Newborn screening for spinal muscular atrophy in Germany: clinical results after 2 years.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since motor neuron injury is usually not reversible, early diagnosis and treatment are essential to prevent major disability. Our objective was to assess the impact of genetic newborn screening for SMA on outcome. METHODS: We provided clinical data from 43 SMA patients, identified via polymerase chain reaction of the SMN1 gene from dried blood spots between January 2018 and January 2020 in Germany. Follow-up included neurophysiological examinations and standardized physiotherapeutic testing. RESULTS: Detection of SMA with newborn screening was consistent with known incidence in Germany. Birth prevalence was 1:6910; 39.5% had 2 SMN2 copies, 23% had 3 SMN2 copies, 32.5% had 4 copies, and 4.5% had 5 copies of the SMN2 gene. Treatment with SMA-specific medication could be started at the age of 14-39 days in 21 patients. Pre-symptomatically treated patients remained throughout asymptomatic within the observation period. 47% of patients with 2 SMN2 copies showed early, presumably intrauterine onset of disease. These patients reached motor milestones with delay; none of them developed respiratory symptoms. Untreated children with 2 SMN2 copies died. Untreated children with 3 SMN2 copies developed proximal weakness in their first year. In patients with ≥ 4 SMN2 copies, a follow-up strategy of "watchful waiting" was applied despite the fact that one of them was treated from the age of 6 months. Two infant siblings with 4 SMN2 copies were identified with a missed diagnosis of SMA type 3. CONCLUSION: Identification of newborns with infantile SMA and prompt SMA-specific treatment substantially improves neurodevelopmental outcome, and we recommend implementation in the public newborn screening in countries where therapy is available. Electrophysiology is a relevant parameter to support the urgency of therapy. There has to be a short time interval between a positive screening result and referral to a therapy-ready specialized treatment center.

One Year of Newborn Screening for SMA - Results of a German Pilot Project.

OBJECTIVE: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. The study was conducted to assess the impact of early detection of SMA by newborn screening (NBS) on the clinical course of the disease. METHODS: Screening was performed in two federal states of Germany, Bavaria and North Rhine Westphalia, between January 2018 and February 2019. The incidence in the screening population was calculated as number of detected patients with a homozygous deletion in the SMN1-gene per number of screened patients. To get an idea about the incidence of newly diagnosed SMA in the year prior to screening a survey covering all neuropediatric centers in the state of Bavaria was conducted, identifying all SMA-cases in 2017 and 2018. Following positive NBS and confirmatory diagnostic test, treatment was advised according to the recommendations of the "American SMA NBS Multidisciplinary Working Group". Immediate treatment with Nusinersen was recommended in children with 2 and 3 SMN2 copies and a conservative strict follow-up strategy in children with ≥4 copies. All children underwent regular standardized neuropediatric examination, CHOP INTEND and HINE-2 testing as well as electrophysiological exams every 2-3 months. RESULTS: 165,525 children were screened. 22 cases of SMA were identified, meaning an incidence rate of 1:7524. SMN2 copy number analysis showed 2 SMN2 copies in 45% of patients, 3 SMN2 copies in 19 % and 4 SMN2 copies in 36%. These findings are confirmed in the most recent statistical data-cut from 31st August 2019 (incidence 1:7089, 2 SMN2 copies in 44%, 3 in 15% and 4 in 38%). Comparison with up-to-date German data on SMA incidence and the Bavarian survey give evidence that NBS did not lead to a relevant increase in incidence. 10 patients with 2 or 3 SMN2 copies were treated with Nusinersen, starting between 15- 39 days after birth, in 7/10 patients before onset of symptoms. Presymptomatically treated patients (age at last examination: 1- 12 months, median 8 months) showed no muscle weakness by the age of one month to one year. One child with 4 SMN2 copies became symptomatic at the age of 8 months. CONCLUSIONS: Newborn screening, resulting in presymptomatic treatment, improves outcome in children with genetically proven SMA. Newborn screening for SMA should be introduced in all countries where therapy is available. An immediate therapy in cases with 4 SMN2 copies should be considered.

In situ assay of fatty acid ß-oxidation by metabolite profiling following permeabilization of cell membranes.

Quantitative analysis of mitochondrial FA ß-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve ß-oxidation function. The aim was to develop a new assay to quantify ß-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on ß-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO.

Newborn screening for isovaleric acidemia using tandem mass spectrometry: data from 1.6 million newborns.

BACKGROUND: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used in the Bavarian newborn screening (NBS) program since 1999. The use of ESI-MS/MS has led to the inclusion of isovaleric acidemia (IVA) into NBS. We retrospectively evaluated data on more than 1.6 million newborns screened during 9.5 years. METHODS: Acylcarnitines from whole blood spotted on filter paper were converted to their corresponding butyl esters, and the samples were analyzed by use of ESI-MS/MS with stable isotope labeled internal standards. RESULTS: A total of 24 individuals with IVA were detected by use of a multiparametric threshold criteria panel including isovalerylcarnitine (C5) and the ratios of C5 to octanoyl-, butyryl-, and propionylcarnitine. A cutoff set at the 99.99th percentile for isolated C5 or at the 99th percentile for C5 plus at least 2 ratios resulted in a positive predictive value for IVA screening of 7.0% and an overall recall rate of 0.024%. Adjusted reference ranges for age and birth weight were applied, and the incidence of IVA in the study population was calculated to be 1 in 67,000. Missed cases were not brought to our attention. IVA was also detectable in cord blood and early postnatal blood samples. CONCLUSIONS: IVA can be reliably detected in NBS through acylcarnitine analysis in dried blood spots by using multiparametric threshold criteria. Further improvement (positive predictive value 13.0%, recall rate 0.01%) can be achieved by using more stringent recall criteria. In view of the potentially life-threatening natural course of IVA in early life, presymptomatic diagnosis may thus prevent mortality and morbidity.

Neonatal screening for metabolic and endocrine disorders.

BACKGROUND: Neonatal screening for treatable endocrinopathies and inborn errors of metabolism is an important preventive measure. Advances in the diagnosis and treatment of these diseases have made it necessary to expand the screening program. METHODS: This article is based on a selective literature review and our clinical experience. RESULTS: In 2005, neonatal screening in Germany was expanded from 3 to 14 diseases, as mandated by the responsible governmental authority (the Gemeinsamer Bundesausschuss, i.e., Joint Federal Committee). From 2005 to 2008, screening revealed diseases requiring treatment in 1932 out of a total of 2,758,633 newborns (prevalence, 1 in 1428). The expansion of the screening program resulted in a 57% increase in the overall number of cases detected and a 92% increase for metabolic diseases alone. CONCLUSION: The German neonatal screening program for treatable endocrinopathies and inborn errors of metabolism is a complex and integrated preventive measure that has become markedly more effective as a result of its expansion in 2005.

Correlation of HPV16 L1 capsid protein expression in cervical dysplasia with HPV16 DNA concentration, HPV16 E6*I mRNA and histologic outcome.

OBJECTIVE: To determine whether the absence ofHPV1i6 LI capsid protein is a prognostic parameter for the histologic outcome of cervical intraepithelial neoplasia (CIN) 2+ in cytologically diagnosed cervical dysplasia. STUDY DESIGN: Papanicolaou-stained microscopic slides of 95 HPV16-positive cervical samples that had a cytologic diagnosis of cervical dysplasia or borderline cytology were immunostained using an HPV16-specific anti-L1 viral capsid antibody. In parallel to the cytologic examination, HPV16 DNA and E6*I mRNA were quantitated using real-time polymerase chain reaction. Expression of L1 protein was correlated with relative levels of HPV16 DNA and E6*I mRNA as well as histologic diagnoses/cytologic follow-up. RESULTS: Thirty-five cases with a histologic diagnosis of CIN 2+ (61%) were negative for HPV16 L1 protein; 22 (39%) were positive. Of the cases that either were CIN 1 or regressed to normal cytology, 10 cases (26%) were positive for HPV16 L1 protein, while 28 (74%) were negative. L1-negative and L1-positive cases showed no statistically significant difference (p = 0.22) in their histologic diagnosis/cytologic follow-up. The positive predictive value for CIN 2+ was 56% if L1 protein was absent; the negative predictivre value for CIN 1/regression to normal cytology was 31% if L1 protein was present. HPV16 Ll-positive cases had significantly higher HPV16 DNA concentrations than L1-negative cases (p < 0.001), while levels of HPV16 E6*I mRNA were comparable in both types of cases (p = 0.36). CONCLUSION: The expression of HPV16 L1 capsid protein is correlated with viral DNA load but does not predict the histologic outcome of HPV16-positive cervical dysplasia.

Stability of acylcarnitines and free carnitine in dried blood samples: implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency.

OBJECTIVE: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in newborn screening programs. Acylcarnitine profiles from dried blood spots (DBS) are used to detect fatty acid oxidation disorders, carnitine cycle disorders, and organic acidurias. Stored dried blood is also a valuable source for postmortem investigations to unravel the cause of unexplained death in early childhood. However, diagnostic uncertainties arising from the unknown stability of acylcarnitines and free carnitine during prolonged storage have not yet been studied in a systematic manner. METHODS: Whole blood spiked with acylcarnitines was stored either at -18 degrees C or at room temperature up to 1000 days. At regular time intervals 3.2 mm spots of these samples were extracted with 150 microL of methanol. Free carnitine and acylcarnitines were converted to their corresponding butyl esters and analyzed by ESI-MS/MS. RESULTS: At -18 degrees C acylcarnitines are stable for at least 330 days. If stored for prolonged periods at room temperature (>14 days), acylcarnitines are hydrolyzed to free carnitine and the corresponding fatty acids. The velocity of decay is logarithmic and depends on the chain length of the acylcarnitines. Short-chain acylcarnitines hydrolyze quicker than long-chain acylcarnitines. CONCLUSION: The data indicate that stored filter cards should only be used for retrospective quantitation of acylcarnitines if appropriate correction for sample decay during storage is applied. Free carnitine increases upon storage but can reliably be quantitated under standardized derivatization conditions. Furthermore, carnitine transporter (OCTN2) deficiency can reliably be diagnosed by examining acylcarnitine profiles, which can supplement free carnitine levels as a discriminatory marker.

Determination of EDTA in dried blood samples by tandem mass spectrometry avoids serious errors in newborn screening.

Newborn screening programs use whole blood dried on filter paper as the standard specimen. Metabolites are reasonably stable and can easily be sent to screening laboratories by regular mail. The recommended sample collection procedure is to spot native blood without anticoagulants onto the filter paper, because anticoagulants can interfere with the different laboratory methods. However, visual examination of the blood spots cannot always detect contamination. In this study, whole blood was drawn by venous puncture from a healthy volunteer, spiked with the corresponding metabolites and EDTA, and spotted onto filter paper. TSH and 17alpha-hydroxyprogesterone were determined by time resolved fluoroimmunoassays with the AutoDelfia system. Total galactose, biotinidase activity, and galactose-1-phosphate uridyltransferase activity were measured photometrically or fluorometrically. Succinyl acetone was estimated indirectly through the inhibition of porphobilinogen synthase activity (PBGS assay). EDTA, amino acids, and acylcarnitines were converted to the corresponding butyl esters, after extraction with methanol, and analysed by LC-MS/MS. EDTA contamination gives falsely elevated 17-OHP values and falsely reduced TSH and PBGS values. The inclusion of an EDTA determination in routine screening revealed that at least 0.06% of newborn screening samples were contaminated with EDTA. In conclusion, non-conformity during the pre-analytical phase is a source of false positive and false negative screening results. Determination of EDTA from NBS blood spots can reliably identify these samples and prevent screening errors.

Newborn screening for inborn errors of metabolism and endocrinopathies: an update.

Newborn screening for inborn errors of metabolism and endocrinopathies has expanded during the last two decades, mainly owing to the introduction of new technologies such as tandem mass spectrometry and DNA analysis. However, every expansion of the screening panel requires critical review, discussion, and pilot studies. Different legal regulations and ethical concerns may lead to different decisions. Without claiming to be comprehensive, this review tries to give an overview of newborn screening, including its main problems and target diseases.

Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment.

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions. We elaborated the rationale for decision making in 3-methylcrotonyl-coenzyme A (CoA) carboxylase deficiency (MCCD), which afflicts leucine catabolism, with reported outcomes ranging from asymptomatic to death. In Bavaria, we screened 677,852 neonates for 25 conditions, including MCCD, based on elevated concentrations of 3-hydroxyisovalerylcarnitine (3-HIVA-C). Genotypes of MCCA (MCCC1) and MCCB (MCCC2) were assessed in identified newborns, their relatives, and in individuals (n = 17) from other regions, and correlated to biochemical and clinical phenotypes. NBS revealed eight newborns and six relatives with MCCD, suggesting a higher frequency than previously assumed (1:84,700). We found a strikingly heterogeneous spectrum of 22 novel and eight reported mutations. Allelic variants were neither related to biochemical nor anamnestic data of our probands showing all asymptomatic or benign phenotypes. Comparative analysis of case reports with NBS data implied that only few individuals (< 10%) develop symptoms. In addition, none of the symptoms reported so far can clearly be attributed to MCCD. MCCD is a genetic condition with low clinical expressivity and penetrance. It largely represents as nondisease. So far, there are no genetic or biochemical markers that would identify the few individuals potentially at risk for harmful clinical expression. The low ratio of benefit to harm was pivotal to the decision to exclude MCCD from NBS in Germany. MCCD may be regarded as exemplary of the ongoing controversy arising from the inclusion of potentially asymptomatic conditions, which generates a psychological burden for afflicted families and a financial burden for health care systems.

Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay.

We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.

Population spectrum of ACADM genotypes correlated to biochemical phenotypes in newborn screening for medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent inherited defect of fatty acid oxidation, with a significant morbidity and mortality in undiagnosed patients. Adverse outcomes can effectively be prevented by avoiding metabolic stress and following simple dietary measures. Therefore, prospective newborn screening (NBS) is being proposed for this condition. However, technical validation of MCADD population screening and assessment of its overall benefit require broadening of the as-yet-scarce knowledge of the MCADD genetic heterogeneity unraveled by NBS and its phenotypic consequences. Here, we describe the entire spectrum of sequence variations occurring in newborns with MCADD in the population of Bavaria, Germany, in relation to the biochemical phenotype. Among 524,287 newborns, we identified 62 cases of MCADD, indicating a birth incidence of 1 in 8,456. In all of the 57 newborns available for analysis, two alterations within the MCADD gene (ACADM) were identified. The most prevalent alteration c.985A>G (Lys329Glu) occurred in 27 (47%) newborns in the homozygous and in 18 (32%) in the heterozygous state (63% of defective alleles). The mild folding variant c.199T>C (Tyr67His) was identified in nine individuals, six of them being compound heterozygous with c.985A>G (Lys329Glu). Neither of the prevalent alterations were found in the remaining nine newborns. A total of 18 sequence variations were identified; 13 of them were novel: eight missense mutations, one nonsense mutation, two splice variants, and two small deletions. The remaining five were previously reported in MCADD patients. The ACADM heterogeneity uncovered was larger as anticipated from previous c.985A>G (Lys329Glu) carrier screening data. In addition, we show that MCADD appears to occur as frequently in Turkish newborns as in the native German population. Our data validate that biochemical NBS for MCADD is a highly specific procedure for disease detection, with the identification of a significant share of milder biochemical phenotypes, such as c.199T>C (Tyr67His). These show statistically lower acylcarnitine markers, allowing us to distinguish subgroups within the spectrum of ACADM sequence variations that correlate to biochemical MCADD disease expression. Our data might provide technical and medical guidance for decision making in the worldwide efforts to introduce MCADD population screening.

Rapid second-tier molecular genetic analysis for congenital adrenal hyperplasia attributable to steroid 21-hydroxylase deficiency.

BACKGROUND: Neonatal screening for steroid 21-hydroxylase (CYP21) deficiency is performed to identify congenital adrenal hyperplasia (CAH). The immunologic assay for 17alpha-hydroxyprogesterone (17-OHP) has a high rate of false positives. We assessed the potential for increasing the specificity for CAH by use of a second step involving analysis of the CYP21 gene. METHODS: Between January 1999 and December 2003, a total of 810,000 newborns were screened. Of these, 7920 had to be retested because their 17-OHP values were above the cutoff of the assay. Sixty-one had positive 17-OHP values in their recall samples and were diagnosed as having CAH. We used a rapid assay for common mutations of the CYP21 gene to analyze these 61 samples. In a prospective study, 198 consecutive samples that had increased 17-OHP and 100 samples that had normal 17-OHP concentrations were genotyped. RESULTS: Fifty-nine of 61 cases diagnosed as having CAH were confirmed genetically as CYP21 deficiencies. One patient had a 3beta-hydroxysteroid dehydrogenase deficiency, and one patient carried no CYP21 mutations. The 198 increased 17-OHP results were designated as false positives after immunologic testing of recall samples. None of these samples exhibited the genetic pattern consistent with CYP21 deficiency. CONCLUSIONS: If samples with increased 17-OHP values were screened genetically, the number of retests would decrease by approximately 90%, but the overall sensitivity of CAH screening would remain the same. Adding a second-tier genetic step would require a modest increase in costs, but is counterbalanced by fewer recalls, less clinical follow-up, and a reduction in unnecessary worry for families.

Simple technique for internal control of real-time amplification assays.

BACKGROUND: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. METHODS: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe-control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. RESULTS: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. CONCLUSIONS: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.

Screening for congenital adrenal hyperplasia: adjustment of 17-hydroxyprogesterone cut-off values to both age and birth weight markedly improves the predictive value.

Newborn screening procedures for congenital adrenal hyperplasia (CAH) are still suboptimal because of low specificity, particularly in premature infants. This study evaluated a multitiered strategy for reporting abnormal 17-hydroxyprogesterone screening values that simultaneously takes into account not only birth weight but also age at sampling. A cautious three-tiered cut-off scheme was used during the initial 24 months of CAH screening in Bavaria. Data were then reanalyzed using five birth weight classes to reflect more precisely the markedly higher values in low-birth-weight newborns. Because 17-hydroxyprogesterone values apparently decline with increasing age, these classes were then further subdivided into a total of 21 groups according to the age at sampling. Based on this reanalysis, we defined new multitiered cut-off levels and used them for the next 18 months. A total of 538466 newborns were screened from January 1999 to June 2002; 51 CAH cases were detected. Application of the new threshold values resulted in a 35% reduction of the total recall rate (from 1.13% to 0.74%) and an increase in the positive predictive value from 0.84% to 1.29% without reducing diagnostic sensitivity. This improvement of CAH screening can be achieved by simply using request forms that ask for both age and birth weight at the time of sampling.

Advances in analytical mass spectrometry to improve screening for inherited metabolic diseases.

UNLABELLED: Gas chromatography/mass spectrometry became available more than 30 years ago and has subsequently profoundly contributed not only in the identification of a wide range of inborn errors but also as a key tool for clinical diagnostic screening of genetic metabolic disease. Due to extraordinary advances in liquid chromatography and mass spectrometry (MS) developed in the last decade, the utilisation of MS and the potential number of applications for the purpose of metabolic screening is currently undergoing considerable expansion. CONCLUSIONS: This overview aims to describe only current new developments in clinically most relevant applications, in particular with focus on low molecular weight compounds.

Type-specific detection of human papillomaviruses in a routine laboratory setting--improved sensitivity and specificity of PCR and sequence analysis compared to direct hybridisation.

Human papillomaviruses (HPV) are known to cause cervical dysplasia and cervical carcinoma. We used a 3-step PCR protocol that allows rapid type-specific HPV testing in a routine laboratory setting: HPV-16-positive samples were determined using a specific LightCycler PCR; HPV-16-negative samples were amplified by nested PCR and typed by sequence analysis. During a period of 7 months, 1275 PCR-based HPV tests were performed. Of the 1275 samples, 829 samples tested negative for HPV and 446 tested positive, including 124 positives found in the initial HPV-16-specific LightCycler assay. Sequence analysis of 132 samples detected 18 HPV types that are not included in the widely used Hybrid Capture II assay. For comparison, the first 100 cervical specimens were tested in parallel using PCR and direct hybridisation (Hybrid Capture II assay). PCR detected HPV DNA in 23 samples that tested negative in the Hybrid Capture assay. Four out of 37 samples that tested positive for HPV in the Hybrid Capture test may be false positives, because sequence analysis detected HPV types not included in the probe mixtures. As rare and novel HPV types may also confer an oncogenic risk, highly sensitive and specific PCR assays will help in understanding cervical HPV infection and cervical cancer of unknown causes.