MI). The regeneration is dependent upon cardiac differentiation of the delivered stem cells. We hypothesized that timing of the stem cell delivery determines the extent of cardiac differentiation as cell differentiation is dependent on matrix properties such as biomechanics, structure and morphology, and these properties in cardiac extracellular matrix (ECM) continuously vary with time after MI. In order to elucidate the relationship between ECM properties and cardiac differentiation, we created an in vitro model based on ECM-mimicking fibers and a type of cardiac progenitor cell, cardiosphere-derived cells (CDCs). A simultaneous fiber electrospinning and cell electrospraying technique was utilized to fabricate constructs. By blending a highly soft hydrogel with a relatively stiff polyurethane and modulating fabrication parameters, tissue constructs with similar cell adhesion property but different global modulus, single fiber modulus, fiber density and fiber alignment were achieved. The CDCs remained alive within the constructs during a 1week culture period. CDC cardiac differentiation was dependent on the scaffold modulus, fiber volume fraction and fiber alignment. Two constructs with relatively low scaffold modulus, ∼50-60kPa, most significantly directed the CDC differentiation into mature cardiomyocytes as evidenced by gene expressions of cardiac troponin T (cTnT), calcium channel (CACNA1c) and cardiac myosin heavy chain (MYH6), and protein expressions of cardiac troponin I (cTnI) and connexin 43 (CX43). Of these two low-modulus constructs, the extent of differentiation was greater for lower fiber alignment and higher fiber volume fraction. These results suggest that cardiac ECM properties may have an effect on cardiac differentiation of delivered stem cells.

Comparison of frequency of significant electrocardiographic abnormalities in endurance versus nonendurance athletes.

Most data assessing the accuracy of electrocardiographic (ECG) screening in identifying cardiac pathology in athletes are derived from relatively unselected cohorts of subjects involved in competitive sports. We hypothesized that the prevalence of ECG abnormalities may be greater in athletes performing the greatest combination of exercise intensity and duration, namely professional endurance athletes. A total of 1,007 male and 254 female elite adult athletes underwent cardiovascular screening inclusive of an electrocardiogram, interpreted using the 2010 European Society of Cardiology guidelines. Training-related ECG changes (group 1) were more common in endurance athletes (EAs) than nonendurance athletes (NEAs; 90.8% vs 86.0%, p = 0.04), as were multiple (≥2) training-related changes (78.9% vs 53.5%, p <0.0001). Group 2 ECG changes (previously considered uncommon and training unrelated) were seen in 18.1% of subjects and were twice as prevalent in EAs compared with NEAs (29.9% vs 15.1%, p <0.0001). Right ventricular hypertrophy (4.4% EAs vs 1.5% NEAs, p <0.005) and deep right precordial T-wave inversion (14.3% EAs vs 4.7% NEAs, p <0.0001) were 3 times as common in EAs. Both group 1 and group 2 changes were similarly prevalent among elite male and female athletes and were more common in EAs regardless of gender. In conclusion, ECG abnormalities are very common in elite athletes and are more common in EAs than NEAs. Right ventricular hypertrophy and deep right precordial T-wave inversion are particularly common in EAs, possibly because of increased structural and/or electrical right ventricular remodeling in this subgroup. The predictive value of ECG screening and criteria for abnormal findings in elite EAs requires specific appraisal.

The Seattle Criteria increase the specificity of preparticipation ECG screening among elite athletes.

BACKGROUND: In 2010, the European Society of Cardiology (ESC) released recommendations for the interpretation of the 12-lead ECG in athletes, dividing changes into group 1 (training related) and group 2 (training unrelated). Recently, the 'Seattle Criteria', a series of revisions to these recommendations, was published, with the aim of improving the specificity of ECG screening in athletes. OBJECTIVES: First, to assess the prevalence of ECG abnormalities in a cohort of elite Australian athletes using the 2010 ESC recommendations and determine how often group 2 ECG changes correlate with the evidence of significant cardiac pathology on further investigation. Second, to assess the impact of the 'Seattle Criteria' in reducing the number of athletes with ECG abnormalities in whom further cardiac testing is unremarkable ('false positives'). DESIGN: 1197 elite athletes underwent cardiovascular screening between 2011 and 2012, of whom 1078 aged 16-35 years volunteered and were eligible to participate. RESULTS: 186 (17.3%) had an abnormal ECG according to ESC recommendations and a further 30 (2.8)% had unclassified changes. Three athletes (0.3%) were found to have a cardiac abnormality on further investigation. Using the Seattle Criteria, the number of athletes classified as abnormal fell to 48 (4.5%, p<0.0001) and the three with an underlying cardiac abnormality were still identified. The improved specificity was due to reclassification of 71 athletes (6.6%) with an equivocal QTc interval, 42 (3.9%) with T wave inversion isolated to V1-2 and 22 (2%) with either isolated right axis deviation or right ventricular hypertrophy on voltage criteria. CONCLUSIONS: The 'Seattle Criteria' reduced the false-positive rate of ECG screening from 17% to 4.2%, while still identifying the 0.3% of athletes with a cardiac abnormality.

Factors associated with decreased kidney function in HIV-infected adults enrolled in the MTCT-Plus Initiative in sub-Saharan Africa.

BACKGROUND: Pre-existing kidney disease in HIV-infected patients may necessitate dose modification of antiretroviral therapy (ART). Despite increasing ART availability, there are few prevalence studies of chronic kidney disease in HIV-infected individuals across multiple African countries. METHODS: Routine laboratory data obtained before ART initiation were used to evaluate prevalence and predictors of decreased creatinine clearance (CrCl) in participants of the MTCT-Plus Initiative from 7 sub-Saharan countries. Cockcroft-Gault equation was used to estimate CrCl and logistic regression modeling to identify factors associated with CrCl <50 mL/min. RESULTS: Of 2495 individuals evaluated, median age was 30 years (interquartile range: 27-35); 70% were women. Median CD4+ cell count was 295 (interquartile range: 173-450); 78% were World Health Organization stage 1/2. Median CrCl was 95 mL/min. Overall, 3.4% [95% confidence interval (CI): 2.7 to 4.1] of patients had a CrCl <50 mL/min. Age >30 years (odds ratio = 2.06; 95% CI: 1.23 to 3.45) and CD4+ count <50 cells per cubic millimeter (odds ratio = 5.4 for CD4+ <50, 95% CI: 2.5 to 11.9) were associated with CrCl <50 mL/min. CONCLUSIONS: The prevalence of clinically significant kidney disease was low in this relatively healthy population of HIV-infected adults, and few participants would have required ART dose reductions. These findings support recent World Health Organization guidelines to initiate ART without routine laboratory screening. Our findings suggest that available laboratory resources could be targeted to older persons and those with very low CD4+ cell count.

Adult clinical and immunologic outcomes of the national antiretroviral treatment program in Rwanda during 2004-2005.

BACKGROUND: By December 2007, over 48,000 persons had initiated antiretroviral treatment (ART) at 171 clinics in Rwanda. Assessing national ART program outcomes is essential to determine whether programs have the desired impact. METHODS: We conducted a retrospective cohort study to assess key 6- and 12-month outcomes among a nationally representative, stratified, random sample of 3194 adults (> or =15 years) who initiated ART from January 1, 2004, through December 31, 2005. FINDINGS: At ART initiation, the median patient age was 37 years and 65% were female. Overall, the baseline median CD4 cell count was 141 cells per microliter. At 6 and 12 months after ART initiation, 92% and 86% of patients, respectively, remained on ART at their original site. By 6 months, 3.6% were dead and 3.4% were lost to follow-up; by 12 months, 4.6% were dead and 4.9% were lost to follow-up. Among patients with available follow-up CD4 cell count data, median CD4 cell counts increased by 98 cells per microliter and 119 cells per microliter at 6 and 12 months after ART initiation, respectively. CONCLUSIONS: Rwanda's national ART program achieved excellent 6- and 12-month retention and immunologic outcomes during the first 2 years of rapid scale-up. Routine supervision is required to improve compliance with clinical guidelines and data quality.

A novel single-base deletion in ROR2 causes atypical brachydactyly type B1 with cutaneous syndactyly in a large Chinese family.

Mutations in ROR2, encoding the receptor tyrosine kinase-like orphan receptor 2, cause two distinct skeletal diseases: autosomal dominant brachydactyly type B1 (BDB1) and autosomal recessive Robinow syndrome. In a large Chinese family with a limb phenotype, consisting of atypical BDB1 and cutaneous syndactyly of varying degrees, we performed a two-point linkage analysis using microsatellite markers on 2q33-q37 and 9q22.31, and found a significant linkage to the ROR2 locus. We identified a novel single-base deletion in ROR2, c.2243delC (p.W749fsX24), and confirmed its segregation with the limb phenotype in the family. This deletion is predicted to produce a truncated ROR2 protein with an additional C-terminal polypeptide of 24 amino-acid residues. To the best of our knowledge, the deletion represents the second ROR2 mutation associated with a BDB1-syndactyly phenotype.

Copy-number mutations on chromosome 17q24.2-q24.3 in congenital generalized hypertrichosis terminalis with or without gingival hyperplasia.

Congenital generalized hypertrichosis terminalis (CGHT) is a rare condition characterized by universal excessive growth of pigmented terminal hairs and often accompanied with gingival hyperplasia. In the present study, we describe three Han Chinese families with autosomal-dominant CGHT and a sporadic case with extreme CGHT and gingival hyperplasia. We first did a genome-wide linkage scan in a large four-generation family. Our parametric multipoint linkage analysis revealed a genetic locus for CGHT on chromosome 17q24.2-q24.3. Further two-point linkage and haplotyping with microsatellite markers from the same chromosome region confirmed the genetic mapping and showed in all the families a microdeletion within the critical region that was present in all affected individuals but not in unaffected family members. We then carried out copy-number analysis with the Affymetrix Genome-Wide Human SNP Array 6.0 and detected genomic microdeletions of different sizes and with different breakpoints in the three families. We validated these microdeletions by real-time quantitative PCR and confirmed their perfect cosegregation with the disease phenotype in the three families. In the sporadic case, however, we found a de novo microduplication. Two-color interphase FISH analysis demonstrated that the duplication was inverted. These copy-number variations (CNVs) shared a common genomic region in which CNV is not reported in the public database and was not detected in our 434 unrelated Han Chinese normal controls. Thus, pathogenic copy-number mutations on 17q24.2-q24.3 are responsible for CGHT with or without gingival hyperplasia. Our work identifies CGHT as a genomic disorder.

Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis.

Marie Unna hereditary hypotrichosis (MUHH) is an autosomal dominant form of genetic hair loss. In a large Chinese family carrying MUHH, we identified a pathogenic initiation codon mutation in U2HR, an inhibitory upstream ORF in the 5' UTR of the gene encoding the human hairless homolog (HR). U2HR is predicted to encode a 34-amino acid peptide that is highly conserved among mammals. In 18 more families from different ancestral groups, we identified a range of defects in U2HR, including loss of initiation, delayed termination codon and nonsense and missense mutations. Functional analysis showed that these classes of mutations all resulted in increased translation of the main HR physiological ORF. Our results establish the link between MUHH and U2HR, show that fine-tuning of HR protein levels is important in control of hair growth, and identify a potential mechanism for preventing hair loss or promoting hair removal.

Prospect of medical genetics in China from a historical point of view.

The history of medical genetics is briefly reviewed. It is evident that medical genetics with its inseparable part, clinical genetics, started out as a clinical science from the very beginning. Its robust development in the developed countries is the result of a close interaction between the basic sciences and clinical genetics. In China, however, clinical genetics has not received due emphasis and medical genetics is still not recognized as one of the medical specialties. This is in marked contrast to the situation in the West It is high time to acknowledge that medical genetics is a medical specialty and to promote clinical genetics service in qualified hospitals in our country.

Novel point mutations in GDF5 associated with two distinct limb malformations in Chinese: brachydactyly type C and proximal symphalangism.

Growth/differentiation factor 5 (GDF5) is a secreted growth factor that plays a key regulatory role in embryonic skeletal and joint development. Mutations in the GDF5 gene can cause different types of skeletal dysplasia, including brachydactyly type C (BDC) and proximal symphalangism (SYM1). We report two novel mutations in the GDF5 gene in Chinese families with distinct limb malformations. In one family affected with BDC, we identified a novel nonsense mutation, c.1461T > G (p.Y487X), which is predicted to truncate the GDF5 precursor protein by deleting 15 amino acids at its C-terminus. In one family with SYM1, we found a novel missense mutation, c.1118T > G (p.L373R), which changes a highly conserved amino acid in the prodomain of GDF5. We transfected COS-7 cells with retroviral constructs to express human wild-type or mutant GDF5 cDNAs. The mature GDF5 protein was detected, as in the wild-type, in supernatant derived from the p.L373R mutant GDF5 transfected cells, but not in the supernatant from the p.Y487X mutant transfected cells, indicating that the two mutations led to different fates of the mutant GDF5 proteins, thereby producing distinct limb phenotypes.

Mutations in HOXD13 underlie syndactyly type V and a novel brachydactyly-syndactyly syndrome.

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A-->G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.

A novel deletion in TNNI2 causes distal arthrogryposis in a large Chinese family with marked variability of expression.

Distal arthrogryposis (DA) is composed of a group of clinically and genetically heterogeneous disorders, characterized by multiple congenital contractures of the limbs. Point mutations in three genes encoding contractile fast-twitch myofibers, TPM2, TNNI2 and TNNT3, were recently identified in DA type 1 (DA1; MIM 108120) and DA type 2B (DA2B; MIM 601680). We have described a large Chinese DA family in which different individuals had phenotypes similar to DA1 or DA2B. To map the disease locus in this family, two-point linkage analysis was first performed using microsatellite markers selected from the genomic regions close to the TPM2, TNNI2/TNNT3 and TNNC2 genes. A positive LOD score of 3.61 at theta = 0 was obtained with the marker close to the TNNI2/TNNT3 genes, corresponding to the genetic mapping site of DA2B. Direct sequencing of the PCR-amplified DNA fragment spanning exon 8 of the TNNI2 gene showed a heterozygous deletion, c.523_525delAAG (p.K175del), in the proband. This novel mutation was confirmed to cosegregate with the DA phenotype in affected individuals but not detected in all unaffected individuals of the family and not in 50 healthy controls. In summary, we have found a novel TNNI2 mutation in a Chinese family with DA2B. Our work represents the first report on the link between TNNI2 and the DA phenotype in Chinese.

[A DNA duplication at chromosome 10q24.3 is associated with split-hand split-foot malformation in a Chinese family].

OBJECTIVE: To identify the disease-causing genetic alteration of split-hand/split-foot malformation (SHFM) in a Chinese family. METHODS: Three of the 5 affected individuals from a four-generation Chinese SHFM family were examined physically and radiologically. Peripheral blood samples were collected from Digital photographs of the malformed hands and feet were taken. Peripheral blood samples were collected from 2 affected individuals, and lymphocytes were isolated to undergo high resolution G-banding. Genomic DNA was extracted from the whole blood samples of 4 available family members, including the 3 affected individuals. All 16 exons and their flanking intronic sequences of the TP63 gene were amplified using polymerase chain reaction (PCR) and sequenced directly. Microsatellite markers from the five SHFM loci were analyzed in the available family members by PCR, polyacrylamide gel electrophoresis and silver staining. For semi-quantitative determination of the allele copy number, the polymorphic PCR-amplified fragments representing genetic markers from the SHFM3 locus at chromosome 10q24.3 were sequenced in the affected individuals using normal individuals with identical genotypes as controls. RESULTS: All 3 existing affected individuals showed absence of 3 radial fingers, 2 affected individuals had a deep central cleft and central ray deficiency in the feet, and 1 affected individual had a fibular monodactyli, all limb malformations being bilateral and consistent with the phenotype of typical SHFM. G-banding showed normal karyotypes in the 3 affected individuals and no visible cytogenetic abnormality was found. Moreover, no mutation was identified in the TP63 gene. While no haplotype sharing was observed in the markers from loci SHFM1, SHFM4 and SHFM5, potential haplotype sharing was detected in the markers from two loci, SHFM2 and SHFM3, indicating possible causative mutation at SHFM2 or SHFM3. Furthermore, obviously biased silver density toward the allele fragments shared by the 3 affected individuals was observed in the markers from the SHFM3 locus. Comparative sequencing showed roughly one-fold increase of fluorescent signal of the shared fragments in the affected individuals. These results suggested a large-scale DNA duplication within the SHFM3 locus. CONCLUSION: A large-scale DNA duplication within the SHFM3 locus at chromosome 10q24.3 has been identified as the pathogenic genetic change in Chinese patients with SHFM.

[A de nono I462S mutation in the KRT6A gene is associated with pachyonychia congenita type I].

OBJECTIVE: To analyze the KRT6A gene mutation and mutating patterns in a sporadic Chinese patient with Pachyonychia congenita (PC)-1 so as to provide a basis for gene diagnosis and genetic counseling of this disorder. METHODS: Genomic DNA was extracted from whole blood by standard methods from a female patient with PC-1 and her parents, and from 50 normal, unrelated individuals. Primers for specific amplification of the structural KRT6A gene without co amplification of homologous genes were designed and synthesized. All exons of the gene and their flanking intronic sequences were amplified using polymerase chain reaction (PCR) and subjected to automatic DNA sequencing. The mutation was confirmed by Mbo I restriction digestion of the KRT6A-specific PCR products. RESULTS: Direct sequencing of the PCR products revealed a novel heterozygous missense mutation, I462S in the KRT6A gene, which resulted from T to G transversion at nucleotide 1385 (1385T > G) in exon 7 was detected in the patient. This mutation would result in the substitution of Isoleucine by Serine at codon 462 (I462S) located in the end 2B domain of keratin 6A. No such mutation was found in the patient's parents by sequencing of PCR products and this mutation was confirmed in the patient and excluded from both parents and 50 normal, unrelated controls by restriction analysis of PCR fragments using Mbo I enzyme. CONCLUSIONS: A de novo missense mutation in the KRT6A gene, I462S, has been found in a sporadic PC-1 patient. The identification of this novel mutation in the KRT6A gene provides further evidence that mutation in the KRT6A gene causes PC-1 phenotype.